Neutralization of hepatitis A virus (HAV) by an immunoadhesin containing the cysteine-rich region of HAV cellular receptor-1

Abstract

Hepatitis A virus (HAV) infects African green monkey kidney (AGMK) cells via the HAV cellular receptor-1 (havcr-1), a mucin-like type 1 integral-membrane glycoprotein of unknown natural function. The ectodomain of havcr-1 contains an N-terminal immunoglobulin-like cysteine-rich region (D1), which binds protective monoclonal antibody (MAb) 190/4, followed by an O-glycosylated mucin-like threonine-serine-proline-rich region that extends D1 well above the cell surface. To study the interaction of HAV with havcr-1, we constructed immunoadhesins fusing the hinge and Fc portion of human IgG1 to D1 (D1-Fc) or the ectodomain of the poliovirus receptor (PVR-Fc) and expressed them in CHO cells. These immunoadhesins were secreted to the cell culture medium and purified through protein A-agarose columns. In a solid-phase assay, HAV bound to D1-Fc in a concentration-dependent manner whereas background levels of HAV bound to PVR-Fc. Binding of HAV to D1-Fc was blocked by treatment with MAb 190/4 but not with control MAb M2, which binds to a tag epitope introduced between the D1 and Fc portions of the immunoadhesin. D1-Fc neutralized approximately 1 log unit of the HAV infectivity in AGMK cells, whereas PVR-Fc had no effect in the HAV titers. A similarly poor reduction in HAV titers was observed after treating the same stock of HAV with murine neutralizing MAbs K2-4F2, K3-4C8, and VHA 813. Neutralization of poliovirus by PVR-Fc but not by D1-Fc indicated that the virus-receptor interactions were specific. These results show that D1 is sufficient for binding and neutralization of HAV and provide further evidence that havcr-1 is a functional cellular receptor for HAV.

FIG. 1

Expression of D1-Fc and PVR-Fc immunoadhesins in CHO cells. (A) Schematic representation of the D1-Fc and PVR-Fc immunoadhesins. To construct D1-Fc, the cysteine-rich region of havcr-1 (D1) was tagged at its N terminus with peptide DTKDDDDK (FLAG) and fused to the hinge and Fc regions of human IgG1 (IgG1 Fc). To construct PVR-Fc, the ectodomain of PVR containing the three immunoglobulin-like domains (V1, C1, and C2) was fused to the hinge and Fc regions of human IgG1. Two identical fusion proteins are linked by disulfide bonds (dashed lines), forming homodimers, which are secreted to the cell culture medium as soluble immunoadhesins. (B) D1-Fc and PVR-Fc were purified through protein A columns. The eluted immunoadhesins were analyzed by denaturing SDS-PAGE in a 4 to 20% polyacrylamide gel and stained with Coomassie blue. The arrow points to the fully glycosylated 50-kDa form of D1-Fc. The positions of prestained molecular mass markers and their sizes in kilodaltons are shown on the right.

FIG. 2

Western blot analysis of purified D1-Fc and PVR-Fc immunoadhesins. Protein A-purified D1-Fc (lanes 1 and 3) and PVR-Fc (lanes 2 and 4) were analyzed by denaturing SDS-PAGE in a 4 to 20% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with anti-FLAG MAb M2 (lanes 1 and 2) or anti-human Fc antibodies (lanes 3 and 4). The positions of prestained molecular mass markers and their sizes in kilodaltons are shown on the left.

FIG. 3

Binding of protective MAb 190/4 to D1-Fc. Binding of D1-Fc to MAb 190/4 was determined by capture ELISA. Twofold dilutions of cell culture medium containing D1-Fc (■ and □) or PVR-Fc (● and ○) were bound to 96-well plates coated with MAb 190/4 (■ and ●) or negative control MAb P1B5 (□ and ○). Bound immunoadhesins were stained with peroxidase-labeled anti-human Fc antibodies. TMB was used as substrate, and the absorbance (O.D.) was read at 450 nm (y axis) and plotted against the dilution (x axis). Data are means of results from duplicate wells; duplicate values varied by less than 10%. The results are those of one experiment which was repeated at least twice with approximately 5 to 10% experimental error.

FIG. 4

Binding of HAV to 96-well plates coated with D1-Fc. Twofold dilutions of purified HAV were bound to 96-well plates coated with 10 μg of purified D1-Fc or human normal immunoglobulins (hu normal Ig) per ml. A well coated with 10 μg of human anti-HAV immunoglobulins (hu anti-HAV Ig) per ml was used as a positive control for virus binding. Bound HAV was detected by ¹²⁵I-labeled human anti-HAV antibodies, extensive washing, and direct autoradiography of the 96-well plate.

FIG. 5

Binding of HAV to immunoadhesins attached to protein A-treated beads. Different amounts of purified D1-Fc or PVR-Fc were bound to 25 μl of protein A-Trisacryl beads for 2 h at 4°C. Sucrose-purified HAV (5 × 10⁷ TCID₅₀) was added, and the mixture was incubated with rotation overnight at 4°C. After the mixture was washed three times with PBS at 4°C, bound HAV was eluted with 100 μl of 6 M LiCl₂ for 30 min at room temperature, diluted 40-fold with EMEM, and subjected to titer determination on AGMK GL37 cell monolayers. Values are the log₁₀ of the HAV titers determined by the Reed and Muench method (32), and the standard deviations are shown as error bars.

FIG. 6

Inhibition of binding of HAV to D1-Fc by protective MAb 190/4. Equal amounts (3 μg) of D1-Fc and PVR-Fc were treated with 0, 0.5, 5, or 50 μg of MAb 190/4 or control MAb M2 at 4°C. Protein A-Trisacryl beads (25 μl) were added, and the mixture was incubated for 2 h at 4°C. Sucrose-purified HAV (5 × 10⁷ TCID₅₀) was added, and the mixture was incubated with rotation overnight at 4°C. HAV was eluted and subjected to titer determination as described in the legend to Fig. 5. Values are the log₁₀ of the HAV titers determined by the Reed and Muench method (32), and the standard deviations are shown as error bars.

FIG. 7

Neutralization of HAV by D1-Fc. Different amounts of purified D1-Fc and PVR-Fc (100, 10, and 1 μg) were incubated overnight at 4°C with 10⁵ TCID₅₀ of HAV PI stock. Tenfold dilutions of the neutralization reactions were subjected to titer determination on 96-well plates containing confluent monolayers of AGMK GL37 cells. After 4 h of adsorption at 37°C, the plates were washed three times and incubated for 10 days at 35°C under CO₂. All dilutions and incubations were done in the presence of 10 μg of the respective immunoadhesin per ml. HAV titers were determined by ELISA. Values are the log₁₀ of the HAV titers determined by the Reed and Muench method (32), and the standard deviations are shown as error bars.

FIG. 8

Neutralization of HAV by MAbs. HAV PI stock (10⁵ TCID₅₀) was treated with 50 μg of protein A-purified MAbs K2-4F2, K3-4C8, and VHA 813 for 1 h at 37°C. Tenfold dilutions of the neutralization reaction products were subjected to titer determination on 96-well plates containing confluent monolayers of AGMK GL37 cells. After 4 h of adsorption at 37°C, the plates were washed three times and incubated for 10 days at 35°C under CO₂. HAV titers were determined by ELISA. Values are the log₁₀ of the HAV titers calculated by the Reed and Muench method (32), and the standard deviations are shown as error bars.