Search for: All records

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler’s diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing. 

Abstract Human mesenchymal stem cells (hMSCs) have gained traction in transplantation therapy due to their immunomodulatory, paracrine, immune‐evasive, and multipotent differentiation potential. The inherent heterogeneity of hMSCs poses a challenge for therapeutic treatments and necessitates the identification of robust biomarkers to ensure reproducibility in both in vivo and in vitro experiments. In this study, we utilized dielectrophoresis (DEP), a label‐free electrokinetic phenomenon, to investigate the heterogeneity of hMSCs derived from bone marrow (BM) and adipose tissue (AD). The electrical properties of BM‐hMSCs were compared to homogeneous mouse fibroblasts (NIH‐3T3), human fibroblasts (WS1), and human embryonic kidney cells (HEK‐293). The DEP profile of BM‐hMSCs differed most from HEK‐293 cells. We compared the DEP profiles of BM‐hMSCs and AD‐hMSCs and found that they have similar membrane capacitances, differing cytoplasm conductivity, and transient slopes. Inducing both populations to differentiate into adipocyte and osteoblast cells revealed that they behave differently in response to differentiation‐inducing cytokines. Histology and reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyses of the differentiation‐related genes revealed differences in heterogeneity between BM‐hMSCs and AD‐hMSCs. The differentiation profiles correlate well with the DEP profiles developed and indicate differences in the heterogeneity of BM‐hMSCs and AD‐hMSCs. Our results demonstrate that using DEP, membrane capacitance, cytoplasm conductivity, and transient slope can uniquely characterize the inherent heterogeneity of hMSCs to guide robust and reproducible stem cell transplantation therapies.