Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a

Abstract

Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc-expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50 values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc's when combined individually with AR4A.

Conclusion: Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least three HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV.

Fig. 1

Phylogenetic analysis of the E1/E2 amino acid sequences of the genotype 1-3 HCVcc panel used for in vitro neutralization analysis. Multiple sequence alignment was computed with ClustalW (MEGA5 software). The phylogenetic tree was generated using a neighbor-joining algorithm. The HCV subtype and isolate names are indicated. A novel genotype 3a JFH1-based Core-NS2 recombinant (DH11/JFH1) is indicated with a star. Numbers at phylogenetic branches represent the percentage (≥75%) at which the included sequences cluster together in 1000 replicate multiple alignments through bootstrapping. The scale bar represents the evolutionary distance expressed as amino acid substitutions per site. Percentage ranges indicated to the right represent the amino acid differences among isolates within specific subtypes and genotypes, respectively.

Fig. 2

Dose-response neutralization of the genotype 1-3 HCVcc panel by HMAbs HC84.24 and AR4A. (A-B) Neutralization of genotype 1 Core-NS2 JFH1-based recombinants including H77 (1a), TN (1a), DH6 (1a), J4 (1b), DH1 (1b), and DH5 (1b); (C-D) Genotype 2: J6 (2a), T9 (2a), JFH1 (2a), J8 (2b), DH8 (2b), DH10 (2b), and S83 (2c); (E-F) Genotype 3: DH11 (3a), DBN (3a), and S52 (3a). HMAb concentrations ranged from 0.0012 to 100 μg/ml and were incubated in a 5-fold dilution series with the various virus stocks in a 96 well format. Inhibitory concentrations for 50% virus neutralization (IC₅₀) values for each HCVcc are indicated. Controls for this experiment were isotype-matched antibodies R04 (for HC84.24) and b6 (for AR4A) (see Supplemental Fig. 1). All measurements were performed in 4 replicates and error bars show the standard error of the mean (SEM). For data on all HMAbs see Supplemental Fig. 1.

Fig. 3

Radar chart of HMAb neutralization patterns against the 16 HCVcc JFH1-based Core-NS2 recombinants of the genotype 1-3 panel. Only HMAbs exhibiting the most efficient neutralization ability, i.e. HC84.24, HC84.26, AR3A, AR4A, and HC-1AM are shown. Values in the radar chart display log₁₀(IC₅₀) values, with the maximum measured log(IC₅₀) = 2. HCVcc with IC₅₀ >100 μg/ml were defined as 3. Each data point represents an IC₅₀-value against a specific virus isolate. The color code in the connecting lines represents the various included HMAbs. The selection of antibodies for cooperation analysis was based on minimizing the area towards the center of the graphs and the differences in epitope targets, thus suggesting that two given HMAbs would complement their respective IC₅₀-values.

Fig. 4

Cooperativity in virus neutralization between HMAbs HC84.26 and AR4A against the DH1(1b) recombinant. (A) Dose-response neutralization of HC84.26/AR4A alone and in combination in a constant ratio 2-fold dilution series using concentrations ranging from 2⁻⁴- to 2³-fold of the previously determined IC₅₀-values. (B) Neutralization percentages, represented as a fractional effect (Fa) from 0.01-0.99, were input into the CompuSyn software in relation to the corresponding HMAb dose. The combination index (CI) plot, displaying the calculated CI for the combined HC84.26/AR4A in relation to Fa, was subsequently computed as described previously^,,. The CI value was calculated based on the Fa values from the HC84.26/AR4A combination relative to the Fa values obtained for the HMAbs when tested individually. Blue symbols represent combined HC84.26/AR4A. The various CI-values for HC84.26/AR4A against the HCVcc panel are shown in Table 4. Following the CompuSyn recommendations, a CI-value of 0.1-0.3 was defined as strong synergism; 0.3-0.7 as synergism; 0.7-0.85 as moderate synergism; 0.85-0.9 as slight synergism; 0.9-1.1 as nearly additive; 1.1-1.2 as slight antagonism; 1.2-1.45 as moderate antagonism; 1.45-3.3 as antagonism; 3.3-10 as strong antagonism.