Figure 1.
Design and Analysis of FHV-VWAANTXR2 VLPs
(A) Ribbon diagram of a single FHV coat protein subunit showing the surface-exposed loops into which the VWA domain of ANTXR2 was inserted.
(B) Schematic diagram showing structure of FHV-VWAANTXR2 chimeric proteins 206 (top) and 264 (bottom). Numbers refer to amino acid positions in the FHV coat protein. The assembly-dependent autocatalytic maturation cleavage occurs between Asn 363 and Ala 364.
(C) Electrophoretic analysis of purified VLPs on a 10% Bis-Tris gel stained with Simply Blue (Invitrogen). Lane 1, molecular weight markers; lane 2, wt FHV VLPs; lane 3, FHV-VWAANTXR2 chimera 206; lane 4, FHV-VWAANTXR2 chimera 264.
(D) Electron micrographs of gradient-purified wt and chimeric VLPs negatively stained with uranyl acetate. Bar = 500 Å.
Figure 2.
Dose Response Curve Showing Cell Viability as a Function of Antitoxin Concentration
CHO-K1 cells were incubated with PA-LFNDTA in the presence of increasing concentrations of sANTXR2, chimera 206, or chimera 264. After incubation at 37 °C for 48 h, cell viability was determined with Celltiter-glo (Promega). Data points represent the mean ± SD values for triplicate samples. All data were normalized to untreated cell controls.
Table 1.
In Vivo Intoxication Experiments in Fisher 344 Rats
Figure 3.
Three-Dimensional Models of FHV-VWAANTXR2 VLPs with and without Bound PA83
(A, B) Pseudoatomic models of FHV-VWAANTXR2 chimeras. X-ray coordinates of FHV capsid protein (green) and ANTXR2 VWA domain (yellow) were docked into the cryoEM density of chimera 206 (A) and chimera 264 (B). Panels show surface views of the particles in the absence of the cryoEM density maps. Note the different distributions of the ANTXR2 domains on the surfaces of the VLPs.
(C, D) In silico model of PA83 bound to the surface of FHV-VWAANTXR2 chimeras. PA83 (purple) was modeled onto the surface of chimera 206 (C) and chimera 264 (D) using the known high resolution X-ray structure of the ANTXR2-VWA/PA63 complex as a guide [9]. Panels show surface views of the entire particles to illustrate the difference in occupancy of PA83 on the two chimeras.
Figure 4.
Antibody and LeTx Challenge Response of immunized Rats
(A–D) Rats (four per group) were immunized with FHV-VWAANTXR2–PA83 complex, FHV-VWAANTXR2 chimera alone, PA83, or PBS and boosted 4 wk later. Serum samples were collected prior to as well as 3 and 7 wk after immunization and tested by ELISA for IgG-specific antibody response to (A) PA, (B)VWAANTXR2, and (C) FHV coat protein. Data represent the mean ± SD of animals in the respective groups and are shown for the 1:1,000 serum dilution in panels (A) and (B) and for the 1:100 serum dilution in panel (C). In panel (A) at week 3, * indicates p = 0.003 compared to PBS control and ** indicates p = 0.003 compared to PA83 alone. At week 7, * indicates p = 0.005 compared to PBS control and ** indicates p = 0.012 compared to PA83 alone. (D) Relationship between anti-PA antibody level and survival of individual rats following challenge with 10 MLDs of LeTx.
(E, F) Rats (five per group) were immunized once with FHV-VWAANTXR2–PA83 complex, FHV-VWAANTXR2 chimera alone, PA83, or PBS. Serum samples were collected prior to and 3 wk after immunization, diluted 1:100, and tested for IgG-specific antibody response to PA (E). Data represent the mean ± SD of animals in the repective groups. At week 3, * indicates p < 0.0001 compared to PBS control and ** indicates p = 0.001 compared to PA83 alone. (F) Relationship between anti-PA antibody level and survival of individual rats following challenge with 10 MLDs of LeTx.