Cryo EM structures map a post vaccination polyclonal antibody response to canine parvovirus

Abstract

Canine parvovirus (CPV) is an important pathogen that emerged by cross-species transmission to cause severe disease in dogs. To understand the host immune response to vaccination, sera from dogs immunized with parvovirus are obtained, the polyclonal antibodies are purified and used to solve the high resolution cryo EM structures of the polyclonal Fab-virus complexes. We use a custom software, Icosahedral Subparticle Extraction and Correlated Classification (ISECC) to perform subparticle analysis and reconstruct polyclonal Fab-virus complexes from two different dogs eight and twelve weeks post vaccination. In the resulting polyclonal Fab-virus complexes there are a total of five distinct Fabs identified. In both cases, any of the five antibodies identified would interfere with receptor binding. This polyclonal mapping approach identifies a specific, limited immune response to the live vaccine virus and allows us to investigate the binding of multiple different antibodies or ligands to virus capsids.

Fig. 1. Isolation of canine CPV-binding Fab by affinity chromatography.

a Timeline showing CPV modified live virus (MLV) immunizations (with vaccine names), and day of blood sample collection from the male Beagle analyzed in this study. b Purification strategy for the isolation of specific CPV-binding Fab by affinity chromatography for cryo EM.

Fig. 2. Isolation of total-Fab by mixed-mode chromatography.

a Timeline showing CPV modified live virus (MLV) immunizations (with vaccine names) and day of blood sample collection of the Beagle analyzed in this study. b Strategy for the preparation of total polyclonal Fab from papain-digested total Ig by mixed-mode chromatography. The total-Fab mixture was incubated with purified CPV capsids, complexes concentrated and washed to remove unbound Fab.

Fig. 3. Icosahedral Reconstruction of CPV-affinity purified Fab complexed with CPV.

a Representative micrograph where spherical virus particles can be seen with few Fab decorating the capsids. b 2D classification of particles illustrates the lower density of bound Fab (red arrows). c Central section of icosahedral reconstruction where capsid can be seen with weak Fab densities (white dashed line) bound to the exterior suggesting that the purified Fab was of relatively low concentration, which was overcome by the subvolume reconstruction approach. d The refined sharpened (left) and unsharpened (right) icosahedral reconstruction maps colored radially according to the color key.

Fig. 4. Affinity purified Fab Subparticle Classification and Refinement.

a Icosahedrally averaged, unsharpened map showing Fab density over the A and B sites (boxed in red and cornflower blue, respectively). Note the tiny blob of averaged density corresponding to the Fab bound to the B site. b 3D Classification of B site subparticles, with one Fab density class outlined in bolded blue that was used for the 3D refinement of the B site Fab to 3.6 Å resolution. c 3D Classification of the A site subparticles, with one Fab density classes (bolded red) that was used for the 3D refinement of the A site Fab to 3.7 Å resolution. Based on the classification there are about 12 of each Fab bound per capsid.

Fig. 5. Total-Fab incubated with CPV for complex reconstruction.

a Representative micrograph where spherical virus particles can be seen with Fab decorating the capsids. In this incubation, there is more Fab per capsid than in the previous experiment. b 2D classification of particles illustrates stronger magnitude of Fab density (red arrows) compared to the affinity purified complexes. c Central section of icosahedral reconstruction where capsid can be seen with Fab density (white dashed lines) of slightly lower magnitude bound to the exterior. d Sharpened and unsharpened icosahedral reconstructions are colored radially according to the key (left and right respectively).

Fig. 6. Total-Fab-CPV Subparticle Classification and Refinement.

a Unsharpened map of icosahedral reconstruction where Fab density can be seen over the A and B sites (boxed in orange and light blue respectively). b 3D Classification of the B site subparticles, with two Fab density classes corresponding to Fab B1 (blue) and Fab B2 (cyan) that were refined independently. c 3D Classification of the A site subparticles, with one Fab density class (boxed in orange) that was used for the 3D refinement of the A site Fab. Based on this classification, there are approximately 13 B1 and B2 Fabs per capsid while only about 6A site Fabs per capsid.

Fig. 7. Estimated Polyclonal Fab Binding Footprints.

a, left, The two Fab identified from the affinity purified polyclonal are shown bound to capsid (gray scale) with a single Fab recognizing the B site (cornflower blue) and the A site (red) with corresponding roadmap using same color scheme (b, d). a, right, The three Fab identified from the total-Fab polyclonal are shown bound to capsid (gray scale) with A site Fab (orange) and the Fabs B1 and B2 (blue and cyan) with their corresponding footprints shown in roadmaps (c, e, f). Between the two preparations, the A site antibodies have different footprints onto the virus capsid (b, c). The affinity purified B site footprint is similar to the that of the non-affinity purified B1 footprint, although the heavy chain and light chain have different binding orientations. For the roadmaps the viral surface is shown as a stereographic projection where the polar angles φ and θ represent the latitude and longitude of a point on the viral surface, respectively.