Journal: bioRxiv

Article Title: Simultaneous and sequential multi-species coronavirus vaccination

doi: 10.1101/2022.05.07.491038

Figure Lengend Snippet: (A) Schematics of mRNA vaccine construct design against pathogenic human coronavirus species. Each construct has regulatory elements (5’UTR, 3’UTR and polyA) and spike ORF. The domain structures as well as engineered mutations of translated spike proteins of SARS-CoV-2 Delta variant (Delta), SARS-CoV (SARS) and MERS-CoV (MERS). (B) Engineered mutations in spike protein structures of SARS-CoV-2 Delta, SARS-CoV and MERS-CoV. The N-terminal domain (NTD, blue), receptor binding domain (RBD, green) and S2 subunit (orange) of one protomer along with homologous HexaPro mutations (pink) and Delta variant mutations (red) were highlighted in the spike trimer structures. (C) Schematics of characterization of LNP-mRNA vaccine formulations. Assembly procedure of LNP-mRNA vaccine on NanoAssemblr Ignite and downstream biophysical characterization assays. (D) Histogram displaying radius distribution of LNP-mRNA formulations of SARS-CoV-2 Delta and a Triplex (Delta + SARS + MERS) (abbreviated as Triplex-CoV or Triplex), measured by dynamic light scattering (DLS). The polydispersity index and mean radius of each LNP sample were shown at top left corner. (E) Transmission electron microscope (TEM) images of Delta and Triplex-CoV LNP-mRNAs. (F) Surface expression of functional spike proteins in 293T cells after electroporation of corresponding mRNA, as detected by human ACE2 or human DPP4 Fc fusion protein bound to PE anti-Fc antibody. (G) Schematics of vaccination schedule of the Triplex LNP-mRNA formulations, as well as downstream assays to evaluate the antibody responses and other immunological profiles. (H) Binding antibody titers of plasma samples from mice administered with PBS or different LNP-mRNAs (n = 9) against RBD or ectodomain (ECD) of SARS-CoV-2 wild type (WT, Wuhan/WA-1), Delta variant, SARS and MERS spikes. The binding antibody titers were quantified by area under curve of log 10 -transformed titration curve (log 10 AUC) in . The mice were intramuscularly injected with two doses (x2, 2 weeks apart) of PBS, 1μg SARS-CoV-2 Delta variant LNP-mRNA (delta), 1μg or 3μg equal mass mixture of Delta, SARS and MERS LNP-mRNA (Triplex-CoV). Notes: In the dot-box plots of this figure, each dot represents data from one mouse. Data are shown as mean ± s.e.m. plus individual data points in plots. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. Statistical significance labels: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Non-significant comparisons are not shown, unless otherwise noted as n.s., not significant.

Article Snippet: To detect surface-protein expression, the cells were stained with ACE2–Fc chimera (Genscript, Z03484) or DPP4-Fc (Sino Biological, 10688-H01H) in MACS buffer (D-PBS with 2 mM EDTA and 0.5% BSA) for 30 min on ice.

Techniques: Construct, Variant Assay, Binding Assay, Transmission Assay, Microscopy, Expressing, Functional Assay, Electroporation, Transformation Assay, Titration, Injection

Journal: Viruses

Article Title: Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors

doi: 10.3390/v14020384

Figure Lengend Snippet: Characteristics of recombinant human cell receptors and viral spike proteins.

Article Snippet: hDPP4 , Sino Biological , 10688-H08H , D34 , P766 , 744 , 86.3.

Techniques: Recombinant, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Co-localization of the identified proteins with cell membrane-bound SARS-CoV-2 spike proteins. Representative images of co-localization with SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells were co-stained for SARS-CoV-2 spike protein (green) and the antibodies recognizing ACE2, CD133, Cadherin 17, DPP4, and VAPA (Red). The resulting specimens were subsequently stained with appropriate secondary antibodies and DAPI (Blue), then observed using confocal microscopy (20× objective). Co-localization is indicated in yellow in the “Merge” images. White bar: 10 μm.

Article Snippet: The ORFs of the candidate protein CDH17 and DPP4 were obtained from pCMV3-human CDH17 (HG11360-UT; Sino Biological) and pCMV3-human DPP4 (HG10688-UT; Sino Biological) by KpnI and XbaI digestion.

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Candidate proteins located near SARS-CoV-2 spike proteins. ( A to D ) Morphological observation of SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells observed using electron microscopy. Cultured Caco-2 cells were fixed and co-stained with the SARS-CoV-2 spike protein (indicated as 20 nm particles), and candidate molecules identified. CD133 ( A ), DPP4 ( B ), CDH17 ( C ), and VAPA ( D ) are indicated as 10 nm particles. Red arrows indicate the locations of SARS-CoV-2 spike proteins. Yellow arrow heads indicate the location of each candidate protein. Scale bar; 200 or 500 nm.

Article Snippet: The ORFs of the candidate protein CDH17 and DPP4 were obtained from pCMV3-human CDH17 (HG11360-UT; Sino Biological) and pCMV3-human DPP4 (HG10688-UT; Sino Biological) by KpnI and XbaI digestion.

Techniques: Electron Microscopy, Cell Culture, Staining