(Hadlock, Journal of Virology, 2000), (Keck, Journal of Virology, 2008) CBH-5 Domain B 412, 416, 417, 418, 420, 421, 422, 423, 483, 484, 485, 488,

Transcription

1 Supplementary Table. mabs analyzed mab Antigenic Critical Binding Residues by Citations Domains Alanine Scanning 2 CBH-4B Domain A NA (Hadlock, Journal of Virology, 2), (Keck, Journal of Virology, 24) HC- Domain B 529, 53, 535, 536, 537, 54 (Keck, Journal of Virology, 28) CBH-2 Domain B 425, 426, 43, 434, 436, 523, 529, 53, 535, 536 (Hadlock, Journal of Virology, 2), (Keck, Journal of Virology, 28) CBH-5 Domain B 42, 46, 47, 48, 42, 42, 422, 423, 483, 484, 485, 488, (Hadlock, Journal of Virology, 2), (Owsianka, J. Gen. Virol., 28) 523, 525, 527, 53, 533, 535, 538, 54, 55 CBH-7 Domain C 538, 54, 547, 549 (Hadlock, Journal of Virology, 2), (Owsianka, J. Gen. Virol., 28) HC84.22 Domain D 42, 426, 428, 429, 437, 44, (Keck, PLOSPathogens, 22) 442, 443, 53, 66 HC84.26 Domain D 429, 44, 442, 446, 66 (Keck, PLOSPathogens, 22) HC33.4 Epitope "" 43, 48, 42 (Keck, Journal of Virology, 23) HC33.8 Epitope "" 43, 48, 42 (Keck, Journal of Virology, 23) ARA Antigenic 46, 47, 484, 485, 538, 54, (Law, Nature Medicine, 28) Region 546, 549 AR2A Antigenic 54 (Law, Nature Medicine, 28) AR3B AR3C AR3D Region 2 Antigenic Region 3/Domain B Antigenic Region 3/Domain B Antigenic Region 3/Domain B Antigenic Region 3/Domain B 424, 523, 525, 53, 535, 538, 54 42, 46, 48, 423, 424, 523, 525, 53, 535, , 488, 523, 525, 53, 535, 538, 54 (Law, Nature Medicine, 28) (Law, Nature Medicine, 28) (Law, Nature Medicine, 28) 42, 424, 523, 53, 535 (Law, Nature Medicine, 28) AR4A Antigenic 2, 24, 25, 26, 487, 657, (Giang, PNAS, 22) Region 4 658, 692, 698 AR4B Antigenic NA (Giang, PNAS, 22) Region 4 AR5A Antigenic Region 5 2, 24, 25, 26, 639, 657, 658, 665, 692 (Giang, PNAS, 22) Numbering based on polyprotein position 2 Mutation of critical binding residues reported for HC84.22, HC84.26, HC33.4, HC33.8 to alanine reduce mab binding by at least 6%. Mutation of critical binding residues reported for the remaining mabs reduce binding by at least 5%. NA: Not Available

2 Supplementary Figure AR3D AR3B AR3C HC84.22 CBH-5 HC84.26 CBH-2 CBH-4B a a a a a a a a a a a a b b b b b b b mabs AR4B ARA AR5A AR4A HC33.8 HC33.4 AR2A CBH-7 HC- Lowest (most sensitive) Highest (most resistant) Supplementary Figure. Each neutralizing mab produces a neutralization fingerprint across the library. Eighteen previously-characterized HCVspecific mabs were tested for neutralization of each of the 9 clonal genotype a and b. Four representative mabs are shown in Figure, and neutralization results for all 8 mabs are shown here. infection is calculated as infection in the presence of µg/ml of neutralizing mab relative to infection in the presence of nonspecific IgG. Each value is a mean of duplicate wells. For each mab, relative infection values are colored on a gradient, with lower relative infection values (greatest neutralization) darker green, and higher relative infection values (less neutralization) darker red. Neutralization of pseudoparticles with MLV envelope was measured as a negative control (values not shown).

3 Supplementary Figure 2 infection ().. CBH5 CBH2 HC84.22 HC33.4 HC r=.93 p<.2 HCVcc IC5 (µg/ml) Supplementary Figure 2. Significant correlation between HCVcc IC 5 and relative infection. 5% inhibitory concentrations (IC 5 s) of the six indicated mabs against full-length replication competent HCV (HCVcc) bearing H77 E were compared by Spearman correlation to the relative infection of with H77 E, measured using the same antibodies. Two mabs that did not achieve 5% neutralization of HCVcc at the highest mab concentration tested, 2 µg/ml, were assigned an IC 5 of 4 µg/ml.

4 a Supplementary Figure 3 IC5 CBH-5 ppa6 >5.48 ppb HC84.26 ppa ppb ppb9..3 CBH-2 ppa29 > ppb HC- ppb4 > ppa23 >5.699 ppa HC33.4 ppb ppa ppa ppa ppa ppa AR3C ppa ppa ppa AR4A ppa ppb ppa AR5A ppb ppa b IC5 (µg/ml) r=.9 p<. Supplementary Figure 3. Significant correlation between IC 5 and relative infection. (a) All combinations for which both relative infection at µg/ml of mab and IC 5 were measured in independent experiments. (b) Correlation between relative infection value and IC 5 for the same /mab combination. Each point represents one mab/ combination. MAbs that did not achieve 5% neutralization at the highest mab concentration tested, 5 µg/ml, were assigned an IC 5 of µg/ml. Spearman correlation (r)=.9 with p<..

5 Supplementary Figure 4 E HVR E HVR E HVR b9.9 b34. b38.25 b52.35 b4.36 a72.43 a57.56 b58.57 a38.58 a29.58 a53.64 a6.64 a54.65 a9.67 a3.74 a23.76 a8.8 a42.2 b2.34 CBH-2 b9.3 b34.4 b52.6 a23.2 a54.26 b4.43 a53.89 a38.8 b38.26 b2.222 b58.54 a a3.739 a a a a a8.493 a HC84.26 b9.6 b2. b34.2 a53.7 b38.24 a72.32 b4.38 b52.39 a23.39 a6.48 b58.52 a9.56 a29.59 a8.6 a38.62 a3.65 a54.67 a57.72 a42.88 CBH-5 Supplementary Figure 4. Sequence analysis reveals resistance-associated polymorphisms. Sequence analysis for NC mabs not shown in Figure 3. Clones are grouped into the 5 most sensitive, 7 with intermediate resistance, and 7 with greatest resistance, separated by horizontal black lines. Gray vertical bars indicate positions with a substitution in any resistant E clone but in none of the five most sensitive E clones. Black vertical bars indicate CD8 binding sites in. Blue vertical bars indicate critical binding residues for the indicated mab, determined by alanine scanning. Sites with substitutions in at least two resistant clones but in no sensitive clones are included in the summary panel in the bottom row. Sites marked with vertical red bars are predominantly polymorphic in the seven most resistant clones. Orange vertical bars indicate sites that are polymorphic in an equal number of highly resistant and intermediate resistant clones. Green vertical bars indicate sites that are predominantly polymorphic in the seven clones with intermediate resistance.

6 Supplementary Figure 4 (Continued) E HVR E HVR E HVR E HVR a3. a53.9 b9.2 b38.4 a54.23 b34.3 a57.3 a72.38 a23.43 a8.47 a6.53 a9.5 b4.54 a42.54 b52.59 a38.62 b2.69 b58.73 a29.8 a53.5 b9.6 b38.9 a3.2 a6.26 b34.3 a72.35 a54.37 b4.4 a57.4 a9.53 a8.54 b52.6 a42.6 a38.74 b58.77 a23.77 a29.84 b2.68 AR3B a53. b9.5 a3.7 b38. b34.3 a54.3 b4.5 b2.2 a57.23 a72.27 a6.3 a8.38 b52.4 a9.43 a23.5 b58.53 a42.54 a38.54 a29.65 AR3C a53.9 a6.2 b9.22 b38.3 a3.32 a54.33 a72.4 a57.4 b4.44 b34.48 a9.49 a42.5 a8.55 a29.62 b52.64 b58.7 a38.73 a23.78 b2.6 AR3D

7 Supplementary Figure CBH HC AR3B CBH ppa53 N43D D43E L433I L438I L438V F442I F442L K446E P453S A475T E53A F56Y ppa53 N43D D43E L433I L438I L438V F442I F442L K446E P453S A475T E53A F56Y AR3C HC84.22 Supplementary Figure 5. Introduction of resistance-associated polymorphisms into a second neutralization sensitive E clone (a53) confirms the resistance phenotypes of D43E and F442I. Eleven mutations of interest were introduced by site directed mutagenesis into an E clone that was sensitive to neutralization by each of the NC mabs (clone a53). The dashed line indicates relative infection of using wildtype a53 in the presence of the indicated mab, adjusted to. Each bar indicates the fold change in neutralization resistance after the indicated mutation(s) were introduced into clone a53. Error bars indicate standard deviation between duplicate wells. Black triangles indicate with D43E mutations. Open triangles indicate with F442I or F442L mutations.

8 Supplementary Figure CBH AR3B.2 HC AR3C HC AR3D ML V ppb2 D43N E43D D43N/E43D ppa42 I438L I442F E446K I438L,I442F ML V ppb2 D43N E43D D43N/E43D ppa42 I438L I442F E446K I438L,I442F Supplementary Figure 6. Back-mutation of resistance-associated polymorphisms in E clones where they appear naturally. Each bar indicates relative infection of the indicated in the presence of the indicated mab. b2 is an E clone containing naturally occurring N43D and D43E. The dashed blue line indicates relative infection of ppb2 prior to introduction of D43N, E43D, or both mutations. a42 is an E clone containing naturally occurring L438I, F442I, and K446E. The dashed red line indicates relative infection of ppa42 prior to introduction of I438L, I442F, E446K, or I438L/I442F. Error bars indicate standard deviation between duplicate wells.

9 Supplementary Figure 7.. a b CBH-2 IC5 ppa3 wildtype (D43E) > ppb9/d43e 7.4 ppb2 wildtype (D43E) 4.3 ppb9 wildtype.93 AR3B... c ppb9/d43e IC5 8.2 ppb2 wildtype (D43E) 3.56 ppb9 wildtype.4 ppa3 wildtype (D43E).27 HC84.22 IC5 ppb9/d43e 4.5 ppb9 wildtype 2.7 ppb2 wildtype (D43E).62 ppa3 wildtype (D43E).... [mab] (µg/ml) Supplementary Figure 7. conferred by D43E is E context-specific. IC5 s were measured against using two E clones with naturally-occurring D43E (b2 and a3), a naturally occurring neutralization sensitive E clone (b9), and b9 with D43E introduced by site-directed mutagenesis (b9/d43e). (a) Mutation of D43 to E confers CBH-2 resistance to clone b9, and all E variants with D43E were CBH-2 resistant. (b) Mutation of D43 to E confers AR3B resistance to clone b9, and the b2 clone with naturally occurring D43E is resistant to AR3B as well. Clone a3, which also contains a naturally occurring D43E, is not resistant to AR3B. (c) Mutation of D43 to E also confers HC84.22 resistance to clone b9. However, clones a3 and b2, which also carry D43E, are not resistant to HC Dashed lines indicate 5% neutralization. Error bars indicate standard deviation between duplicate wells.

10 Supplementary Figure ppb2 (D43E) 2 ppb9/d43e ppa29. ppb9/f442i ppb HC84.22 ppa42 (F442I) ppa8 (F442I) Supplementary Figure 8. D43E and F442I polymorphisms are present in outliers on the neutralization correlation plot between mabs HC84.22 and. Each diamond or colored circle indicates the neutralization of a single by mab HC84.22 on the x-axis and mab on the y-axis. D43E confers relatively greater resistance to, while F442I confers relatively greater resistance to HC Solid purple circles indicate clones with naturally occurring D43E polymorphisms. Solid red circles indicate clones with naturally occurring F442I polymorphisms. Open purple and red diamonds indicate clone b9 with D43E or F442I, respectively, introduced by site directed mutagenesis. Two clones (a42 and a8) with greater resistance to HC84.22 than to carry naturally-occurring F442I polymorphisms. A clone (b2) with greater resistance to than to HC84.22 carries a naturally-occurring D43E polymorphism. Two other clones with naturally-occurring D43E (numbered and 2 ) are relatively sensitive to both and HC The E clone from the panel with highest combined resistance to both and HC84.22 (a29) does not carry either D43E or F442I. E chimeras used in Figures 5 and 6 were generated between the circled clones on this plot.

11 Supplementary Figure 9 A entry (RLU) E+8 E+7 E+6 E+5 E+4 mock ppb9 N43D D43E L433I L438I L438V F442I K446E P453S A475T E53A F56Y L438I,F442I LI/FI/A475T LI/FI/P453S/AT B entry (RLU) E+8 E+7 E+6 E+5 E+4 mock a9 a3 a38 a53 a72 a8 a6* a23 a29 a42 a54 a57 b9 b4 b2* b34 b38 b52 b58 Supplementary Figure 9. Additional polymorphisms can compensate for fitness cost of resistance polymorphisms. (A) The effect of site-directed mutations on b9 E fitness (ability to mediate entry). RLU are relative light units, indicating productive entry of into target cells. Values are means of two independent experiments performed in duplicate. Error bars indicate standard deviations. The dotted line indicates entry of with wildtype b9 E. (B) entry mediated by naturally-occurring E clones in the panel. Mock pseudoparticles with no E were used as a negative control. Solid triangles indicate with naturally-occurring D43E polymorphisms. Open triangles indicate with naturally-occurring F442I or F442L. Gray triangles indicated with naturallyoccurring F56Y. Values are the means of four to eleven independent experiments performed in duplicate. Error bars indicated standard deviations. The dotted line indicates median entry of all 9. indicated with asterisks were never frozen prior to testing. The remaining were freeze-thawed once prior to testing.

12 Supplementary Figure NS * * entry (RLU) ppb9 ppb9 I538V Q546L T563V ppa29 ppa29 V538I L546Q V563T Supplementary Figure. Fitness cost of I538V/Q546L/T563V polymorphisms is compensated in a resistant E clone where it arises naturally. Each bar represents entry by with the indicated Es. Values are means of two to six independent experiments performed in duplicate. RLU is relative light units. Error bars are standard deviations. An asterisk indicates p<.5 by T test. NS indicates p value not significant. Introduction of I538V/Q546L/T563V reduces fitness of clone b9, but clone a29, with naturally-occurring I538V/Q546L/T563V, does not exhibit reduced fitness, and reversion of these polymorphisms in clone a29 also reduces fitness. Therefore, fitness cost of these polymorphisms is E context dependent.

13 Supplementary Figure a3 a53 b9 b38 a54 b34 a57 a72 a23 a8 a9 a6 b4 a42 b52 a38 b2 b58 a N..A.D...FV...L...Y..V..A... A.N...D...FV...L...A..A... TYTWGENETDVLILNNTRPPQGNWFGCTWMNSTGFTKTCGGPPC..S...L...R...G.....S..A.D...FV...L...V..A.....N...L...L...S.....N..A.D...FV...L...V..A.....S...FV...L...V..A......FV...L...S...V..A.....N...D...FV...L...V..A.....N...D...V...L...V..A.....N..DSD...FV...L...S...V..A......L...G.....G...D...FV...L...Y..V..A......L...G.....S...D...FV...L...V..A.....S...L.....S...L.....S...D...FV...L...V..A... Supplementary Figure. Neutralization resistance conferred by I538V/Q546L/T563V is E context dependent. Sequence alignment of amino acids for all E clones in the panel. Clones are listed by increasing resistance to mab. Boxes indicate residues 538, 546, and 563. Dots indicate homology to sensitive clone b9 at that site. Arrows indicate genotype b clones. The I538V/Q546L/T563V combination of polymorphisms is common in genotype a isolates in the E panel and not independently associated with NC mab resistance.