Hepatitis A virus-specific immunoglobulin A mediates infection of hepatocytes with hepatitis A virus via the asialoglycoprotein receptor

Abstract

The mechanisms underlying the hepatotropism of hepatitis A virus (HAV) and the relapsing courses of HAV infections are unknown. In this report, we show for a mouse hepatocyte model that HAV-specific immunoglobulin A (IgA) mediates infection of hepatocytes with HAV via the asialoglycoprotein receptor, which binds and internalizes IgA molecules. Proof of HAV infection was obtained by detection of HAV minus-strand RNA, which is indicative for virus replication, and quantification of infectious virions. We demonstrate that human hepatocytes also ingest HAV-anti-HAV IgA complexes by the same mechanism, resulting in infection of the cells, by using the HepG2 cell line and primary hepatocytes. The relevance of this surrogate receptor mechanism in HAV pathogenesis lies in the fact that HAV, IgA, and antigen-IgA complexes use the same pathway within the organism, leading from the gastrointestinal tract to the liver via blood and back to the gastrointestinal tract via bile fluid. Therefore, HAV-specific IgA antibodies produced by gastrointestinal mucosa-associated lymphoid tissue may serve as carrier and targeting molecules, enabling and supporting HAV infection of IgA receptor-positive hepatocytes and, in the case of relapsing courses, allowing reinfection of the liver in the presence of otherwise neutralizing antibodies, resulting in exacerbation of liver disease.

FIG. 1

HAV release from the HAV–anti-HAV IgA complexes during interaction with the murine hepatocyte cell line NCTC 1469. NCTC 1469 cells were inoculated with HAV–anti-HAV IgA immunocomplexes for 2 h at 34°C. As controls, cells were inoculated with HAV in amounts equivalent to those in the immunocomplex inoculum (2¹¹ TCID₅₀/ml). HAV titers were determined by titration of the cell lysates using FRhK-4 cells. (A) Inoculation with and without Ca²⁺. ic, inocula without cell contact. (B) Preincubation with 200 μg of nonspecific (nonsp.) IgA MOPC 315 per ml or inoculation in the presence of 0.45 M sucrose. The data represent means from two independent experiments, and error bars indicate standard deviations.

FIG. 2

Proof of IgA-mediated infection of the murine hepatocyte cell line NCTC 1469 with HAV. NCTC 1469 cells were inoculated for 2 h at 34°C with HAV–anti-HAV IgA immunocomplexes as well as with HAV as a control in amounts equivalent to those in the immunocomplex inoculum (2¹⁶ TCID₅₀/ml) and incubated further for various times. Additionally, HAV-specific IgA was used as a supplement at days 4, 8, 12, and 16 after infection with HAV–anti-HAV IgA. (A) HAV titers were determined by titration of the cell lysates using FRhK-4 cells. Day 0 represents infectious HAV after inoculation for 2 h. (B) Factors by which infectious HAV increased during the course of the incubation in comparison to the virus detected after inoculation for 2 h (day 0). The data are means obtained from two separate experiments, and error bars indicate standard deviations.

FIG. 3

Proof of IgA-mediated infection of the human hepatocyte cell line HepG2 with HAV. HepG2 cells were inoculated for 2 h at 34°C with HAV–human anti-HAV IgA as well as with HAV–human anti-HAV IgG. As controls, cells were inoculated with HAV in amounts equivalent to those in the immunocomplex inocula (3 × 10⁶ TCID₅₀/ml). (A) HAV titers were determined by titration of the cell lysates using FRhK-4 cells. Day 0 represents infectious HAV after inoculation for 2 h. (B) Factors by which infectious HAV increased during the course of the incubation in comparison to the virus detected after inoculation for 2 h (day 0). The data represent means from two independent experiments, and error bars indicate standard deviations.

FIG. 4

Influence of inhibitors of IgA binding on IgA-mediated HAV infection of primary human hepatocytes. Cultured human primary hepatocytes preincubated for 1 h with 200 μg of nonspecific (nonsp.) IgA MOPC 315 (▥) per ml or anti-human ASGPR antibody (▤) were inoculated with HAV–human anti-HAV IgA complexes. As controls, cells were inoculated with HAV in amounts equivalent to those in the immunocomplex inoculum (2¹⁷ TCID₅₀/ml). (A) HAV titers were determined after inoculation for 2 h and 5 days (d) after inoculation by titration of the cell lysates using FRhK-4 cells. (B) TCID₅₀ per milliliter as a percentage of the titers obtained without inhibitors (black bars) for a better view of the inhibitory effect. The data represent means from two separate experiments, and error bars indicate standard deviations.

FIG. 5

Stability of HAV–anti-HAV IgA complexes at different pHs. HAV–anti-HAV IgA complexes were incubated in 0.1 M glycine-HCl buffer at pHs 7, 3.5, 2.5, and 1.5 for 180 min and at pH 1.5 for 30, 60, and 120 min at 37°C. After pH neutralization, infectious virus was determined by titration with FRhK-4 cells. As a control, HAV in amounts equivalent to those in the complexes (10⁵ TCID₅₀/ml) was treated in the same way. Shown are the TCID₅₀ per milliliter as a percentage of the titer obtained with HAV without treatment.