Fig. 1

Genetically engineered murine RBCs covalently linked to VHHs against BoNT/A protect neurons in vitro and mice in vivo against BoNT/A challenge. a Design of the chimeric proteins. Each chimera was assembled from different protein segments shown as colored boxes (yellow: signal peptide of GPA; red: myc epitope; VHH-H7 and VHH-B5: anti-BoNT/A VHHs; grey: spacer; VHH-D10 and VHH-G10: anti-BoNT/B VHHs; GPA human glycophorin A). b RBC potency to neutralize BoNT/A assessed by SNAP25 immunoblot following overnight treatments of primary rat neurons exposed to 20 pM BoNT/A preincubated with the indicated number of myc+ RBCs. The percentage of SNAP25 cleaved by BoNT/A was estimated by image analysis. Left: RBCs expressing either GPA-VNA/A or Kell-VNA/A; Right: RBCs expressing GPA-VNA/B or Kell-VNA/B. c Complete blood counts of control 7-week-old female C57BL/6J mice and mice subjected to bone marrow transplantation with progenitor cells expressing vector or GPA-VNA/A and bled at the indicated time points. (n = 4/group, mean ± S.E.M.). d Myc surface expression on red cells from GPA-VNA/A transplanted mice was measured by flow cytometry at the indicated time points; the percentage of myc+ cells was determined. (n = 4/group, mean ± S.E.M.). e Kaplan–Meier survival plots of mice challenged with BoNT/A. CD1 mice were subjected to bone marrow transplantation with progenitor cells expressing GPA-VNA/A or Kell-VNA/A. After bone marrow reconstitution, these mice were challenged with 10 LD₅₀ BoNT/A and monitored for a week. The surviving mice received increasing doses of BoNT/A in subsequent weeks. The mice bearing Kell-VNA/A RBCs were challenged up to 3 weeks before protection faltered, whereas the mice carrying GPA-VNA/A were challenged for up to 6 weeks with increasing doses as indicated and were still alive at the termination of the study (n = 5/group). All mice with RBCs expressing GPA-VNA/A survived following the final 10,000 LD₅₀ treatment without showing signs of botulism