Characterization of binding interactions of SARS-CoV-2 spike protein and DNA-peptide nanostructures.

Binding interactions of the spike proteins of the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) to a peptide fragment derived from the human angiotensin converting enzyme 2 (hACE2) receptor are investigated. The peptide is employed as capture moiety in enzyme linked immunosorbent assays (ELISA) and quantitative binding interaction measurements that are based on fluorescence proximity sensing (switchSENSE). In both techniques, the peptide is presented on an oligovalent DNA nanostructure, in order to assess the impact of mono- versus trivalent binding modes. As the analyte, the spike protein and several of its subunits are tested as well as inactivated SARS-CoV-2 and pseudo viruses. While binding of the peptide to the full-length spike protein can be observed, the subunits RBD and S1 do not exhibit binding in the employed concentrations. Variations of the amino acid sequence of the recombinant full-length spike proteins furthermore influence binding behavior. The peptide was coupled to DNA nanostructures that form a geometric complement to the trimeric structure of the spike protein binding sites. An increase in binding strength for trimeric peptide presentation compared to single peptide presentation could be generally observed in ELISA and was quantified in switchSENSE measurements. Binding to inactivated wild type viruses could be shown as well as qualitatively different binding behavior of the Alpha and Beta variants compared to the wild type virus strain in pseudo virus models.

Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application.

Curdlan sulfate (CRDS), a sulfated 1→3-ß-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the ß-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies.

Inducible expression of chimpanzee prion protein (PrP) in murine PrP knock-out cells.

In transmissible spongiform encephalopathy (TSE) pathogenesis the cellular prion protein (PrP(C)) is converted into its pathogenic PrP(Sc) isoform. Prion protein gene (Prnp) deficient mice (PrP(0/0)) are resistant to PrP(Sc) infection, but following reconstitution of Prnp they regain their susceptibility to infection. Therefore, it is challenging to simulate this natural situation in a cell culture model. We have previously reported the inducible stable expression of a human PrP(C) in murine 3T3 cells. In this study, we used murine PrP(0/0) cells stably expressing exemplarily the chimpanzee Prnp under the control of inducible tetracycline (Tet) system. The Prnp was integrated using a lentiviral vector. Its expression in the engineered PrP(0/0)Chimp1/Tet-Off cell line was analyzed by Western blot (Wb) and fluorescence activated cell sorting (FACS) analyses. PrP(C) was partially purified by using immobilized metal affinity chromatography (IMAC). Compared to all the other cell systems which possess an endogenous PrP(C) expression, here described cell line contains only an overexpressing species specific PrP(C) expression which is tightly regulated and can be turned-off at any time without showing any endogenous host PrP(C) expression. Consequently, a contamination of the isolated PrP(C) is impossible. This cell line potentially offers a new tool for simulation of mice bioassays widely used in TSE infection studies.