Project description:EPICERTIN is an engineered variant of cholera toxin B subunit (CTB) containing a C-terminal ER-retention motif that promotes mucosal healing in experimental colitis. In this study, RNA sequencing was performed to characterize global transcriptional responses of human peripheral blood mononuclear cell (PBMC)-derived macrophages following treatment with EPICERTIN, CTB, or PBS control. Macrophages were generated by culturing PBMCs with M-CSF and treated with EPICERTIN, CTB, or PBS for 12 hours prior to RNA extraction and sequencing. Comparative transcriptomic analysis revealed that EPICERTIN and CTB induced overlapping transcriptional responses that differed in magnitude and directionality, with EPICERTIN eliciting a moderated inflammatory response and enhanced expression of stress-adaptive and survival-associated genes relative to CTB. These data support a role for EPICERTIN in modulating macrophage activation and survival pathways relevant to mucosal wound healing.
Project description:Proteomic identification and characterization of antibodies comprising the serological response to antigen can provide unique insight into the functional dynamics of adaptive immunity. We have developed a novel method to overcome the technical challenges which previously limited the direct analysis of immunoglobulin proteins in serum, as demonstrated by the identification of human anti-tetanus toxoid (TT) immunoglobulin G (IgG) proteins following booster vaccination. We analyzed the serum IgG repertoire across four time-points corresponding to pre-vaccination, 7 days, 3 months, and 9 months post vaccination. Antigen-specific antibodies were affinity purified against immobilized TT protein and sequenced by bottom-up nanoLC-MS/MS. Interpretation of mass spectra required a custom reference database of IgG heavy and light chain variable sequences determined by NextGen RNA sequencing of the donor's circulating plasmablasts and memory B cells following booster vaccination.
Project description:Rice (Oryza sativa L.) is a candidate crop for production of plant-based vaccines by genetic engineering technologies. MucoRice-CTB has been developed as a rice-seed-based vaccine against cholera by transgenic expression of modified cholera toxin B-subunit (CTB) under the RNA interference (RNAi)-mediated suppression of endogenous seed storage proteins. Here, we performed non-targeted metabolomic profiling of MucoRice-CTB to understand the overall effects of the genetic engineering on rice seed metabolism using gas chromatography/time-of-flight mass spectrometry.
Project description:Oral cholera vaccines (OCVs) have become important components of strategies for cholera control, but the demand for OCVs has outstripped the supply2–4. There is a need for new cholera control measures for the one billion people living in cholera endemic regions5. The use of orally delivered single-domain antibodies has been proposed as a potential approach for the control of gastrointestinal pathogens6. Here, we describe the development of an orally deliverable bivalent VHH construct (BL3.2) that binds to the B-subunit of cholera toxin (CTXB). The epitope of CTXB for binding molecule BL3.2 was mapped by Hydrogen Deuterium Exchange HDX
Project description:Diphtheria, tetanus, and pertussis (DTP) are infectious diseases caused by toxin-producing bacteria that can be fatal, especially in children. Despite the availability of combined DTP vaccines for more than 60 years, a comprehensive understanding of how human antibodies respond to DTP vaccination, which helps further improve the vaccine remains elusive. Here, we aimed to characterize toxin-specific antibody repertoires following DTP vaccination. To this end, CD19+CD27+ antigen-experienced B cells obtained from four healthy donors 7-days post vaccination were sorted for toxin-binding and non-toxin-binding B cells, using each of the DTP recombinant toxins (DT, TT, and PT) as fluorescent bait. 10X single-cell immune profiling was then performed, followed by in silico antibody sequence clustering.
Project description:We observed the expression profile of the total mRNA of crp (TTHA1437) deletion mutant of Thermus thermophilus HB8 strain grown in a rich (TT) medium at 70 degC
