Optimizing variant-specific therapeutic SARS-CoV-2 decoys using deep-learning-guided molecular dynamics simulations.

Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of multiple spike RBD/hACE2 variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W and multi-mutation variants as high-affinity candidates, which we validated in vitro with virus neutralization assays.

A versatile bifunctional dendritic cell targeting vaccine vector.

We have developed an efficient versatile in vivo dendritic cell (DC) targeting vector for delivering different classes of antigens such as proteins, peptide, glycolipids and naked DNA for vaccine applications. A single chain antibody (scFv) that recognizes DEC-205 receptor of DC was fused with a core-streptavidin domain and expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed as a periplasmic soluble protein and affinity-purified in its monomeric form.

Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.

We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column.

Detection of Escherichia coli O157:H7 using chicken immunoglobulin Y.

A sandwich ELISA technique was examined to detect Escherichia coli O157:H7 using chicken anti-E. coli O157:H7 IgY as the capture-antibody and an anti-E. coli O157 mouse mAb conjugated with biotin as the detection antibody.

Antigen targeting to dendritic cells with bispecific antibodies.

We have developed a universal DC targeting vehicle that could be a convenient method to deliver any type of antigen to DC. P125, a quadroma (hybrid-hybridoma) secreting bispecific monoclonal antibodies (bsmAb), with one paratope specific for mouse DC DEC-205 and another paratope specific for biotin, was developed by PEG-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter. The bsmAb were purified using a biotin-Agarose column and the bsmAb activity was demonstrated using ELISA method employing mouse bone marrow DC and biotinylated BSA.

Biotin carboxyl carrier protein co-purifies as a contaminant in core-streptavidin preparations.

We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP).