Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases

Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris

Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, ‘hit-to-lead identification’ remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigenspecific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).Bill & Melinda Gates FoundationUnited States. Defense Advanced Research Projects AgencySpace and Naval Warfare Systems Center San Diego (U.S.) (Contract N66001-13-C-4025)W. M. Keck FoundationNational Institute of Allergy and Infectious Diseases (U.S.) (U19AI090970).National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051

Angiogenic competency of biodegradable hydrogels fabricated from polyethylene glycol-crosslinked tyrosine-derived polycarbonates

Synthetic biomaterials can be used as instructive biological milieus to guide cellular behaviour and function. To further realize this application, we synthesized a series of structurally similar hydrogels and tested their ability to modulate angiogenesis. Hydrogels were synthesized from poly(DTE-co-x% DT carbonate) crosslinked by y% poly(ethylene glycol) (PEG). Hydrogel desaminotyrosyl tyrosine (DT) contents (x%) ranged from 10-100%, and crosslink densities (y% PEG-crosslinker) ranged from 5-80%. The hydrogels were fashioned into porous scaffolds with highly interconnected macro- and micro-pore (>100 and <10 mm in diameter, respectively) architecture using poly(DTE-co-10%DT carbonate) crosslinked with 8% PEG. Under physiological conditions (in vitro), the hydrogels degraded into three major products: desaminotyrosyl-tyrosine ethyl ester (DTE), desaminotyrosyl tyrosine (DT), and poly(ethylene glycol)-di-DT-hydrazide (PEG-di-DT hydrazide). Increasing either DT content or crosslink density brought quickened degradation. Because DT and DTE, two of the three major degradation products, have not demonstrated any noticeable cytotoxicity or angiogenic effect in previous studies, we measured the cytotoxicity of PEG-di-DT hydrazide, the third major degradation product. We found that PEG-di-DT hydrazide only displayed significant cytotoxicity at the high concentration of 100 mg/mL. Interestingly, PEG-di-DT hydrazide and its further degradation product PEG-dihydrazide stimulated in vitro endothelial cell migration and tubulogenesis, which is comparable to results found with FGF-beta treatment. Subcutaneous implantation of the PEG-crosslinked poly(DTE-co-10%DT carbonate) scaffolds into the backs of rats elicited greater tissue growth over time and superior vascularization than poly(DTE carbonate) implantation. These results show that this new class of biomaterials has a strong potential to modulate angiogenesis

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV – recently infected and post-convalescent subjects – and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.National Institutes of Health (U.S.) (U19AI089992)National Institutes of Health (U.S.) (AI091816)National Institutes of Health (U.S.) (R01AI104739)National Institutes of Health (U.S.) (1F32AI112359-01)National Institutes of Health (U.S.) (T15LM07056)Human Immunology Project ConsortiumGillson Longenbaugh Foundatio

Design and synthesis of dual 5-HT1A and 5-HT7 receptor ligands

5-HT(1A) and 5-HT(7) receptors have been at the center of discussions recently due in part to their major role in the etiology of major central nervous system diseases such as depression, sleep disorders, and schizophrenia. As part of our search to identify dual targeting ligands for these receptors, we have carried out a systematic modification of a selective 5HT(7) receptor ligand culminating in the identification of several dual 5-HT(1A) and 5-HT(7) receptor ligands. Compound 16, a butyrophenone derivative of tetrahydroisoquinoline (THIQ), was identified as the most potent agent with low nanomolar binding affinities to both receptors. Interestingly, compound 16 also displayed moderate affinity to other clinically relevant dopamine receptors. Thus, it is anticipated that compound 16 may serve as a lead for further exploitation in our quest to identify new ligands with the potential to treat diseases of CNS origin