Ebola Virus Uses Tunneling Nanotubes as an Alternate Route of Dissemination.

Ebola virus (EBOV) disease is marked by rapid virus replication and spread. EBOV enters the cell by macropinocytosis and replicates in the cytoplasm, and nascent virions egress from the cell surface to infect neighboring cells. Here, we show that EBOV uses an alternate route to disseminate: tunneling nanotubes (TNTs).

Visualizing Filoviral Nucleoproteins Using Nanobodies Fused to the Ascorbate Peroxidase Derivatives APEX2 and dEAPX.

Fusions of single-domain antibodies (sdAbs, nanobodies) to enzymatic reporters make convenient molecular probes to detect the presence of an antigen of interest. We have previously fused the monomeric hyperactive ascorbate peroxidase derivative APEX2 to anti-Ebolavirus and anti-Marburgvirus sdAbs to generate immunoreagents useful in detecting nucleoprotein (NP) on western blots, ELISA, and within cells following transfection of NP expression plasmids or following virus infection. Here we present the methods used to overexpress and purify these sdAb-APEX2 fusion proteins, and to employ them as probes in various scenarios with colorimetric and fluorometric signal development.

Generating Uniformly Cross-Reactive spp. Anti-nucleoprotein Nanobodies to Facilitate Forward Capable Detection Strategies.

It is often challenging for a single monoclonal antibody to cross-react equally with all species of a particular viral genus that are separated by time and geographies to ensure broad long-term global immunodiagnostic use. Here, we set out to isolate nanobodies or single-domain antibodies (sdAbs) with uniform cross-reactivity to the genus by immunizing a llama with recombinant nucleoprotein (NP) representing the 5 cultivated species to assemble a phage display repertoire for mining. Screening sdAbs for reactivity against the C-terminal domain of NP guided the isolation of clones that could perform as both captor and tracer for polyvalent antigen in sandwich assays.

Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness.

Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from , to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library.

Paratope Duality and Gullying are Among the Atypical Recognition Mechanisms Used by a Trio of Nanobodies to Differentiate Ebolavirus Nucleoproteins.

We had previously shown that three anti-Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti-Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti-Zaire ebolavirus sdAb and a totally specific anti-Sudan ebolavirus sdAb were the result of exclusion from this region.

Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among and .

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 periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the genus and cross-reactive within the genus, we show that periplasmic sdAb-APEX2 fusion proteins are easily purified at multi-mg amounts.

Unveiling a Drift Resistant Cryptotope within Nucleoprotein Recognized by Llama Single-Domain Antibodies.

Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays.

Intracellular Crosslinking of Filoviral Nucleoproteins with Xintrabodies Restricts Viral Packaging.

Viruses assemble large macromolecular repeat structures that become part of the infectious particles or virions. Ribonucleocapsids (RNCs) of negative strand RNA viruses are a prime example where repetition of nucleoprotein (NP) along the genome creates a core polymeric helical scaffold that accommodates other nucleocapsid proteins including viral polymerase. The RNCs are transported through the cytosol for packaging into virions through association with viral matrix proteins at cell membranes.

Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA) formulation.

Background: Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP).

Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures.

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein.

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin.

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders.

Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents.

Background: There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype.

Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex.

Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb) and are among the smallest naturally IgG-derived antigen binding units.

Rapid assembly of sensitive antigen-capture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4.

There is a pressing need for rapid and reliable approaches to the delivery of sensitive yet rugged diagnostic assays specific for emerging viruses, to hasten containment of outbreaks when and wherever they occur. Within 3 weeks, we delivered an antigen-capture assay for Marburg virus (MARV) that was based on llama single-domain antibodies (sdAbs) selected at biosafety level 4. Four unique sdAbs were capable of independently detecting MARV variants Musoke, Ravn, and Angola without cross-reactivity with the 4 Ebola virus species.

Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library.

Llamas possess a class of unconventional immunoglobulins that have only heavy chains; unpaired heavy variable domains are responsible for antigen binding. These domains have previously been cloned and expressed as single domain antibodies (sdAbs); they comprise the smallest known antigen binding fragments. SdAbs have been shown to bind antigens at >90 degrees C and to refold after being denatured.