Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice.

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

A paratransgenic strategy to block transmission of Xylella fastidiosa from the glassy-winged sharpshooter Homalodisca vitripennis.

BACKGROUND: Arthropod-borne diseases remain a leading cause of human morbidity and mortality and exact an enormous toll on global agriculture. The practice of insecticide-based control is fraught with issues of excessive cost, human and environmental toxicity, unwanted impact on beneficial insects and selection of resistant insects. Efforts to modulate insects to eliminate pathogen transmission have gained some traction and remain future options for disease control. RESULTS: Here, we report a paratransgenic strategy that targets transmission of Xylella fastidiosa, a leading bacterial pathogen of agriculture, by the Glassy-Winged Sharpshooter (GWSS), Homalodisca vitripennis. Earlier, we identified Pantoea agglomerans, a bacterial symbiont of the GWSS as the paratransgenic control agent. We genetically engineered P. agglomerans to express two antimicrobial peptides (AMP)-melittin and scorpine-like molecule (SLM). Melittin and SLM were chosen as the effector molecules based on in vitro studies, which showed that both molecules have anti-Xylella activity at concentrations that did not kill P. agglomerans. Using these AMP-expressing strains of P. agglomerans, we demonstrated disruption of pathogen transmission from insects to grape plants below detectable levels. CONCLUSION: This is the first report of halting pathogen transmission from paratransgenically modified insects. It is also the first demonstration of paratransgenic control in an agriculturally important insect vector.

Measuring Success in Global Health Training: Data From 14 Years of a Postdoctoral Fellowship in Infectious Diseases and Tropical Medicine.

BACKGROUND.: In modern academic medicine, especially in the fields of infectious diseases and global health, aspiring physician-scientists often wait years before achieving independence as basic, translational, and clinical investigators. This study employed mixed methods to evaluate the success of the Burroughs Wellcome Fund/American Society for Tropical Medicine and Hygiene (BWF/ASTMH) global health postdoctoral fellowship in promoting scientific independence. METHODS.: We examined quantitative data obtained from the National Institutes of Health (NIH) and qualitative data provided by the ASTMH and program participants to assess BWF/ASTMH trainees' success in earning NIH grants, publishing manuscripts, and gaining faculty positions. We also calculated the return on investment (ROI) associated with the training program by dividing direct costs of NIH research grants awarded to trainees by the direct costs invested by the BWF/ASTMH fellowship. RESULTS.: Forty-one trainees received fellowships between 2001 and 2015. Within 3 years of completing their fellowships, 21 of 35 (60%) had received career development awards, and within 5 years, 12 of 26 (46%) had received independent research awards. Overall, 22 of 35 (63%) received 1 or more research awards. BWF/ASTMH recipients with at least 3 years of follow-up data had coauthored a mean of 36 publications (range, 2-151) and 29 of 35 (82%) held academic positions. The return on investment was 11.9 overall and 31.8 for fellowships awarded between 2001 and 2004. CONCLUSIONS.: Between 2001 and 2015, the BWF/ASTMH postdoctoral training program successfully facilitated progress to scientific independence. This program model underscores the importance of custom-designed postdoctoral training as a bridge to NIH awards and professional autonomy.

Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm.

BACKGROUND: Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. RESULTS: Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser)3 linker precipitated at physiological pH 7.4. CONCLUSIONS: This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.

Modeling horizontal gene transfer (HGT) in the gut of the Chagas disease vector Rhodnius prolixus.

BACKGROUND: Paratransgenesis is an approach to reducing arthropod vector competence using genetically modified symbionts. When applied to control of Chagas disease, the symbiont bacterium Rhodococcus rhodnii, resident in the gut lumen of the triatomine vector Rhodnius prolixus (Hemiptera: Reduviidae), is transformed to export cecropin A, an insect immune peptide. Cecropin A is active against Trypanosoma cruzi, the causative agent of Chagas disease. While proof of concept has been achieved in laboratory studies, a rigorous and comprehensive risk assessment is required prior to consideration of field release. An important part of this assessment involves estimating probability of transgene horizontal transfer to environmental organisms (HGT). This article presents a two-part risk assessment methodology: a theoretical model predicting HGT in the gut of R. prolixus from the genetically transformed symbiont R. rhodnii to a closely related non-target bacterium, Gordona rubropertinctus, in the absence of selection pressure, and a series of laboratory trials designed to test the model. RESULTS: The model predicted an HGT frequency of less than 1.14 × 10(-16) per 100,000 generations at the 99% certainty level. The model was iterated twenty times, with the mean of the ten highest outputs evaluated at the 99% certainty level. Laboratory trials indicated no horizontal gene transfer, supporting the conclusions of the model. CONCLUSIONS: The model treats HGT as a composite event, the probability of which is determined by the joint probability of three independent events: gene transfer through the modalities of transformation, transduction, and conjugation. Genes are represented in matrices and Monte Carlo method and Markov chain analysis are used to simulate and evaluate environmental conditions. The model is intended as a risk assessment instrument and predicts HGT frequency of less than 1.14 × 10(-16) per 100,000 generations. With laboratory studies that support the predictions of this model, it may be possible to argue that HGT is a negligible consideration in risk assessment of genetically modified R. rhodnii released for control of Chagas disease.

Module based antibody engineering: a novel synthetic REDantibody.

We describe the facile generation of a stable recombinant antibody with intrinsic red fluorescent properties for qualitative and potentially quantitative immunofluorescence analysis. The REDantibody based on the X-ray crystallographic structures of the anti-sialyl-Tn antibody B72.3 and 3D model of the monomeric red fluorescent protein was designed to retain optimal spatial geometry between the C- and N-termini of the V(H) and V(L) chains respectively to mimic the domains interface pairing in antibody Fab fragments and to incorporate the red fluorescent protein as a bridging scaffold. The model was further validated by assembling a REDantibody based on CA19.9 the anti-sialylated Lewis (Le)(a) blood group antigen and 4D5-8 the anti-p185(HER2) antibodies. The chimeric heavy and light chains containing red fluorescent protein as a bridge were correctly processed and secreted into Escherichia coli periplasm for assembly and disulphide bond formation, further analysis revealed the molecules to be exclusively monomers. Purified anti-glycan proteins were used for an immunofluorescent analysis of Trypanosoma cruzi epimastigotes, and the anti-p185(HER2) used to determine the binding properties. The REDantibody platform facilitates rapid generation of scFv chimeras that could be used for screening antibodies against cell surface markers. Furthermore, such modular assembly should permit the interchange of binding sites and of fluorophores to create robust panels of coloured antibodies.

Genetic transformation of a Corynebacterial symbiont from the Chagas disease vector Triatoma infestans.

Insect-borne diseases have experienced a troubling resurgence in recent years. Emergence of resistance to pesticides greatly hampers control efforts. Paratransgenesis, or the genetic transformation of bacterial symbionts of disease vectors, is an alternative to traditional approaches. Previously, we developed paratransgenic lines of Rhodnius prolixus, a vector of Chagas disease in Central America. Here, we report identification of a Corynebacterial species as a symbiont of Triatoma infestans, a leading vector of Chagas disease in South America. We have modified this bacterium to produce an immunologically active single chain antibody fragment, termed rDB3. This study establishes the basis for generating paratransgenic T. infestans as a strategy for control of Chagas disease.

Transformation of Rhodococcus rhodnii, a symbiont of the Chagas disease vector Rhodnius prolixus, with integrative elements of the L1 mycobacteriophage.

Elimination of vector populations through the use of insecticides is the principal means of controlling Chagas disease. Because of the limitations of insecticide use, we have been developing a new potential method of control, to be used in conjunction with insecticide programs, a method which utilizes genetically modified symbiotic bacteria. These transformed bacteria can express anti-parasitic agents in the gut of the bug where the trypanosomes also are found. Previous studies have shown that it is possible to transform Rhodococcus rhodnii with a shuttle plasmid that contains the gene for cecropin A, an insect anti-microbial peptide. The bacteria expressed this peptide and reduced or eliminated the number of trypanosomes in the bug Rhodnius prolixus [Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3274]. In an effort to improve efficacy and transformation stability, we have begun using plasmids that contain integrative elements from the L1 mycobacteriophage to insert DNA into the genome of the bacterium. The integrative plasmid pBP5 contains the attachment site (attP) and integrase gene (int) of the L1 mycobacteriophage, an antibiotic resistance gene and the lacZ gene. After transforming R. rhodnii with pBP5, nine positive clones were obtained and six different insertions sites were identified. In each clone, the integrative plasmid is inserted only once, the lacZ gene is expressed intensely and, all clones but one, remained stable for 100 generations of culture in the absence of antibiotic selection. In addition, the construct remains stable throughout the life cycle of the bug. These data demonstrate that L1 mycobacteriophage integrative plasmids are significantly more stable than episomally located plasmids used in previous studies and will be greatly beneficial for use in the transformation of symbiotic bacteria of Chagas disease vectors.

Bacterial symbionts of the triatominae and their potential use in control of Chagas disease transmission.

Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi and transmitted by insects in the family Reduviidae, subfamily Triatominae, commonly known as kissing bugs. Because these insects feed throughout their entire developmental cycle on vertebrate blood, they harbor populations of symbiotic bacteria in their intestinal track that produce nutrients that are lacking in the insects' limited diet. It is possible to cultivate these bacteria, genetically modify them, and place them back into their insect host, thus generating a paratransgenic insect. This procedure has allowed the expression of antitrypanosomal gene products in the insect gut, thereby resulting in insects that are incapable of transmitting Chagas disease. A method has been developed that would allow introduction and spread of genetically modified symbionts into natural populations of kissing bugs, thus leading potentially to a transgenic intervention tool for use as a part of an integrated vector control approach.