Wei-Gang Hu

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with KD of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

A recombinant humanized monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus

A recombinant humanized antibody to Venezuelan equine encephalitis virus (VEEV) was constructed in a monocistronic adenoviral expression vector with a foot-and-mouth-disease virus-derived 2A self-cleavage oligopeptide inserted between the antibody heavy and light chains. After expression in mammalian cells, the heavy and light chains of the humanized antibody (hu1A4A1IgG1-2A) were completely cleaved and properly dimerized. The purified hu1A4A1IgG1-2A retained VEEV binding affinity and neutralizing activity similar to its parental murine antibody. The half-life of hu1A4A1IgG1-2A in mice was approximately 2 days. Passive immunization of hu1A4A1IgG1-2A in mice (50 microg/mouse) 24 h before or after virulent VEEV challenge provided complete protection, indicating that hu1A4A1IgG1-2A has potent prophylactic and therapeutic effects against VEEV infection.

Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system.

A recombinant single-chain fragment variable antibody (scFv) to botulinum A neurotoxin (BoNT/A) was developed. BALB/C mice were immunized with BoNT/A. Splenomic RNA was isolated from the hyperimmune mice and used to prepare a cDNA library, from which the variable regions of the heavy and light chain antibody genes were generated and connected by a DNA linker. The resulting scFv genes were cloned into the phagemid vector pCANTAB5 in order to construct phage display scFv libraries. Individual anti-BoNT/A phage clones were isolated from the phage display libraries by immunoaffinity selection using immobilized BoNT/A and further evaluated by enzyme-linked immunosorbant assay, immunoprecipitation and Western blotting. Forty-eight clones were found to be BoNT/A-reactive. The most reactive clone, designated D12, was selected for further study. The scFv gene of D12 was subcloned into a Pichia pastoris vector, and expression in yeast was evaluated.

Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (K D values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 105Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (Kd) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with Kd value of 10−7 M.

Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to venezuelan equine encephalitis virus

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody (MAb), but also possessed streptavidin-binding activity. This experimental approach can eliminate the need for chemical biotinylation of antibodies and the risk associated of antibody denaturation and can provide a stable and reproducible reagent for rapid and efficient immunoassay of VEE when detected by horseradish peroxidase (HRP)-conjugated streptavidin.

Engineered Mesenchymal Cells Improve Passive Immune Protection Against Lethal Venezuelan Equine Encephalitis Virus Exposure

Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof‐of‐concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito‐borne pathogen affecting humans and equines and can be used in bio‐warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short‐lived, method of providing immediate protection against a pathogen. We compared the protective efficacy of human umbilical cord perivascular cells (HUCPVCs; a rich source of MSCs), engineered with a transgene encoding a humanized VEEV‐neutralizing antibody (anti‐VEEV), to the purified antibody. In athymic mice, the anti‐VEEV antibody had a half‐life of 3.7 days, limiting protection to 2 or 3 days after administration. In contrast, engineered HUCPVCs generated protective anti‐VEEV serum titers for 21–38 days after a single intramuscular injection. At 109 days after transplantation, 10% of the mice still had circulating anti‐VEEV antibody. The mice were protected against exposure to a lethal dose of VEEV by an intramuscular pretreatment injection with engineered HUCPVCs 24 hours or 10 days before exposure, demonstrating both rapid and prolonged immune protection. The present study is the first to describe engineered MSCs as gene delivery vehicles for passive immunity and supports their utility as antibody delivery vehicles for improved, single‐dose prophylaxis against endemic and intentionally disseminated pathogens.

Active immunity induced by passive IgG post-exposure protection against ricin.

Therapeutic antibodies can confer an instant protection against biothreat agents when administered. In this study, intact IgG and F(ab’)2 from goat anti-ricin hyperimmune sera were compared for the protection against lethal ricin mediated intoxication. Similar ricin-binding affinities and neutralizing activities in vitro were observed between IgG and F(ab’)2 when compared at the same molar concentration. In a murine ricin intoxication model, both IgG and F(ab’)2 could rescue 100% of the mice by one dose (3 nmol) administration of antibodies 1 hour after 5 × LD50 ricin challenge. Nine days later, when the rescued mice received a second ricin challenge (5 × LD50), only the IgG-treated mice survived; the F(ab’)2-treated mice did not. The experimental design excluded the possibility of residual goat IgG responsible for the protection against the second ricin challenge. Results confirmed that the active immunity against ricin in mice was induced quickly following the passive delivery of a single dose of goat IgG post-exposure. Furthermore, it was demonstrated that the induced active immunity against ricin in mice lasted at least 5 months. Therefore, passive IgG therapy not only provides immediate protection to the victim after ricin exposure, but also elicits an active immunity against ricin that subsequently results in long term protection.

Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis virus.

An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and by different operators demonstrated that the assay was reproducible (coefficient of variation 4.7-18.5% month-to-month; 3.3-8.8% person-to-person). The VEEV ECL assay exhibited no cross-reactivity with two closely related alphaviruses or with 21 heterologous biological agents. A genetically biotinylated recombinant VEEV antibody, MA116SBP, was evaluated for utility for detection of rE2; although functional in the ECL assay, the LOD was two log units higher (100 ng/ml vs 1 ng/ml) using MA116SBP than when chemically biotinylated antibody was used. The ECL assay detected VEEV at the lowest LOD (highest sensitivity) hitherto reported in the published literature and ECL assay results were generated in ∼60 min compared to a 6-8h period required for ELISA. Results have demonstrated a sensitive, rapid, and fully automated ECL immunoassay for detection and identification of VEEV.

A Novel Approach to Development of Monoclonal Antibodies Using Native Antigen for Immunization and Recombinant Antigen for Screening

The production of monoclonal antibodies (MAb) specific to microbes is rapidly growing. Finding an appropriate antigen to screen hybridoma clones has become increasingly important. However, the conventional method, in which the purified antigen from the microbe is routinely used for screening, cannot avoid selection of false positive hybridoma clones, since even highly purified antigen is found to be contaminated with some other proteins from the microbe. In this study, MAbs against anthrax protective antigen (PA), the central component of the three-part toxin secreted by Bacillus anthracis were developed using a pair of the roughly purified native PA as an immunogen and the recombinant PA as a screening antigen without any possibility of false selection, since the recombinant PA was produced by a gene engineering approach and impossible to be contaminated with any other proteins from B. anthracis. In total, nine stable hybridoma clones secreting anti-PA MAbs were developed. All of them had the same type of heavy and light chains, IgG1/kappa. The binding profiles for these anti-PA MAbs were investigated by ELISA. This novel approach to the development of MAbs should be applicable to the production of MAbs to other microbes, especially to those from which antigens can hardly be purified to a high degree.