Identification of aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection, virus neutralization, and immune complex formation

Abstract

Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.

FIG. 1

Shaded surface representation of the ADV-G_VP2 three-dimensional reconstruction viewed perpendicular to a twofold icosahedral axis, obtained by the method of McKenna et al. (41). The following features are depicted on the reconstruction: twofold (“2”), threefold (“3”), and fivefold (“5”) icosahedral axes; the mounds adjacent to the threefold axis (3-fold mounds); the dimple or depression at the twofold axis (2-fold dimple); and the canyon surrounding the fivefold axis (5-fold canyon). An asymmetric icosahedral unit is superimposed on the structure as a triangle. The resolution of the image is 22 Å.

FIG. 2

Immunoblot with antibodies prepared against ADV VP2 peptides. The indicated rabbit polyclonal (P) or mouse monoclonal (M) antibody preparations were reacted in immunoblot against a whole-cell lysate of ADV-G-infected CrFK cells. The rabbit polyclonal sera were used at a 1/100 dilution, except for the anti-ADV-G capsid serum, which was used at 1/1,000; the mouse monoclonal antibodies were at 1 μg/ml. The positions of the ADV capsid proteins VP1 and VP2 are marked.

FIG. 3

Immunoelectron microscopy with antibodies prepared against ADV VP2 peptides. Antibody preparations were tested for the capacity to react with ADV-G_VP2 capsids either in decoration or aggregation and were scored as detailed in Materials and Methods. (A and B) Decoration (A) and aggregation (B), both at +++ levels (anti-VP2:428-446 antibodies) and at negative levels (control antibodies) are depicted. (C) Tabulation of results from all sera tested. No difference was noted between rabbit polyclonal (used at a 1/100 dilution) or mouse monoclonal (used at 100 μg/ml) antibodies directed against the same peptide.

FIG. 4

Antibody-dependent enhancement of ADV infection for K562 cells mediated by antibodies prepared against ADV VP2 peptides. Dilutions of the indicated rabbit polyclonal and murine monoclonal antibodies (1 mg/ml, undiluted) were incubated with ADV-G at 37°C for 1 h. The mixture was added to K562 cells. After culture for 72 h at 31.8°C, 5 × 10⁴ cells were cytocentrifuged onto slides, acetone fixed, and stained for ADV antigens. The number of positive cells was counted and compared to control cultures receiving ADV mixed with medium alone.

FIG. 5

Immunofluorescent staining of ADV-G-infected CrFK cells with antibodies prepared against ADV VP2 peptides. Acetone-fixed cytocentrifuged preparations of ADV-G-infected CrFK cells were reacted with rabbit polyclonal or murine monoclonal antibodies with the indicated specificities. Staining was revealed with FITC-conjugated anti-rabbit IgG or anti-mouse IgG. Nuclei were counterstained with propidium iodide. As indicated in the text, no difference was observed between monoclonal and polyclonal antibodies with the same specificity.

FIG. 6

Location of ADV VP2 peptides on ADV capsid. VP2:428-446 (green), VP2:455-470 (blue), and VP2:487-501 (red). (A) Predicted surface-exposed residues from peptides are indicated on a roadmap representation of the surface of the ADV-G_VP2 pseudo-atomic structure. The view is down a twofold icosahedral axis. (B) Predicted locations of ADV VP2 peptides are projected onto a shaded-surface representation of ADV-G_VP2 viewed down a twofold icosahedral axis.

FIG. 7

Side-view models of ADV-G_VP2 particles complexed with IgG Fab fragment (depicted as van der Waals balls). The position of the icosahedral threefold related mounds and residues involved in host range and pathogenicity (VP2:352, VP2:395, VP2:434) (14, 31) is noted. (A) Fab fragment (yellow) docked with VP2:428-446 (green) in a side view perpendicular to an icosahedral twofold axis. (B) Fab fragment (yellow) docked with VP2:455-470 (blue) in a side view perpendicular to an icosahedral threefold axis. (C) Fab fragment (yellow) docked with VP2:487-501 (red) in a side view perpendicular to an icosahedral threefold axis.