INTRODUCTION

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The interactions between virus and antiviral antibodies play a crucial role in the pathogenesis of Aleutian mink disease parvovirus (ADV) infections (4, 15, 18, 51). Adult mink infected with pathogenic isolates of ADV develop a persistent infection associated with high levels of antiviral antibodies and hypergammaglobulinemia (4, 15, 17, 18, 51). In spite of this robust immune response, virus is not eliminated in vivo (15, 30, 33, 49) and severe immune complex disease and vasculitis develop (51, 53). In fact, complexes containing infectious virus have been demonstrated, denoting the direct involvement of antiviral antibody in this syndrome (50). Furthermore, antiviral antibody enables ADV to infect cells such as macrophages or the monocytic cell line, K562, via an Fc-receptor-dependent mechanism termed antibody-dependent enhancement (ADE) of infection (29, 35). Macrophages are the target cells for persistent ADV infection in vivo, and their infection may play a role in the genesis of the immune disorder (15, 34, 36, 42). Finally, as might be anticipated from these observations, vaccination of mink or the presence of preexisting antiviral antibody does not protect adult mink from ADV infection but, rather, leads to an accelerated form of disease upon challenge (1, 52).

Antiviral antibodies in some circumstances can also play a beneficial role in ADV infections. For example, antibody is able to neutralize ADV infectivity for Crandell feline kidney (CrFK) cells in vitro (1, 35, 59). In addition, antiviral antibody has a mitigating effect on ADV infection in mink kits (2, 10, 11), where presence of natural or passively administered antibody prevents the fulminant, fatal pneumonitis associated with the permissive infection of type II alveolar cells by ADV (9, 10, 15). The mechanism for this effect is unclear, although at the level of the individual cell, the antibody converts permissive infection into a restricted infection (10, 11).

ADV infections stand in sharp contrast to infections of mink with another nondefective parvovirus, mink enteritis virus (MEV), which is a viral host range variant of feline panleukopenia virus (46, 47, 48). Capsid-based vaccines against MEV promptly induce neutralizing antibody and prevent infection and disease (22, 39). In addition, persistent infections do not develop. Consequently, the atypical picture observed during ADV infections cannot be ascribed simply to a generic response of mink to parvoviruses.

The ADV capsid consists of 60 individual capsid proteins. In native capsids from ADV-infected cells, ca. 90% is the 647-amino-acid major capsid protein, VP2 (8, 23). The minor capsid protein, VP1, contains the entire VP2 sequence but has 43 additional unique residues at the N terminus (8, 23, 24, 63). When VP2 from the ADV-G isolate is expressed in either recombinant vaccinia viruses (24) or baculoviruses (23, 63), the proteins assemble into empty capsids. Recent work with prokaryotic expression vectors has localized immunodominant targets for the antibody response to specific regions of the VP2 capsid protein (16, 28). The most immunoreactive region spans VP2 residues 429 to 524 (VP2:429-524) (16, 28). Polyclonal rabbit antibodies directed against this region neutralize ADV infectivity for CrFK cells and strongly react with capsids in immunoelectron microscopy (16). Infected mink also recognize a major epitope in VP2:428-446, a sequence contained within this region (16, 28).

Taken together, these findings from antigenic mapping and pathogenesis studies indicate the importance of understanding the capsid structure. A three-dimensional model of the T=1 ADV capsid has been built into electron density determined to 22 Å resolution by cryoelectron microscopy and image reconstruction (Fig. 1) (41). Although the structure shares canonical features with more prototypical parvoviruses, such as canine parvovirus (CPV), feline panleukopenia virus, and minute virus of mice (5, 7, 41, 61), several prominent differences are evident (Fig. 1). Most notably, there are three knob-like mounds decorating the viral icosahedral threefold axes of symmetry and wider ridges that separate the dimple-like depression at the icosahedral twofold axes from the canyon-like depression surrounding the icosahedral fivefold axes. The differences can largely be accounted for by short stretches of amino acids inserted into the flexible loop segments of the VP2 protein molecules in ADV compared to all other parvoviruses (16, 20, 41). Several of these insertions are located in the highly immunoreactive VP2:429-524 segment. The additional peptide sequences may be potential binding sites for antibodies involved in ADV pathogenesis.

View larger version (156K): [in this window] [in a new window]   FIG. 1.   Shaded surface representation of the ADV-GVP2 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.

By in vitro monitoring of immune complex formation, virus neutralization in CrFK cells, and ADE in K562 cells as model systems for the involvement of antibody in ADV pathogenesis, several questions may be formulated. First, are the same antigenic determinants of ADV implicated in these phenomena? Second, at what location on the virus particle do these determinants reside? Third, do specific structural features on the ADV capsid contribute to pathogenicity and the immune response it engenders?

To address these questions, we prepared rabbit polyclonal and mouse monoclonal antibodies against several peptides contained within the VP2:429-524 portion of VP2. We have characterized these antibodies with respect to their capacity to participate in vitro in immune complex formation, to neutralize infectivity, and to mediate ADE. Our results suggested that antibodies to a short sequence in the VP2 capsid protein can be implicated in all three phenomena. Other antibodies were able to mediate ADE but failed to neutralize virus or generate immune complexes. These findings indicate that antibodies to specific capsid sequences are important in the pathogenesis of ADV infections. The observation that the same amino acid sequence can host both virus neutralization and ADE may explain the inability of capsid-based vaccines to protect against ADV infections in adult animals.

	MATERIALS AND METHODS

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Viruses and cells. Crandell feline kidney cells (CrFK), K562 cells and the Spodoptera frugiperda insect cell line (Sf9) were maintained according to previously reported methods (14, 29, 41, 63). The procedure for propagating the molecularly cloned, cell culture-adapted ADV-G has also been described (14). For production of recombinant ADV capsids containing only VP2 (ADV-G_VP2), Sf9 cells were infected with recombinant baculovirus in suspension cultures at a multiplicity of 1 PFU/cell for 72 h (41, 63).

Peptides. Peptides, based on the ADV-G VP2 capsid protein sequence (16, 43) and conjugated to keyhole limpet hemocyanin through an additional N-terminal cysteine, were obtained from a commercial source (Princeton Biomolecules, Columbus, Ohio) and characterized by the Structural Biology Section of the National Institute of Allergy and Infectious Diseases (NIAID) (27). Purity was estimated to be >75%. The peptide designations and amino acid sequences were as follows: VP2:428-446, (C)SNYYSDNEIEQHTAKQPL (28); VP2:455-470, (C)KIDSWEEGWPAASGTH; and VP2:487-501, (C)EQELNFPHEVLDAA. The peptides were dissolved in phosphate-buffered saline (PBS) at 0.5 mg/ml. The VP2:429-524 region is contained in a prokaryotic fusion protein (16).

Production of polyclonal and monoclonal antibodies. Heterologous polyclonal antipeptide antisera were prepared in rabbits according to a published protocol (27).

Immune blotting. Immune blots were performed by using lysates of ADV-G-infected CrFK cells as antigen (16). The blocked membranes were clamped into a multichannel manifold, and individual primary antibodies and developing reagents were applied to separate channels. The following slight modifications yielded very low backgrounds; blocking and dilution of primary and secondary antibodies was in PBS-0.5% Tween 20-5% nonfat dry milk. Signals from rabbit sera and mouse monoclonal antibody samples were developed by using, respectively, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated rabbit anti-mouse immunoglobulins.

Immune electron microscopy. The ability of antibodies to bind native ADV-G or ADV-G_VP2 capsids immobilized on Parlodion-coated grids was evaluated precisely as detailed in an earlier study (16). Antibody that decorated virus was revealed by use of goat anti-rabbit IgG or rabbit anti-mouse IgG, both conjugated to 5-nm gold particles. Reactions were graded as follows: , negative; +, <10% of the capsids had 1 or more gold particles; ++, 30 to 70% of the capsids decorated with fewer gold particles per capsid; +++, heavy gold particle decoration of >90% of capsids.

ADE of infection. The method for monitoring ADE was modified slightly from previous reports (29, 35). Briefly, individual wells of a 24-well culture plate were seeded with 5 × 10⁵ K562 cells in 0.25 ml of complete medium. Equal volumes (0.4 ml) of serial 10-fold dilutions of sera or monoclonal antibodies were incubated at 37°C for 1 h with a standard dilution of ADV-G. The mixture was added to the cells and cultured for 72 h at 31.8°C. At this time, 5 × 10⁴ cells were cytocentrifuged onto slides, acetone fixed, and stained for ADV antigens with fluorescein isothiocyanate (FITC)-conjugated IgG from ADV-infected mink. The number of positive cells was counted and compared to control cultures receiving ADV mixed with medium alone. No enhancement was observed when normal serum or an isotype-matched monoclonal antibody was used.

Virus neutralization. The assay for antibody neutralization of ADV-G infectivity for CrFK cells was performed as detailed in earlier work (16). Any serum dilution that caused a >70% reduction in the number of ADV-positive cells was considered positive.

IFA. Indirect immunofluorescence assay (IFA) was done on acetone-fixed cytocentrifuged samples of CrFK cells, either uninfected or infected 72 h previously with ADV-G (44). The secondary antibodies were FITC-conjugated goat anti-rabbit IgG or FITC-conjugated rabbit anti-mouse immunoglobulins. After the final wash, a drop of propidium iodide was placed on the cells for 10 s in order to delineate the nucleus. Images were captured by laser scanning confocal microscopy as reported elsewhere (44).

Structural modeling. An isodensity map of the ADV-G_VP2 capsid derived by cryoelectron microscopy, image reconstruction, and comparison with the CPV capsid atomic structure has been reported (41). In an effort to evaluate possible antibody interactions of these ADV peptide regions, Fab fragments were docked into approximate binding sites by using available lysozyme-Fab complex structures as a guide (45). Specifically, antigenic epitopes of lysozyme in the complexes were superimposed onto the ADV peptides to position the Fab fragments at allowable antigen-antibody interaction distances and orientations relative to antigenic sites (21, 56, 58). The lysozyme coordinates were then deleted from the complexes, leaving the Fab in contact with the ADV capsid.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Immunoblot analysis. To evaluate reactivity of the antipeptide antibodies against the full-length ADV capsid proteins, antibodies were tested in immunoblot against lysates of ADV-G-infected CrFK cells (Fig. 2). All three polyclonal sera reacted against both of the overlapping capsid proteins, VP1 and VP2, as did the anticapsid serum. The monoclonal antibodies against VP2:428-446 and VP2:487-501 also recognized both capsid proteins. The polyclonal antibody against the VP2:429-524 expression protein (16) also reacted with VP1 and VP2 and, in addition, recognized a smaller protein that is likely a degradation product (3). All antibodies reacted specifically with their cognate peptides by ELISA (data not shown). Thus, immunization of rabbits and mice with the peptides induced antibodies that were reactive with denatured full-length capsid proteins.

View larger version (50K): [in this window] [in a new window]   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.

Immunoelectron microscopy. Antiviral antibody interacts with ADV in animals to form immune complexes, which are a key feature in the pathogenesis (15, 51, 53). To determine whether the antipeptide antibodies can bind and aggregate capsids, we used immunoelectron microscopy. Antibodies were assessed for their ability both to decorate immobilized capsids and to aggregate particles, the latter being a surrogate measure for immune complex formation (Fig. 3). Both the polyclonal and monoclonal anti-VP2:428-446 antibodies bound densely to immobilized capsids, as evidenced by the heavy decoration with gold-labeled secondary reagents (Fig. 3A). Furthermore, these antibodies aggregated soluble capsids to produce obvious immune complexes (Fig. 3B), in which antibody bridging was noted and the capsid margins were indistinct. Some of these complexes were very large, incorporating hundreds of particles.

View larger version (62K): [in this window] [in a new window]   FIG. 3.   Immunoelectron microscopy with antibodies prepared against ADV VP2 peptides. Antibody preparations were tested for the capacity to react with ADV-GVP2 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.

Antibody-dependent enhancement of infection. Antiviral antibody can mediate ADV infection of the monocytic cell line, K562, via the FcRII receptor (29). This phenomenon of ADE of infection may play a role in the immunopathogenesis of ADV infections in adult mink (15, 29, 35, 36). To determine if antibodies to specific regions of the capsid could enable Fc-receptor-dependent ADE, we assayed the capacity of the antipeptide rabbit polyclonal and mouse monoclonal antibodies to mediate ADE (Fig. 4).

View larger version (17K): [in this window] [in a new window]   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.

In vitro virus neutralization assay. Although ADV is not eliminated from infected adult animals, antibody can mitigate the severity of ADV infections in mink kits and neutralize infectivity for CrFK cells in vitro (2, 11, 16, 59). Our previous studies show that in vitro neutralizing determinants are present in the segment spanning residues, i.e., VP2:429-524 (16). To extend this work, we tested the antibodies generated against smaller peptides contained within this region (Table 1). The rabbit polyclonal antibodies prepared against both capsids and VP2:429-524 and VP2:428-446 peptide neutralized ADV-G. No other polyclonal antibody had detectable neutralizing activity. The mouse monoclonal antibodies, tested either separately or as a cocktail, did not exhibit any neutralizing activity. Thus, a neutralizing determinant was located in the peptide sequence VP2:428-446.

Indirect immunofluorescence analysis. The results in the previous sections indicated that the antibodies against the ADV VP2 peptides exhibited different reactivities in several assays. We also used these antibodies in indirect immunofluorescence to determine whether they produced different staining patterns on ADV-G-infected CrFK cells and to determine whether the specific epitopes might also be differentially available for staining.

View larger version (45K): [in this window] [in a new window]   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.

Structural considerations. The three ADV peptide regions examined for their ability to elicit antibodies capable of immune complex formation, virus neutralization, and ADE were mapped onto a two-dimensional "roadmap" (19) representation of the ADV-G_VP2 capsid surface by using an available pseudo-atomic model (41, 61) (Fig. 6A). All three peptides contain capsid surface-exposed residues (Fig. 6A). In addition, the location of the peptides was depicted on a three-dimensional reconstruction (Fig. 6B), which gives a perspective on the disposition of the peptides on the actual particle.

View larger version (70K): [in this window] [in a new window]   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-GVP2 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-GVP2 viewed down a twofold icosahedral axis.

View larger version (56K): [in this window] [in a new window]   FIG. 7.   Side-view models of ADV-GVP2 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.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Our previous studies localized immunoreactive sites and in vitro neutralizing epitopes to a specific region of the ADV VP2 capsid protein (VP2:429-524) containing variable loops that partially comprise the surface of the virus particle (16, 41). In the present study, we analyzed antibodies against several short peptides within this region for their capacity to support immune complex formation, neutralization of ADV for CrFK cells, and Fc-receptor-mediated ADE of infection, three features implicated in ADV pathogenesis. We found that a determinant located in VP2:428-446 exhibited these three properties. Therefore, this short sequence in the capsid protein gene is involved in three key aspects of ADV pathogenesis.

Attempts to protect adult mink with capsid-based ADV vaccines have uniformly failed, even though vaccinated animals develop antiviral antibodies (1, 49, 52). Indeed, upon challenge, vaccinated animals suffer a hyperacute disease with enhanced tissue lesions (1, 52). We observed that injection of rabbits with VP2:428-446 induced both neutralizing and ADE-mediating antibody (Fig. 4; Table 1). Whereas virus neutralization by antibody may afford protection from infection, ADE facilitates the infection of macrophages that is central to the resulting immune disorder (15, 29, 35). Antibodies to VP2:428-446 have been demonstrated in ADV-infected adult mink (28) and, if these antibodies function to mediate both ADE and neutralization in vivo, any protective effect might be counteracted or supervened. Thus, our results provide possible explanations for the failure of capsid-based ADV vaccines (1, 52). As noted in the introduction, capsid-based vaccines against MEV are highly effective in mink (22, 39). In fact, peptide vaccines based on the MEV capsid sequence also induce neutralizing antibodies and confer protection (39). This indicates that a peptide vaccine can be efficacious in mink. Consequently, it will be extremely interesting to determine whether mink injected with the VP2:428-446 peptide will produce both neutralizing and ADE-mediating antibody.

Our results suggested that at least one epitope within VP2:428-446 was surface exposed and readily available for antibody binding (Fig. 6 and 7). This observation is consistent with the roadmap representation of the ADV-G_VP2 pseudo-atomic structure which revealed exposed residues of this peptide on the twofold dimple walls (VP2:428-429; Fig. 6, green) and adjacent to the mounds offset from the icosahedral threefold axes (VP2:439-441) (41). Both the polyclonal and the monoclonal antibodies to VP2:428-446 aggregated virus particles (Fig. 3), but only the polyclonal antibody had neutralizing activity (Table 1). This observation supports data from CPV that virus cross-linking is not sufficient for parvovirus neutralization (6, 60, 62). Other possible mechanisms for neutralization include occlusion of the receptor attachment site (6, 38, 40, 55, 60) and stabilization of capsids against genome uncoating (13, 21, 37, 55). The modeled docking of Fab fragments over the exposed amino acids of VP2:428-446 (Fig. 7A) suggests that an antibody binding to either exposed terminus could occlude the region where a putative binding site for a CPV sialic acid receptor is located (6, 12) (Fig. 7). Thus, an antibody to the VP2:428-429 region might neutralize by impeding cell binding (Fig. 6). The same region is close to ADV VP2:395 and other residues implicated in host range and pathogenicity (Fig. 7A) (14, 31). It is currently unclear which of these mechanisms is operative for neutralization of ADV for CrFK cells by the VP2:428-446 antibodies.

The monoclonal antibody to VP2:428-446 cross-linked particles and formed immune complexes (Fig. 3). This mandated that the epitope be located in a position such that a single IgG molecule could bridge two particles. By using previously determined allowable antigen-antibody interaction distances and orientations relative to antigenic sites (45, 21, 56, 58), we modeled potential interactions between an antibody molecule and the surface-exposed portions of VP2:428-446 (Fig. 7). The results suggested that the epitope should be located near the icosahedral twofold axes where the particle has the smallest cross-sectional diameter (41). Furthermore, binding of the Fab arms from a single monoclonal antibody to two virus particles should be readily accommodated (21, 55-58), resulting in particle bridging. A direct evaluation of this prediction can be done by analyzing cryoelectron microscopy-based reconstructions of particles complexed with Fab fragments. These experiments are currently under way.

Structural mapping of antigens for CPV and several other parvoviruses places major neutralizing domains adjacent to the icosahedral threefold axes and on the shoulder of the wall between the fivefold canyon-like region and the icosahedral twofold dimple (6, 38, 60, 62). The mounds on the ADV capsid, which contain the VP2:455-470 and VP2:487-501 peptides, would alter the general capsid topography where major neutralizing epitopes of CPV map (6, 41). It is possible that these mounds might disrupt potential major neutralizing domains and direct the antibody response toward targets that mediate ADE and immune complex formation. This might contribute significantly to the distinct immunopathogenesis of ADV infections.

The failure of the antibody to VP2:455-470 to aggregate, neutralize, or mediate ADE was at first somewhat surprising, because the peptide is mostly surface exposed near the apex of the mounds (Fig. 6 and 7B). The reasons for this are unclear at present; however, upon inspection of the ADV-G_VP2 pseudo-atomic model, the region around VP2:455-470 has significant secondary structure (41). If VP2:455-470 is part of a complex conformational structure, it may not be recognized by the antipeptide antibody in all its configurations. This might also explain the limited reactivity of this antibody in our IFA studies (Fig. 5).

The limited ability of antibodies to VP2:487-501 to aggregate particles and mediate ADE is consistent with the surface exposure of the linear peptide residues (Fig. 6). The low degree of particle aggregation and gold particle decoration (Fig. 3) may be explained by the observation that only a single Fab arm can be accommodated in the "cirque-like" gap between the threefold mounds where three VP2:487-501 epitopes would reside (41) (Fig. 7C). Thus, the expected occupancy would only be one-third of that predicted for VP2:428-446 where each of the 60 epitopes on the capsid is available.

The pathogenicity and host range of ADV are controlled by coordinate interactions among a small number of capsid amino acids (14, 31, 43). One of the residues controlling host range (VP2:434) is located in the VP2:428-446 peptide. Other involved amino acids also are found close to this site (Fig. 6 and 7). The fact that these residues cluster suggests that this small part of the capsid is important not only in the immunopathogenesis of ADV infections but also in viral host range and pathogenicity.

In summary, we have implicated short peptide sequences in key aspects of ADV pathogenesis and in the adverse response of mink to capsid-based vaccines (1, 52). In addition, unique features of the ADV capsid may also play a role in the unusual nature of infections with this virus. Clearly, our studies do not address the entire capsid protein sequence or potential conformational epitopes (43, 54). Consequently, the results leave open the possibility that complex functional classes of epitopes exist on the ADV capsid: some mediating only neutralization, others involved with ADE and immune complex formation, and others having mixed characteristics. It is possible that nonneutralizing epitopes might dominate by inducing antibodies that interfere with neutralization and accentuate disease (25, 32).