Project description:Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, our studies demonstrate that SFTSV surface glycoprotein (Glycoprotein N, Gn) is a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies play a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages including small molecular weight, high affinity, low immunogenicity and convenient production by gene engineering, etc. In this study, we combined next generation sequencing (NGS) with proteomics technology and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, and selected 6 of them for recombinant protein expression and purification. Among these 6 anti-Gn nanobodies, nanobody 57493 was validated to be highly specific for Gn.
Project description:The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development.
Project description:Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the WHO most dangerous pathogens, has 12-30% fatality rates with a characteristic thrombocytopenia syndrome. With a majority of clinically diagnosed SFTSV patients older than ~50 years, age is a critical risk factor for SFTSV morbidity and mortality. Here, we report an age-dependent ferret model of SFTSV infection and pathogenesis that fully recapitulates the clinical manifestations of human infections. While young adult ferrets (≤2 years old) did not show any clinical symptoms and mortality, SFTSV-infected aged ferrets (≥4 years old) demonstrated severe thrombocytopenia, reduced white blood cells, and high fever with 93% mortality rate. Moreover, significantly higher viral load was observed in aged ferrets. Transcriptome analysis of SFTSV-infected young ferrets revealed strong interferon-mediated anti-viral signaling, whereas inflammatory immune responses were markedly upregulated and persisted in aged ferrets. Thus, this immunocompetent age-dependent ferret model should be useful for anti-SFTSV therapy and vaccine development.
Project description:Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is a life-threatening systemic autoimmune disease that frequently presents as crescentic glomerulonephritis (GN) leading to kidney failure1. Despite recent advances2, treatment for most patients with ANCA-associated GN relies on unspecific immunosuppressive agents that are hazardous and only partially efficacious. To develop an anti-T-cell cytokine treatment strategy based on the immunopathogenesis of the individual patient, we established the combination of rapid single-cell immune profiling with standard histopathological analyses of renal biopsies. We report two patients with biopsy-proven ANCA-associated GN who presented with renal failure and active urinary sediment despite immunosuppressive therapy. Our data suggest that the integration of single-cell immune profiling with clinical and histopathological information facilitates personalized anti-cytokine therapies, such as ustekinumab in ANCA-GN, and warrants further investigation.
Project description:Ticks are vectors of arboviruses in many parts of the world. The rising incidence and emergence of tick-borne arboviral infections across human populations indicates that further transmission control strategies including those based on vectors, will be required to reduce the burden of disease. However, arbovirus-tick interactions at the cellular level remain poorly understood in general, and particularly neglected for negative strand RNA arboviruses. In this study we developed a proteomics informed by transcriptomics approach to characterize the cellular response of Rhipicephalus microplus-derived cell cultures to infection with the tick-borne pathogen severe fever with thrombocytopenia syndrome virus (SFTSV, Phenuiviridae). For this, we generated the first de novo transcriptomes and confirmed proteomes of SFTSV- or mock-infected tick cell cultures derived from a vector species that transmits the virus in nature. Through comprehensive annotation of genes, proteins and pathway analysis, we identified core host responses and regulatory processes mediated in response to SFTSV infection. Moreover, examining the interactome of the virally encoded nucleoprotein (N) allowed us to integrate host responses with the analysis of cellular factors required for viral replication. The influence of specific host genes on SFTSV replication was systematically assessed through dsRNA-mediated gene silencing. This functional genomics approach pinpointed two tick-derived RNA helicases as critical antiviral factors capable of restricting SFTSV infection: the DexD/box helicase (DHX9) and the Up-Frameshift Protein 1 (UPF1). Collectively, our findings enrich the repository of resources available for understanding the antiviral response to SFTSV infection in Rh. microplus vector cells and support the identification of SFTSV-antiviral restrictions factors.
Project description:In this study, transcriptomic profiling identified PPM1K as a prominently induced host factor in SFTSV-infected THP-1 cells. We further demonstrate that SFTSV infection promotes endoplasmic reticulum–mitochondria coupling, enabling the viral glycoprotein GC to interact with mitochondrial PPM1K and enhance its expression at both transcriptional and protein levels. Mechanistically, PPM1K dephosphorylates Bcl-2 at Ser248, decreases Bcl-2 ubiquitination, and stabilizes its anti-apoptotic activity, thereby suppressing the BAX–Caspase-3 signaling cascade. This anti-apoptotic remodeling markedly limits apoptosis in infected cells and facilitates efficient SFTSV replication. These findings uncover a previously unrecognized viral strategy in which SFTSV hijacks host PPM1K-dependent mitochondrial signaling to evade apoptosis and promote replication
Project description:In order to better understand bacterial aggregates mechanisms we used RNA seq and a specific setup of E. coli K12 strains expressing either the natural self-recognizing adhesin Ag43 or nanobodies based synthetic adhesins. We studied the changes in gene expression between aggregates and planktonic cells.
Project description:T cell engagers, which bind tumor-associated antigens and T cell specific molecules, represent a promising class of immunotherapies for enhancing targeted immune responses. Here, we introduce a “plug-and-display” platform for engineering T cell nanoengagers by anchoring antibody fragments into lipid-based nanoparticles. This approach utilizes a genetically engineered lipoprotein fused with single-chain variable fragments (scFv) and nanobodies, which spontaneously integrated into lipid bilayer of the nanoparticles, achieving a high surface density of at least 0.102 scFv/nm2 (approximately 3200 scFv per particle). We designed modular bi-specific (Lipo-BiTE) and tri-specific (Lipo-TriTE) immunoliposomes to enhance anti-tumor T cell immune responses. The Lipo-BiTE, integrating anti-CD3 and anti-HER2 scFv at an optimized surface density of 1.28 × 10-3 scFv/nm2, exhibited enhanced CD8+ T cell-mediated cytotoxicity in HER2-positive tumor models by simultaneously engaging tumor cells and T cells. Incorporating anti-PD-L1 nanobodies to create Lipo-TriTE further addressed T cell exhaustion. This modular platform provides a robust foundation for designing immune cell engagers, with broad applications in targeted immunotherapy.