Selection pressure from neutralizing antibodies drives sequence evolution during acute infection with hepatitis C virus

Abstract

Background & aims: Despite recent characterization of hepatitis C virus-specific neutralizing antibodies, it is not clear to what extent immune pressure from neutralizing antibodies drives viral sequence evolution in vivo. This lack of understanding is particularly evident in acute infection, the phase when elimination or persistence of viral replication is determined and during which the importance of the humoral immune response has been largely discounted.

Methods: We analyzed envelope glycoprotein sequence evolution and neutralization of sequential autologous hepatitis C virus pseudoparticles in 8 individuals throughout acute infection.

Results: Amino acid substitutions occurred throughout the envelope genes, primarily within the hypervariable region 1 of E2. When individualized pseudoparticles expressing sequential envelope sequences were used to measure neutralization by autologous sera, antibodies neutralizing earlier sequence variants were detected at earlier time points than antibodies neutralizing later variants, indicating clearance and evolution of viral variants in response to pressure from neutralizing antibodies. To demonstrate the effects of amino acid substitution on neutralization, site-directed mutagenesis of a pseudoparticle envelope sequence revealed amino acid substitutions in hypervariable region 1 that were responsible for a dramatic decrease in neutralization sensitivity over time. In addition, high-titer neutralizing antibodies peaked at the time of viral clearance in all spontaneous resolvers, whereas chronically evolving subjects displayed low-titer or absent neutralizing antibodies throughout early acute infection.

Conclusions: These findings indicate that, during acute hepatitis C virus infection in vivo, virus-specific neutralizing antibodies drive sequence evolution and, in some individuals, play a role in determining the outcome of infection.

Figure 1

E1E2 amino acid substitutions occur during acute HCV infection. E1E2 envelope sequences, depicted as horizontal lines, are shown aligned to the block diagram representing the corresponding region of the HCV polyprotein. Each horizontal line represents a sequence used to generate HCVpp for autologous neutralization studies*, with connecting vertical lines representing amino acid substitutions occurring between test-visits of a given subject. Substitutions were determined using the initial test-visit from each subject as the reference. For clarity, the amino acid sequences of the HVR1 region of E2 are shown on the far right. Outcome refers to the primary infection (s11 and s26 did not have clear outcomes as described in the text). Sequences from s18 consistently maintained a 2 aa insertion at the beginning of HVR1. Numbers refer to the HCV polyprotein location according to the reference strain H77. *For s13 (1.3 months), an infectious HCVpp clone was not isolated and the depicted sequence represents a consensus of six envelope clones from that visit.

Figure 2

HCV pseudoparticle infectivity levels. HCVpp were produced in HEK-293T cells, harvested, and immediately used to infect target Hep3B cells. Infectivity levels, as determined by measuring luciferase activity, are reported in relative light units (RLU). Results of two separate HEK-293T cell transfections are reported as the mean + SD. For positive and negative controls, pp expressing the murine leukemia virus (MLV) envelope protein or no envelope protein (Mock) were used, respectively.

Figure 3

Autologous neutralization during acute HCV infection. HCVpp expressing envelope sequences from sequential timepoints* in acute infection were incubated with two-fold dilutions of autologous plasma, and results reported as ID₅₀ titers. If 50% neutralization was not detected at the starting plasma dilution of 1:50, the result was recorded as one half this value, a titer of 1:25. Results for each subject represent a single experiment or median values from two experiments, in which all autologous HCVpp were assayed concurrently with the full array of plasma samples. The dotted line represents the limit of detection for the HCV RNA assay (50 IU/mL), and negative values were plotted as one half this value (25 IU/mL). The cross-hatched areas for s11 and s26 represent the time period between detection of phylogenetically distinct virus strains. *Only a single infectious HCVpp could be generated for s13.

Figure 4

Heterologous versus autologous HCVpp neutralization results. Autologous neutralization of HCVpp expressing the earliest envelope clone for each subject was compared to heterologous neutralization of HCVpp expressing an H77 envelope sequence. HCVpp were incubated with two-fold dilutions of plasma, and results are reported as ID₅₀ titers. If 50% neutralization was not detected at the starting plasma dilution of 1:50, the result was recorded as one half this value, a titer of 1:25. Results for each subject represent a single experiment or median values from two experiments, in which each HCVpp was assayed with the full array of plasma samples. The dotted line represents the limit of detection for the HCV RNA assay (50 IU/mL), and negative values were plotted as one half this value (25 IU/mL). The cross-hatched areas for s11 and s26 represent the time period between detection of phylogenetically distinct virus strains.

Figure 5

Heterologous neutralization sensitivity decreases as the autologous E1E2 sequence diverges from H77. For each subject, over the entire time of follow-up, we calculated geometric mean titers (GMT) for heterologous (H77) and autologous (early variant) HCVpp neutralization results, including titers from all time points where both HCVpp variants were tested and neutralization was detectable against at least one of the two. The difference in GMTs [log₁₀(GMT_autologous) − log₁₀(GMT_heterologous)] was plotted against the % amino acid similarity of the subject-specific E1E2 sequence to the heterologous H77 E1E2 sequence. Boxed points represent s28, s29, and s110, who displayed only modest differences in heterologous versus autologous neutralization sensitivity and represent the autologous sequences most closely related to H77. The open circle denotes s11 (infected with a genotype 1b virus), representing the only non-genotype 1a sequence.

Figure 6

Hierarchy of neutralization against autologous and heterologous HCVpp. Combining results shown in Figures 4 and 5, neutralization titers against autologous (early and late variants) and heterologous HCVpp are shown for s110. Taken together, the results demonstrate a hierarchy of neutralization, such that neutralization of autologous (2.9 month) > heterologous (H77) > autologous (9.2 month) HCVpp.

Figure 7

Mapping neutralization determinants using autologous HCVpp. A) For s110, neutralization of 2.9 month pp (early variant) compared to 9.2 month pp (late variant) by plasma from 9.2 months into infection showed a significant decrease in ID₅₀ titer, as denoted by the arrow. B) Amino acid substitutions in the 9.2 month pp sequence were back-mutated to express amino acids present in the 2.9 month pp. Mutant and wild-type HCVpp were asessed for neutralization by 9.2 month plasma. Results of 1–4 experiments are reported as both % neutralization (1:50 dilution of serum, shown as mean + SD), and as median ID₅₀ titers. When 50% neutralization was not detected at the starting plasma dilution of 1:50, ID₅₀s were reported as 1:25, represented by the dotted line. Numbers refer to the HCV polyprotein location according to the reference strain H77.

Figure 7

Mapping neutralization determinants using autologous HCVpp. A) For s110, neutralization of 2.9 month pp (early variant) compared to 9.2 month pp (late variant) by plasma from 9.2 months into infection showed a significant decrease in ID₅₀ titer, as denoted by the arrow. B) Amino acid substitutions in the 9.2 month pp sequence were back-mutated to express amino acids present in the 2.9 month pp. Mutant and wild-type HCVpp were asessed for neutralization by 9.2 month plasma. Results of 1–4 experiments are reported as both % neutralization (1:50 dilution of serum, shown as mean + SD), and as median ID₅₀ titers. When 50% neutralization was not detected at the starting plasma dilution of 1:50, ID₅₀s were reported as 1:25, represented by the dotted line. Numbers refer to the HCV polyprotein location according to the reference strain H77.