Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb (original) (raw)
Non-neutralizing anti-VP6 IgA mAb 7D9 effectively protects mice from rotavirus infection. Previously, in a “backpack” model in which mice were transplanted with hybridoma cells secreting selected mAb’s, mAb 7D9 was shown to protect mice from virulent rotavirus challenge, while mice treated with other hybridoma cells failed to be protected (17). To rule out the possibility that the lack of protection was caused by variation in viability or Ig production in vivo by the transplanted hybridoma cells, we treated mice daily with 3 mg of selected mAb injected intraperitoneally. The treated animals were challenged with wild-type murine rotavirus after 2 days of pretreatment as described. The protective efficacy of the antibodies expressed as the percent reduction of total fecal antigen shedding as compared with noninjected controls is presented in Figure 1. The non-neutralizing anti-VP6 IgA 7D9 completely protected mice from challenge, confirming our prior studies using the “backpack” model and a cell transfer approach (Figure 1). Even after a 1:50 dilution of ascites (0.06 mg daily dose), mice treated with 7D9 shed significantly less viral antigen than did controls (62% reduction of shedding). Treatment with other non-neutralizing antibodies to VP6 or NSP4, including mAb 8D3, 1E11, SGI-255/60, and B4-2, did not significantly reduce fecal viral antigen shedding after challenge (Figure 1). Of note, VP7 serotype G3–specific neutralizing mAb 159 also failed to protect mice, even though the in vitro neutralizing titer against tissue culture–adapted EC virus was greater than 1:500,000 (Table 1). The protective efficacy of 7D9 was, however, affected by the dose size of challenge virus. When 7D9-treated mice received 105 ID50 of EC virus rather than 102 ID50, no reduction of shedding was observed (data not shown). These findings confirm and extend our previous observations and indicate that the protective effect of 7D9 is not dependent on the transfer of living hybridoma cells to recipient mice but is dependent on the titer of the challenge dose administrated.
Viral protein specificity and isotypes of mAb’s used in the study
Lipofection-mediated intracellular neutralization. Because 7D9 is directed against the rotavirus intermediate layer protein VP6, which is only exposed on DLPs and is not the target of classical neutralization activity (Table 1 and ref. 17), we reasoned that the antibody may neutralize rotavirus only after the virus has entered the cell and has shed the outer capsid protein VP4 and VP7. Previous studies have shown that “noninfectious” DLPs were rendered infectious if introduced into the cytoplasm of permissive cells (30). Based on these results, we developed a lipofection-mediated intracellular neutralization assay to determine whether antibodies to VP6 had antiviral activity if introduced into the cytoplasm (see Methods). The intracellular neutralization titers of selected antibodies against a panel of rotaviruses including the challenge strain EC are provided in Table 2. Only mAb 7D9 effectively neutralized viruses at high titer in this lipofection-based assay. The neutralization titers of 7D9 were similar against the different virus strains tested, which represented a variety of serotype and subgroup specificities (Table 2). The nonprotective VP6 IgA antibody 8D3 had low intracellular neutralization titers, and this low-level neutralization was not consistently detectable. Antibodies 1E11 and B4-2 did not neutralize virus intracellularly, while mAb SGI-255/60 showed low neutralization activity against RRV only (a subgroup I virus). mAb 159, which effectively neutralizes infectious serotype G3 viruses in classical neutralization assay (Table 1), did not inhibit DLP viruses (Table 2) or TLP viruses (data not shown) in the lipofection assay. The reciprocal titers of the mAb’s detected inside MA104 cells after transfection (data not shown) were between 2560 and 10,240, suggesting that transfection efficiencies for different antibodies were similar and that the differences in intracellular neutralization activity were not due to differences in the amount of antibody transfected.
Lipofection-mediated intracellular neutralization titers by selected mAb’s
Kinetic of intracellular neutralization. To investigate at which stage of infection rotaviruses were being inhibited by IgA 7D9, we infected cells with RRV TLPs and, at selected times after infection, transfected purified 7D9 (0.025 mg/ml) into the infected monolayers (Figure 2). When 7D9 was transfected between 0 and 2 hours after infection, it significantly inhibited virus infection. However, by 3 hours after infection, reduction was less than 50% (below neutralizing titer cutoff) (Figure 2). The intracellular neutralizing activity became undetectable when 7D9 was transfected 4 hours after infection. Since the antibody was effective even when added to cells after infection, its effects are not likely to be due to extracellular aggregation.
Efficiency of lipofection-mediated intracellular neutralization by 7D9 at different time points after infection. MA104 cells were infected with TLPs of RRV. At indicated time points after infection, 7D9 mAb was transfected into cells (0.025 mg/ml) using lipofectin. Viral antigen in the cells was detected 10 hours after initial RRV infection by immunostaining. Data are expressed as percentage of focus reduction as compared with control. *Focus reduction was below 50% and significantly different from that at time 0 (P < 0.05). Results were calculated from triplicate measurements and represent two separate experiments.
In vitro inhibition of viral RNA transcription. The kinetic studies indicated that IgA 7D9 neutralizes rotavirus at an early stage of viral replication following viral infection. We hypothesized that IgA 7D9 may function, at least in part, by inhibiting viral mRNA transcription after the virus uncoats in the cytoplasm. To initially evaluate the inhibitory effects of selected rotavirus-specific antibodies on viral mRNA transcription, we incubated various antibodies with transcriptionally competent DLPs of RRV and EC and measured the incorporation of 32P into viral mRNA transcripts (Figure 3, a and b). The level of transcription of RRV and EC in the presence of antibodies B4-2 and 159 was not significantly different from that of the control with no antibody added. The mRNA transcription of DLPs treated with antibodies 8D3 and 1E11 was significantly greater than that of the control. SGI slightly suppressed mRNA transcription of RRV only (62%, P < 0.05). IgA 7D9, however, completely suppressed the in vitro rotavirus mRNA transcription of both RRV and EC (Figure 3, a and b; _P_ > 0.01). This finding is consistent with the hypothesis that IgA 7D9 mediates its antiviral effect by blocking early-phase transcription of the rotavirus replication cycle.
Effects of selected mAb’s on in vitro rotavirus transcription. RRV (a) and EC (b) were treated with EDTA and mixed with selected mAb’s. Transcription assay was conducted using a Riboprobe system at 40°C for 1 hour as described. Transcripts were labeled with 32P rCTP and measured by liquid scintillation counter. Data are expressed as cpm. As control, reactions with no virus or with virus but no antibody were also included. *Mean cpm in this group was significantly different from that of nonantibody control (P < 0.05). Results were calculated from four replicates and represent four separate experiments.
Three-dimensional structural analysis of 7D9 DLP:IgA complexes. To directly visualize the interactions between 7D9 IgA and the VP6 capsid layer, we imaged DLP:IgA complexes in vitreous ice by electron cryomicroscopy (data not shown), computed the three-dimensional structure to a resolution of about 21 Å from 137 independent particle images, and compared it with the structure of the native DLP (Figure 4). The VP6 capsid is made up of 260 trimers arranged on a T=13 (levo) icosahedral lattice. This arrangement of capsomers defines 132 channels that penetrate the VP6 layer. In the outer regions of the VP6 capsid layer, the trimers are well separated from one another, while in the lower regions, near the VP2 layer, the trimers form a network of interactions with one another.
(a and b) Surface representations of the three-dimensional structure of native DLP (a) and 7D9 DLP:IgA complex (b), viewed along the icosahedral threefold axis. The VP2 capsid layer is shown in green, VP6 in blue, and IgA in pink. Scale bar, 200 Å. (c and d) Surface representations of the mRNA release channel computationally isolated from the native DLP (c) and the 7D9 DLP:IgA complex (d). Scale bar, 50 Å.
In the surface representation of the 7D9 DLP:IgA complex (Figure 4b), a total of 240 individual Fab domains (pink) were clearly visible on the viral surface. The Fab domains were attached at the tops of the VP6 trimers, with the epitope positioned along the edge. The Fab domains were oriented vertically on the viral surface and did not adversely affect the width of any of the channels penetrating the VP6 capsid layer.
The Fab domains had the expected bi-lobed morphology seen in the atomic model of IgA (40), and the molecular envelope was also similar to that observed for IgGs at a comparable resolution (22). Though bivalent IgA molecules were bound to the viral surface, only the Fab domains were visible in the three-dimensional structure. The most reasonable explanation for this is that both the Fc domains and the extended hinge regions, being highly flexible, were disordered and hence were averaged away during the reconstruction process.
Surrounding each icosahedral threefold axis was a cluster of three small spheres of mass density. The volume of each cluster was equivalent to that of an individual Fab domain, suggesting that one Fab domain is also bound to the VP6 trimer located at each of the 20 icosahedral threefold axes, though the morphology cannot be visualized in the three-dimensional structure. A Fab domain interacting with a trimer at the icosahedral threefold axis may adopt any one of three different orientations, depending upon which underlying VP6 monomer is specifically bound. The disrupted morphology of these Fab domains in the three-dimensional structure is likely a consequence of the fact that only one of three symmetrically equivalent positions is occupied around each icosahedral threefold axis. Taken together, these results indicate that a total of 260 IgA antigen-binding domains, or one per VP6 trimer, are bound to the VP6 surface at saturation.
Comparison of the VP6 capsid region in the 7D9 DLP:IgA complex and the native DLP. The attachment of various ligands, such as the VP7 capsid protein or anti-VP6 Fab’s, to the VP6 surface has been reported to introduce architectural changes in the body of the VP6 capsid layer (22). In these studies, mass translocations were observed within the network of trimer-trimer interactions present near the VP6-VP2 interface region in the vicinity of the mRNA release channels at the icosahedral fivefold axes. To investigate whether the attachment of 7D9 IgA causes similar structural changes in VP6, we compared the distribution of mass in the native DLP and the 7D9 DLP:IgA complex at various radii within the VP6 capsid region.
In the vicinity of the mRNA release channels (Figure 5), structural differences were evident at nearly all radii examined, and the types of domain movements were very similar to those observed in previous structural studies of DLP-ligand complexes (22). As in previous studies, the most striking mass translocations were observed within the network of trimer-trimer interactions extending between the radii ∼275 Å to ∼310 Å. In this region, individual molecules of VP6 appeared to be shifted with respect to each other, with some displacements exceeding 10 Å (Figure 5a, section III). As a result of these movements, some of the intermolecular interactions within trimers appeared to be weakened, and some of the intermolecular interactions between trimers appeared to grow stronger. Furthermore, the movement of the VP6 molecules closest to the icosahedral fivefold axes caused the diameter of the mRNA release channel to narrow by about 10 Å in the middle of this region. Mass translocations were not observed at the present resolution in the VP6-VP2 interface (∼260 Å; Figure 5a, section V) or the VP2 capsid layer (data not shown).
(a) Contoured cross sections of mass density from the three-dimensional structures of native DLP (blue) and 7D9 DLP:IgA complex (red), viewed perpendicular to the icosahedral fivefold axis. Each contour represents a mass density difference of about 0.37 ς. The cross sections, labeled I–V, were extracted from radii between 261 Å and 325 Å (b). The upper panel of cross sections depicts the distribution of mass surrounding the mRNA release channel, with the icosahedral fivefold axis passing through the center. The lower panel of cross sections provides a close-up view of the mass distribution in one of the five trimers (indicated in the corresponding upper panel by a dashed box). The arrowheads in cross section III, lower panel, denote representative intermolecular interactions that are different in the two structures. Measurements of three specific mass translocations are also given. Scale bars, 50 Å. (b) Graphical representation of the radii from which cross sections were extracted in a. The cross sections are shown as semitransparent gray planes. The five VP6 trimers surrounding the mRNA release channel in the native DLP are colored in blue, and the underlying VP2 capsid layer is shown in green. (c) A portion of the surface representation of the native DLP, viewed along the icosahedral fivefold axis. The position of the fivefold axis along which the sections shown in a were taken is marked. The pair of VP6 trimers surrounding the twofold axis is also indicated.
Interestingly, the sorts of mass translocations observed within the VP6 trimers surrounding the mRNA release channels did not occur in other regions of the capsid (data not shown). The distribution of mass within the VP6 trimers surrounding the icosahedral twofold axes was virtually identical in both the native DLP and the 7D9 DLP:IgA complex structures. Likewise, few structural changes were observed within the VP6 trimers located at other positions examined. The preponderance of structural changes in the regions of VP6 immediately adjacent to the mRNA release channels suggests that the trimers near the icosahedral vertices are more flexible than trimers at other locations and are more susceptible to alteration upon ligand attachment.
Analysis of transcription products in 7D9 DLP:IgA complex. In previous structural and biochemical studies of DLP-ligand complexes, the appearance of architectural rearrangements in the VP6 capsid layer directly correlated with an inhibition of genome transcription at the stage of transcript elongation (21, 22). Our initial studies indicated that 7D9 efficiently inhibited transcription of RRV and EC while other anti-VP6 mAb’s did not. To determine whether the attachment of 7D9 IgA to the DLP surface likewise blocks elongation but not initiation of transcription, we next compared the length distribution of transcription products in native DLPs and 7D9 DLP:IgA complexes following a period of incubation in a standard in vitro transcription reaction buffer (Figure 6). While native DLPs, as expected, were able to produce full-length mRNA transcripts (lane 1), 7D9 DLP:IgA complexes synthesized predominantly short oligonucleotide transcripts having an apparent length of about six nucleotides and only a small quantity of full-length mRNA (lane 2). The transcriptional behavior of the 7D9 DLP:IgA complex is similar to that of the mature TLP, which is not able to elongate mRNA transcripts beyond about 6 bases in length (lane 3). Given the similarities between the mature TLP and the 7D9 DLP:IgA complex in regard to the architecture of the VP6 capsid region, these results suggest that 7D9 IgA causes an inhibition of genome transcription by the same mechanism as does VP7 in the mature particle.