Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

NanoNet: Rapid end-to-end nanobody modeling by deep learning at sub angstrom resolution

Antibodies are a rapidly growing class of therapeutics. Recently, single domain camelid VHH antibodies, and their recognition nanobody domain (Nb) appeared as a cost-effective highly stable alternative to full-length antibodies. There is a growing need for high-throughput epitope mapping based on accurate structural modeling of the variable domains that share a common fold and differ in the Complementarity Determining Regions (CDRs). We develop a deep learning end-to-end model, NanoNet, that given a sequence directly produces the 3D coordinates of the Cα atoms of the entire VH domain. For the Nb test set, NanoNet achieves 1.7&Aring overall average RMSD and 3.0&Aring average RMSD for the most variable CDR3 loops. The accuracy for antibody VH domains is even higher: overall average RMSD < 1&Aring and 2.2&Aring RMSD for CDR3. NanoNet runtimes allow generation of ~1M nanobody structures in less than an hour on a standard CPU computer enabling high-throughput structure modeling.

A heterohexameric protein consisting of six linked single-domain antibodies is highly protective for BoNT/A, BoNT/B and BoNT/E exposures

Botulinum neurotoxin (BoNT) serotypes A, B and E cause the vast majority of human botulism cases and pose the greatest bioterrorism threats. We previously identified multiple camelid single-domain antibodies (VHHs) that each neutralize BoNT/A, BoNT/B or BoNT/E. We also demonstrated that heterodimers of linked toxin-neutralizing VHHs are much more potent than VHH monomer pools in preventing BoNT intoxication. In this study, we expressed two different heterohexamer proteins (VNA1-ABE and VNA2-ABE) of ~100 kDa secreted from mammalian host cells, each containing the same six linked anti-BoNT VHH components ordered in two different combinations. Each heterohexamer contained two VHHs that neutralize BoNT/A, BoNT/B or BoNT/E. Both heterohexameric antitoxins displayed similar strong binding properties for the three targeted BoNT serotypes by ELISA. One ug of each heterohexameric antitoxin fully protected groups of mice co-administered with 100 LD50 of BoNT/A, BoNT/B or BoNT/E, or a pool containing 100 LD50 of each of the three toxins. The results demonstrate that long chains of at least six different linked VHHs can be expressed such that all component VHHs in the multimer retain their target binding activities. These findings make more feasible the development of a BoNT antitoxin product consisting of a small pool of proteins that, in combination, neutralize all known BoNT serotypes and subtypes.Key ContributionHeteromultimeric proteins consisting of six linked, VHH antibodies, and including VHHs that neutralize BoNT/A, BoNT/B and BoNT/E, retain high potency to protect mice challenged with high doses of all three of these BoNT serotypes.

Production of monoclonal antibody against Ompa protein of Candidatus Liberibacter asiaticus causing citrus greening disease

Citrus Greening, also known as HuangLongbing (HLB), is considered one of the most dangerous citrus diseases, and limiting the production of citrus trees all over the world. Production of antibodies against Ompa protein of Candidatus Liberibacter asiaticus (CLas) for detection of citrus greening disease is considered as promising research direction. In this study, for the purpose of producting antibodies against Ompa of CLas, we firstly used the camel VHH antibody library for screening VHH antibodies against Ompa using phage-display technique. Next, phages which had strong interaction with Ompa as shown in ELISA were selected for phagemid isolation and the DNA fragments encoding VHH antibodies were sequenced. The DNA fragment encoding the best VHH antibody was then selected and inserted into the expression vector pET-21a (+), then cloned in Ecoli DH5α strain and expressed in BL21 (DE3) strain. The expression of VHH antibodies against Ompa was optimized at different temperatures with an inductive concentration of 0.1 M IPTG. Anti-Ompa VHH antibodies were purified under denatured conditions then re-folded. The biological activity of the VHH antibody with Ompa antigen was assessed by indirect-ELISA reaction. Results indicated that the VHH antibody had a very strong interaction with the Ompa antigen. This opens up the prospect of applying VHH antibody in the detection of citrus greening disease.

Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.

Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

SARS-CoV-2 is a newly emergent coronavirus that causes COVID-19, which has adversely impacted human health and has led to a pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction to the ACE2 human receptor. In this study, we then used computer-aided designed and constructed multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs show favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

One-step Preparation of a VHH-based Immunoadsorbent for the Extracorporeal Removal of β2-microglobulin

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of β2-microglobulin (β2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of β2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove β2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with β2-m. In this study, we modified the anti-β2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of β2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed β2-m from blood, therefore opening a new avenue for efficient hemodialysis.