Identification of novel neutralizing determinants for protection against HCV

Abstract

Background and aims: HCV evasion of neutralizing antibodies (nAb) results in viral persistence and poses challenges to the development of an urgently needed vaccine. N-linked glycosylation of viral envelope proteins is a key mechanism for such evasion. To facilitate rational vaccine design, we aimed to identify determinants of protection of conserved neutralizing epitopes.

Approach and results: Using a reverse evolutionary approach, we passaged genotype 1a, 1b, 2a, 3a, and 4a HCV with envelope proteins (E1 and E2) derived from chronically infected patients without selective pressure by nAb in cell culture. Compared with the original viruses, HCV recombinants, engineered to harbor substitutions identified in polyclonal cell culture-passaged viruses, showed highly increased fitness and exposure of conserved neutralizing epitopes in antigenic regions 3 and 4, associated with protection from chronic infection. Further reverse genetic studies of acquired E1/E2 substitutions identified positions 418 and 532 in the N1 and N6 glycosylation motifs, localizing to adjacent E2 areas, as key regulators of changes of the E1/E2 conformational state, which governed viral sensitivity to nAb. These effects were independent of predicted glycan occupancy.

Conclusions: We show how N-linked glycosylation motifs can trigger dramatic changes in HCV sensitivity to nAb, independent of glycan occupancy. These findings aid in the understanding of HCV nAb evasion and rational vaccine design, as they can be exploited to stabilize the structurally flexible envelope proteins in an open conformation, exposing important neutralizing epitopes. Finally, this work resulted in a panel of highly fit cell culture infectious HCV recombinants.

Graphical abstract

FIGURE 1

Reverse evolution of HCV without neutralizing antibody (nAb) pressure resulted in increased HCV infectivity titers and multiple genetic changes. HCV recombinants based on the Japanese Fulminant Hepatitis 1 (JFH1) strain with Core‐NS2 of genotype(isolate) 1a(H77), 1b(J4), 2a(J6), 3a(S52), 4a(ED43), and 6a(HK6a) were serially passaged in Huh7.5 cells. (A) Peak HCV infectivity titers in initial and late passages were determined as log10 focus forming units (FFU)/ml and are means of three replicate determinations with SEM. The late passage (passage number indicated for each recombinant) was analyzed by next‐generation sequencing (NGS): substitutions >70%, number of polyprotein substitutions showing >70% frequency in NGS; E1–E2 substitutions >70%, number of envelope protein substitutions showing >70% frequency in NGS; substitutions 10%–70%, number of polyprotein substitutions showing 10%–70% frequency in NGS; E1–E2 substitutions 10%–70%, number of envelope protein substitutions showing 10%–70% frequency in NGS. (B) Schematic overview of JFH1‐based genotype 1, 2, 3, 4, and 6 genomes with substitutions identified following passage in cell culture. Blue bars indicate previously identified substitution engineered in originally developed viruses.‐ Green bars indicate substitution previously identified in NS5A and NS5B of a high‐yield 5a(SA13) virus. Red bars indicate novel substitutions with >70% frequency in NGS. Yellow bars indicate novel substitutions with 10%–70% frequency in NGS. *Novel substitutions with >70% frequency in NGS were engineered, except for 1a(H77), for which V34A, N532D, and V866I present in 10%–70%, as well as N2034D and S2996G found in two of six subclones, were engineered for technical reasons. Substitution numbers relate to the H77 polyprotein reference sequence (GenBank accession number AF009606).

FIGURE 2

Substitutions selected during reverse evolution in the absence of nAb mediated increased viral fitness. Comparison of fitness of viruses with original envelope proteins (original), engineered viruses with all substitutions identified during in vitro passage in >70% of viral genomes (mutated), and polyclonal viruses resulting from in vitro passage (polyclonal), all tested in a first viral passage kinetic experiment inoculated at multiplicity of infection (MOI) of 0.003. HCV infectivity titers are means of three replicate determinations with SEM. *Cell culture was closed due to HCV‐induced cell death. NGS of passaged viruses did not reveal additional mutations in the open reading frame (ORF) present in >20% of genomes.

FIGURE 3

Substitutions selected during reverse evolution in the absence of nAb resulted in increased sensitivity to nAb. In vitro neutralization assays were carried out using original, mutated, and polyclonal genotype 1, 2, 3, 4, and 6 HCV and human monoclonal antibodies (mAb) AR3A (A) and AR4A (B). Data points are means of three replicate determinations with SD; concentration‐response curves were fitted and half maximal effective concentration (EC50) values were calculated using top and bottom constraints of 0% and 100%, respectively, and the formula y = top/(1 + 10^{[log10EC50 − X] × hillslope}) with GraphPad Prism (version 9). Fold increase values indicate increase in neutralization sensitivity and were calculated as (EC50 of original virus)/(EC50 of mutated virus [or polyclonal virus]). The envelope protein sequence of the used virus stocks was confirmed by Sanger sequencing.

FIGURE 4

Specific envelope protein substitutions mediated increased neutralization sensitivity for genotype 1, 2, 3, and 4 HCV. Sensitivity to neutralization with human mAb AR3A (A,F), AR2A (B), AR4A (C), AR5A (D), and human polyclonal antibody preparation C211 (E) for the specified 1a(H77), 1b(J4), 2a(J6), 3a(S52), and 4a(ED43) viruses. (A–F) For each genotype, the respective original virus (original), the mutated virus (mutated) harboring all substitutions identified during in vitro passage, and variants with the specified envelope protein modifications, which were based on the respective original viruses, are shown. (A) Variants with the combination of all envelope protein substitutions identified during in vitro passage of the respective virus (all envelope protein substitutions) are shown for 1a(H77), 2a(J6), and 3a(S52). For 2a(J6), variants based on the mutated 2a(J6) virus (suffix “mutated‐x”) with reversion of the specified envelope protein substitutions are shown. (A–F) EC50 were calculated with GraphPad Prism (version 9) based on concentration‐response curves shown in Figure S3 (A–E) or Figure S4 (F). Fold‐increase values indicate increase in neutralization sensitivity and were calculated as (EC50 of original virus)/(EC50 of mutated virus). The envelope protein sequence of the used virus stocks was confirmed by Sanger sequencing. na, not applicable.

FIGURE 5

Specific envelope protein substitutions localizing to conserved glycosylation motifs and inducing increased neutralization sensitivity mediated switching from a closed to an open envelope protein conformational state. (A) Breathing capacity for specified 2a(J6) and 3a(S52) viruses, determined in in vitro neutralization assays using human mAb AR3A at 4°C, 37°C and 40°C, as described in the “Experimental procedures” section. EC50 values and 95% CI were calculated in GraphPad Prism (version 9) based on concentration‐response curves shown in Figure S5. (B) Temperature stability of the specified 2a(J6) and 3a(S52) viruses evaluated by incubation at 40°C, 37°C, and 4°C. Data points are means of nine values derived from three replicate experiments, evaluated by FFU determinations in triplicates with SD. One‐phase decay curves were fitted and half‐lives were calculated using the formula y = (Y0‐Plateau)*exp(‐K*X) + Plateau with GraphPad Prism (version 9). Scavenger receptor class B type 1 (SR‐BI) (C) and cluster of differentiation 81 (CD81) (D) dependency of specified 2a(J6) and 3a(S52) viruses, determined in receptor‐blocking assays using specific antibodies. Data points are means of three replicate determinations with SD; concentration‐response curves were fitted and EC50 were calculated with GraphPad Prism using top and bottom constraints of 0% and 100%. The envelope protein sequence of all used virus stocks was confirmed by Sanger sequencing.

FIGURE 6

Envelope protein substitutions contributing to increased neutralization sensitivity mediated increased viral fitness. Comparison of fitness of the specified 2a(J6) and 3a(S52) viruses in first passage kinetic experiments inoculated at MOI 0.003. HCV infectivity titers for 2a(J6) (A) and 3a(S52) (B) variants with original envelope proteins, all substitutions (mutated), all envelope substitutions, or addition of single substitutions identified following serial passage. HCV infectivity titers are means of three replicate determinations with SEM. *Cell culture was closed due to HCV‐induced cell death. The passaged viruses were genetically stable, revealed by Sanger sequencing or NGS of the ORF.