Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

Abstract

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance-associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.

Figure 1

HCV genotype 1‐6 PI escape viruses harbored NS3P RASs with PI concentration‐dependent substitution patterns. (A) Putative RASs were identified by NGS. NS3P aa positions with putative RASs found in more than 5% of the viral population for at least one virus and one PI were included and numbered relative to NS3P of the H77 reference strain; H77 aa residues are specified. •, aa residues identical to that of H77; single letter, nonidentical aa residue; letters separated by dash, putative RASs indicated by the original and the mutated residues, color coded depending on the PI under which they were selected. For detailed data including NGS, see Supporting Figs. S3‐S11. (B) NS3P NGS substitution linkage analysis revealed haplotype distributions for 1a(TN), 2a(JFH1), and 3a(DBN) grazoprevir escape viruses (Supporting Figs. S3, S5, S8, S14A‐C). Haplotypes constituting greater than 2% of the viral population are included in bars; haplotypes greater than 5% are highlighted on bars.

Figure 2

Persistence of 156‐RASs in HCV genotypes 1a and 2a was facilitated by further evolution under PI treatment. Haplotype frequencies were determined by NGS and substitution linkage analysis; haplotypes constituting more than 2% of the viral population are included in the bars; haplotypes with A156T/V/L constituting more than 0.5% of the viral population are included in values above the bars; haplotypes constituting more than 20% of the viral population are highlighted on the bars. (A) 1a(TN), escaping 64‐fold EC50 of grazoprevir (64 × esc.; Fig. 1B and Supporting Figs. S2 and S3) was passaged 3 times without treatment; nontreated first and third passage (NT1P, NT3P) were analyzed. (B) 1a(TN) escaping 64‐fold EC50 of grazoprevir was passaged 6 times under treatment with 64‐fold EC50 of grazoprevir and subsequently 6 times without treatment; treated third and sixth passage (64 × T3P, 64 × T6P) and nontreated ninth and twelfth passage (NT9P, NT12P) were analyzed. (C) 2a(JFH1) escaping 64‐fold EC50 of grazoprevir (different experiment than in Fig. 1B and Supporting Figs. S2 and S5) was passaged 3 times without treatment; nontreated third passage (NT3P) was analyzed (for 64 × esc., NGS was not successful; Sanger sequencing confirmed dominance of A156V). (D) 2a(JFH1) escaping 64‐fold EC50 of grazoprevir was passaged 6 times under treatment with 64‐fold EC50 of grazoprevir and subsequently 6 times without treatment; treated first, third, and sixth passage (64 × T1P, 64 × T3P, 64 × T6P) and nontreated ninth and twelfth passage (NT9P, NT12P) were analyzed. (E) 2a(JFH1) with engineered A156V, transfected in Huh7.5 cells, and escaping 64‐fold EC50 of grazoprevir was passaged as in (D); treated third and sixth passage (64 × T3P, 64 × T6P) and nontreated ninth and twelfth passage (NT9P, NT12P) were analyzed.

Figure 3

Evolution of a multimechanistic genome‐wide substitution network facilitated the persistence of A156T for HCV genotype 1a. (A) Genome‐wide linkage analysis of coding substitutions evolving in the 1a(TN) population during passage with/without treatment (Fig. 2B), based on SNP frequency development in serial viral passage (Supporting Fig. S14A). Abbreviations: N, node (major step in evolution). *NS3P linkage analysis confirmed linkage of the indicated substitution to A156T. NS4B_G1824D, NS5B_N2651H, and NS5B_E2860G are linked. NS3H_V1656A is linked to the substitution groups in blue and cyan, but not in purple. (B) Schematic overview of the 1a(TN) genome and engineered 1a(TN) recombinants. (C) Engineered recombinants were transfected in Huh7.5 cells; extracellular infectivity titers given as focus forming units per milliliter (FFU/ml) are means of triplicates with SEM. (‐) A156T reverted after first or (+) persisted after second passage. (D) Engineered recombinants were transfected in CD81‐deficient S29‐cells. Intracellular (IC) and extracellular (EC) Core levels (percentage, relative to Core concentration determined 4 hours following transfection) and intracellular infectivity titers (FFU/ml) are means of duplicates; extracellular infectivity titers (FFU/ml) are means of triplicates with SEM. *Single determinations. The bar colors in (C) and (D) match the colors of recombinants in (B). 2a(JFH1)‐GND (gray checkerboard) and 2a(JFH1) (gray stripes) are negative and positive controls, respectively. (E) 1a(TN) recombinants with NS3 substitutions engineered as indicated in the legend were transfected in Huh7.5 cells in the same experiment as the recombinants in (C); 1a(TN) and 1a(TN)A156T are identical in (C) and (E); extracellular infectivity titers (FFU/ml) are means of triplicates with SEM. (‐) A156T reverted after first or (+) persisted after second passage. Abbreviations: FFU/ml, focus forming units per milliliter; na, not applicable.

Figure 4

NS3P variants with 156‐RASs showed high PI resistance. For 1a(TN) (A), 2a(JFH1) (B), 3a(S52) (C), and 3a(DBN) (D) 156‐variants, PI concentration‐response experiments were carried out using grazoprevir, paritaprevir, glecaprevir, and voxilaprevir as described in the Materials and Methods section. Fold‐resistance values were calculated by relating EC50 of the indicated variants to that of the original viruses included in the same experiment; numbers are rounded off. When possible, 156‐variants were engineered as recombinants (rec) and first‐passage virus stocks were used. For 1a(TN)N2 rec, 1a(TN)N3 rec, and 1a(TN)A156T+NS3H rec (including NS3H_V1656A), second‐passage virus stocks were used (Fig. 3). For 3a(S52)A156L rec, 3a(DBN)A156T rec, and 3a(DBN)A156V rec, NS3P (Supporting Fig. S12), and for 2a(JFH1)A156L rec and 3a(DBN)A156L rec (Supporting Fig. S14E), the complete ORF was sequenced. Otherwise, 156‐variants were grown as polyclonal virus stocks (pVS) from nontreated first passages (NT1P) or from nontreated twelfth passages (NT12P) of viruses from grazoprevir escape experiments (Fig. 2A,B,D,E). Abbreviations: na, not applicable; nd, not done. ^aResults were from a separate experiment in which the EC50s for 1a(TN) were 2 nM (grazoprevir) and 8 nM (paritaprevir). ^bResults were from a separate experiment in which EC50s for 1a(TN) were 1 nM (grazoprevir) and 6 nM (paritaprevir). ^cViruses were not fully inhibited by the highest PI concentration tested; EC50s are estimates given by GraphPad Prism. ^dViruses were not inhibited by at least 50% by the highest PI concentration tested; EC50s could not be estimated.

Figure 5

Pre‐existing A156T facilitated 1a(TN) escape from combination treatment. 1a(TN)N3 harboring A156T and D168E as well as the original 1a(TN) were subjected to mono or combination treatment with grazoprevir and elbasvir (A) or glecaprevir and pibrentasvir (B) or mono or triple treatment with voxilaprevir, velpatasvir, and sofosbuvir (C) at the indicated fold‐EC50 until viral escape, control, or suppression occurred (see Materials and Methods section). Viral control occurred for 1a(TN) under double treatment and for 1a(TN)N3 under triple treatment. Viral suppression occurred for 1a(TN) under triple treatment. (D) Sanger sequencing of DAA targets of escape variants revealed acquisition of additional RASs in NS3P (brown) and NS5A domain I (purple). ^aThe RAS L31V in NS5A was detected following viral spread to most of the culture cells on day 45 (data not shown), following treatment termination on day 31.

Figure 6

Analysis of HCV sequences of genotype 1a–infected patients failing DAA treatment and harboring A156G or V. Patients A, B, C, D, and E were treated with glecaprevir + piprentasvir (A,D), grazoprevir + elbasvir (B), or paritaprevir + ombitasvir + dasabuvir (C,E). Posttreatment samples were obtained at the given month following end of treatment: patient A, 0 months; patient B, 4.8 months; patient C, 4.6 months; patient D, 3.1 months; and patient E, information not available. Bar graphs to the left in each panel show NS3P haplotype frequencies determined by NGS and substitution linkage analysis before treatment (pre) and after treatment (post). Haplotypes constituting more than 2% of the viral population are included in the bars; haplotypes constituting more than 20% of the viral population are highlighted in the bars. Patient B had the NS3P RAS Q80K in all haplotypes detected before and after treatment. In (A‐C), original refers to the pretreatment consensus sequence. In (D), original refers to the consensus sequence without A156V. The three line graphs to the right in (A‐C) show ORF‐wide NGS, revealing SNP frequencies along the ORF. Pre, SNP frequencies in pretreatment sequences mapped against their consensus sequence (known RASs occurring in more than 0.5% of reads are specified); Post‐all, SNP frequencies in posttreatment sequences mapped to the pretreatment consensus sequence (known RASs occurring in greater than 20% of reads are specified); Post de novo selection, frequencies of de novo selected SNPs, found in less than 0.5% of reads in pretretament sequences, mapped to the pretreatment consensus sequence (all substitutions occurring in greater than 20% of reads are specified). Known RASs are indicated in bold for NS3P using relative NS3P numbers (brown), for NS5A using relative NS5A numbers (purple), and for NS5B using relative NS5B numbers (orange). Other de novo SNPs outside the drug targets are in regular font (black), with numbers relating to the polyprotein of the H77 reference strain (GenBank identifier AF009606).