Development of Etf-3-specific nanobodies to prevent Ehrlichia infection and LNP-mRNA delivery in cellular and murine models

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Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages and causes human monocytic ehrlichiosis. Ehrlichia translocated factor-3 (Etf-3) is a type IV secretion system effector that binds host-cell ferritin light chain and induces ferritinophagy, thus increasing cellular labile iron pool for Ehrlichia proliferation. To further characterize roles of Etf-3 in Ehrlichia infection, we produced immune libraries of Etf-3-specific nanobodies (Nbs). Based on distinct complementarity-determining region 3 sequences, we identified 16 and 15 families of anti-Etf-3 Nbs that could specifically bind the N- and C-terminal halves of Etf-3, respectively. Transfection with plasmids encoding the anti-Etf-3 Nbs N48 and N51, but not N59, significantly inhibited E. chaffeensis infection in HEK293 cells. All three Nbs colocalized with Etf-3-GFP in co-transfected RF/6A cells, but N48 and N51 had significantly higher binding affinities for recombinant Etf-3. Etf-3-GFP transfection-induced ferritinophagy and endogenous ferritin degradation was abrogated in HEK293 cells co-transfected with N48 or N51, but not with N59. To efficiently express Nbs in the infected host-cell cytoplasm, lipid nanoparticles-encapsulated mRNAs (LNP-mRNAs) encoding N48, N51, or N59 were created for delivery into cells or mice. Incubation of HEK293 cells or inoculation of mice with LNP-mRNA-N48 or LNP-mRNA-N51 significantly inhibited E. chaffeensis infection compared to those with LNP-mRNA-N59 or without LNP-mRNA. Our results demonstrate that Etf-3-specific Nbs delivered via LNP-mRNAs can inhibit Etf-3 functions and Ehrlichia infection.