Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>

• Main Authors: Laura J. Sherwood, Andrew Hayhurst
• Format: Article
• Language: English
• Published: MDPI AG 2019-04-01
• Series: Viruses
• Subjects: peroxidase; APEX2; VHH; sdAb; nanobody; Filovirus; Ebola; Marburg; nucleoprotein; nanoluciferase
• Online Access: https://www.mdpi.com/1999-4915/11/4/364

We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the <i>E. coli</i> periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous <i>E. coli</i> outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the <i>Marburgvirus</i> genus and cross-reactive within the <i>Ebolavirus</i> genus, we show that periplasmic sdAb&#8211;APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb&#8211;APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-<i>Marburgvirus</i> sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕngl&#224; virus genome. Antibody&#8211;antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕngl&#224; NP C-terminal domain. The sdAb&#8211;APEX2 fusions also enabled live <i>Marburgvirus</i> and <i>Ebolavirus</i> detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb&#8211;APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.