Generation, lyophilisation and epitope modification of high titre filovirus pseudotyped lentiviruses for use in antibody neutralisation assays

Purpose: Filoviruses, such as Ebolavirus, are zoonotic pathogens causing disease outbreaks with high mortality rates, requiring scarce high containment facilities for research. Nevertheless, pseudotyped viruses (PV), consisting of a lentiviral core (plus luciferase reporter) and the envelope glycoprotein (GP), allow basic and translational virology to be conducted under low containment. Consequently, filovirus PVs were generated and viability assessed after lyophilisation and long-term storage. Next, antibody neutralisation tests were performed using native and hybrid GPs to assess differentiation between genera and species. Methods & Materials: PVs were produced using a 3-plasmid transfection system (representing core, reporter and envelope) in HEK293T/17 cells, and supernatant titrated. Supernatants were then lyophilised in sucrose cryoprotectant solution, stored under various conditions, reconstituted and titrated. For antibody neutralisation tests, serially diluted, polyclonal convalescent sera (NIBSC, UK) or anti-GP monoclonal antibodies (Xiangguo Qiu, PHA, Canada; Erica Saphire, Scripps, USA) were incubated with PV for 1 h at 37 °C, prior to titration. To create artificial GP antigens, EBOV neutralising epitopes were inserted into the GP of another genus (Cuevavirus; LLOV) by mutagenesis, PVs generated and infectivity and neutralisation assessed. Results: High titre PVs were produced with titres between ∼1 × 108 RLU/mL (Ebolavirus/Cuevavirus)and ∼1 × 1010 RLU/mL (Marburgvirus). Lyophilised PV titres remained constant stored at −20 °C and 4 °C for 12 months, while PVs kept at room temperature (22.5 °C) demonstrated titre decreases of up to 3 orders of magnitude after 6 months. At 37 °C, five log (Marburgvirus) or three log (Ebolavirus and Cuevavirus) decreases occurred after one month. Zaire Ebolavirus (EBOV) antibodies showed no cross reactivity with native LLOV PVs. Furthermore, EBOV epitopes inserted into the LLOV GP and expressed on PVs had no significant impact on PV infectivity, and EBOV neutralising epitopes were successfully reconstituted in these chimeric antigens Conclusion: In this study, high titre PVs were generated and found to be amenable to lyophilisation and long-term storage. Reconstituted PVs retained their function in neutralisation assays suggesting their structure is not compromised during freeze-drying. Insertion of epitopes in heterologous GPs did not impact infectivity or functionality. This data suggests a PV-based serological kit could be utilised in resource-limited countries for serological studies, after simple refrigeration storage

Predicting Phospholipidosis Using Machine Learning

Phospholipidosis is an adverse effect caused by numerous cationic amphiphilic drugs and can affect many cell types. It is characterized by the excess accumulation of phospholipids and is most reliably identified by electron microscopy of cells revealing the presence of lamellar inclusion bodies. The development of phospholipidosis can cause a delay in the drug development process, and the importance of computational approaches to the problem has been well documented. Previous work on predictive methods for phospholipidosis showed that state of the art machine learning methods produced the best results. Here we extend this work by looking at a larger data set mined from the literature. We find that circular fingerprints lead to better models than either E-Dragon descriptors or a combination of the two. We also observe very similar performance in general between Random Forest and Support Vector Machine models.</p

A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research

Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines

Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions

Microneedle-based drug and vaccine delivery via nanoporous microneedle arrays

Drug Delivery Technolog