Antibodies recognize non-linear epitopes on soluble E2.

<p>(A) Antibody binding to denatured E1E2 was tested by ELISA. A cell lysate of 293T cells transfected with E1E2 (H77) was treated with 20 mM Dithiothreitol and 0.4% Sodium dodecyl sulfate (denatured E1E2) or directly added on GNA pre-coated plate (native E1E2). Subsequently the antibodies were added at 1 μg/mL. A lysate of non-transfected cells, a native E1E2 cell lysate, AP33 an antibody specific for a linear epitope on E2 and D25 were used as negative and positive controls. (B) The binding of antibodies to H77 derived E2 was tested by ELISA. The antibodies (1 μg/mL) were added to wells pre-coated with His6 tagged E2 (E2-his). Phosphate buffered saline (PBS) coated wells and D25 were used as negative controls. In A) and B) the y-axis indicates the mean Optical density (OD) at 450nm and the errors bars represent one SD. The assay was performed in duplicate wells and repeated in at least one separated experiment.</p

Antibodies compete CD81 binding to E2.

<p>Cells transfected E1E2 from isolate H77 were pre-incubated with different concentrations of the indicated antibodies before CD81-LEL at 0.05 μg/mL was added. (A) D25 was used as isotype negative control. The y-axis indicates the percentage inhibition of CD81 and the errors bars represent one SD. (B) Antibody concentration to reach 50% inhibition of CD81 binding (IC₅₀) in μg/mL. The IC₅₀ was determined using non-linear regression analysis. The assay was performed in duplicate and at least repeated once.</p

Antibody epitope mapping.

<p>To determine the more exact antibody-binding epitope single alanine amino acid substitutions were introduced in the E1E2 sequence from isolate H77 and antibody binding was tested by ELISA. E1E2 containing cell lysates were incubated on GN lectin pre-coated wells before adding an antibody (1 to 0.0002 μg/mL). Antibody concentrations needed to get 50% binding (EC₅₀) was determined using non-linear regression analysis. The relative binding was calculated by dividing the EC₅₀ obtained on wild-type protein versus alanine-mutant protein times 100. The gray scale indicates the relative binding level: Black 0% -25%, grey 25% -50% and white 50% -150%. The E2 mutants (rows) were designated X123Y where 123 is the residue position, X indicates the wild-type amino acid residue in H77 and Y indicates the replacing amino acid. The EC₅₀ which could not be calculated because of very weak binding were indicated by <10%. The data are the mean values of two experiments performed in duplicate.</p

Affinity of the antibodies for HCV E2.

<p>Affinity of the antibodies for HCV E2.</p

Antibody competition.

<p>(A) Antibody competition assay was performed by SPR using H77 derived E2-his. When the antibodies (indicated in rows) were immobilized on the chip and had captured E2, a second antibody (indicated in columns) was injected. Absence of binding after injection of the second antibody indicated the antibodies competed for E2 binding (grey); if the injected antibody bound antibody-captured E2, this was considered to be not competing (white). The assay was performed in triplicate and repeat in one separated experiment. (B) Antibody competition assay was performed by flow cytometry using cells transfected with E1E2 from isolate H77. Cells were pre-incubated with 20 μg/mL of the indicated antibodies before Alexa Fluor 647 conjugated antibodies at their EC₅₀ were added. The percentage of binding was calculated using the percentage of Alexa Fluor 647 positive cells from wells incubated without antibodies. The gray scale indicates the relative binding level: white 100% -75%, light grey 75% -50%, dark grey 50% -25% and black 25%–0%. D25 was used as isotype negative control. The mean value of three duplicate experiments is shown.</p

Characteristics of participant D18926.

<p>Characteristics of participant D18926.</p

Breadth of cross-reactive antibodies against E1E2 glycoproteins.

<p>Antibodies were tested for binding to E1E2 protein derived from different genotypes by ELISA. E1E2 containing cell lysates were incubated on GNA pre-coated wells before antibodies were added at 1 μg/mL. A lysate from non-transfected cells and the RSV F protein specific antibody D25 were used as negative controls. The y-axis indicates the mean optical density (OD) at 450 nm and the errors bars represent one standard deviation (SD). E1E2 sequences are indicated between brackets and assays were performed in duplicate.</p