Cell Based Assays

The design of an immunogenic scaffold that serves a role in treating a pathogen, and can be rapidly and predictively modeled, has remained an elusive feat. Here, we demonstrate that SARS-BLOCK™ synthetic peptide scaffolds act as antidotes to SARS-CoV-2 spike protein-mediated infection of human ACE2-expressing cells. Critically, SARS-BLOCK™ peptides are able to potently and competitively inhibit SARS-CoV-2 S1 spike protein receptor binding domain (RBD) binding to ACE2, the main cellular entry pathway for SARS-CoV-2, while also binding to neutralizing antibodies against SARS-CoV-2. In order to create this potential therapeutic antidote-vaccine, we designed, simulated, synthesized, modeled epitopes, predicted peptide folding, and characterized behavior of a novel set of synthetic peptides. The biomimetic technology is modeled off the receptor binding motif of the SARS-CoV-2 coronavirus, and modified to provide enhanced stability and folding versus the truncated wildtype sequence. These novel peptides attain single-micromolar binding affinities for ACE2 and a neutralizing antibody against the SARS-CoV-2 receptor binding domain (RBD), and demonstrate significant reduction of infection in nanomolar doses. We also demonstrate that soluble ACE2 abrogates binding of RBD to neutralizing antibodies, which we posit is an essential immune-evasive mechanism of the virus. SARS-BLOCK™ is designed to "uncloak" the viral ACE2 coating mechanism, while also binding to neutralizing antibodies with the intention of stimulating a specific neutralizing antibody response. Our peptide scaffolds demonstrate promise for future studies evaluating specificity and sensitivity of immune responses to our antidote-vaccine. In summary, SARS-BLOCK™ peptides are a promising COVID-19 antidote designed to combine the benefits of a therapeutic and vaccine, effectively creating a new generation of prophylactic and reactive antiviral therapeutics whereby immune responses can be enhanced rather than blunted.

Aims: The mouse bioassay (MBA) is the biologically relevant gold standard method for assessment of BoNTs. However, the aspiration for replacement, reduction, and refinement – the 3Rs principles – of animal use in potency assays entail development of alternative methods. Pluripotent embryonic stem cells provide a continuous source of appropriate cells that allow evaluation of receptor binding, internalisation, translocation, and catalytic activity of BoNTs. We aim to develop a cell-based assay that captures all biological functions of BoNTs and is equally sensitive as the MBA.
Methods: Directed differentiation of murine ES cells, strain E14TG2a, into motoneurons was achieved in two steps: 1) embryoid bodies (EBs) were generated and treated with RA (1 μM), SHH (100 ng/ml) and purmorphamine (1 μM); 2) dissociated EBs were differentiated on laminin coated plates in neurobasal medium. Mixed population of mature cholinergic and adrenergic motoneurons were identified by immunolabeling against HB9, MAP-2, tau, synaptophysin, PSD-95, choline-acetyltransferase, and noradrenaline. We demonstrate that they express BoNT/A receptor SV2A and its intracellular target SNAP25. Motoneurons were intoxicated with a range of LD50 values of purified (Metabiologics) and formulated (NIBSC reference) BoNT/A1 samples for 12 to 72 hours. Time and dose dependent changes in the level of cleaved intracellular SNAP25 were evaluated using in-house developed sandwich-ELISA assay: 96 well ELISA plates were coated overnight with affinity purified capture Ab (-amino acids 190-197) specific to epitope exposed upon BoNT/A cleavage of intact SNAP25. Total cell lysates were prepared and added to plates for 90 minutes. Binding of cleaved SNAP25 to coated Ab was detected by addition of a mixture of sheep anti-SNAP25 Abs directed to SNAP25 amino acids 1–57 and 111–157, and reaction was visualised with commercially sourced rabbit anti-sheep HRP conjugated Ab.
Results: Differentiation procedure was optimised to enhance sensitivity of cells to BoNT/A. Optimal sensitivity was achieved when 50,000 cells/well in 96-well plates were cultured for nine days and intoxicated for 48 hours. The preliminary results indicate that intoxication with purified (Metabiologics) BoNT/A1 results in dose dependent proteolysis of SNAP25 respectively within 12 h, 24 h, 48 h and 72 h with EC50s of 10, 3.5, 1, and 0.7 LD50 mL, respectively.
Keywords: botulinum neurotoxins; bioassay; SNA; proteolysis.

Prostate cancer is a leading cause of death in males aged fifty and over.Glutathione transferase (GST) activity is depressed in prostate cancer cells. The aim of this study was to assess GST reactivation in LNCaP prostate cancer cells treated with curcumin or 5-azacitidine (5-Aza) which is a known hypomethylation  agent. GST activity was determined using monochlorobimane (MCB). Cell viability was assessed with resazurin (Vision blue TM) or 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium- bromide (MTT). From the results, treatment with >5 μM of curcumin or 5-Aza for 3 or 6 days depressed LNCaP cell viability. The concentrations of curcumin leading to 50% reduction of LNCaP cell viability (IC50) was 10-25 μM or 2-3 μM for 3 days or 6 days of treatment, respectively. The IC50 with 5-Aza was 17-23 μM (3 days) or 50-52 μM (6 days).Combination treatment using curcumin and 5-Aza showed complimentary interactions affecting cell viability. Low levels of curcumin or 5-Aza had no effect on GST activity. By contrast, cytotoxic doses of curcumin or 5-Aza increased GST activity by 450-750 % (3days) or 161-2800 % (6days). In conclusion, GST reactivation was feasible but only when LNCaP prostate cancer cells were treated with cytotoxic doses of curcumin or 5-azacytidne.

Keywords: Curcumin, 5-Azacytidine, prostate cancer, LNCaP, glutathione-Stransferase (GST), epigenetics.