Journal: bioRxiv

Article Title: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

doi: 10.1101/2021.12.10.472112

Figure Lengend Snippet: (A) Schematic depicting S-triggered barrier dysfunction measured by a trans-endothelial/epithelial electrical resistance assay (TEER; left) and an endo/epithelial glycocalyx layer (EGL) assay (right). (B) Time course TEER assay measuring the barrier function of HPMEC monolayers over time with the indicated treatments, including DENV2 NS1 (5 μg/mL), VEGF (50 ng/mL), and SARS-CoV-2 S (10 μg/mL). Data are from n=2 biological replicates. (C) A TEER assay measuring the barrier of monolayers of HPMEC and HPMEC/ACE2 at 24 hours after the indicated treatments. VEGF positive control (50 ng/mL). Data are from n=3 biological replicates. (D) Same as C but treated with the indicated VSV pseudotyped particles at the indicated dilutions. VSV-bald and VSV-G are diluted 1:30. Data are from n=3 biological replicates. (E) Same as C but treated with the indicated concentrations of SARS-CoV-2 RBD. Data are from n=3 biological replicates. (F) Same as C but measuring the barrier of Calu-3 cell monolayers. Data are from n=2 biological replicates. (G) A TEER inhibition assay measuring the capacity of a cocktail of anti-S antibodies to inhibit S-mediated endothelial hyperpermeability. S (10 μg/mL) and the antibody cocktail (15 μg/mL for each antibody; 1A9 [Genetex] and CR3022 [Absolute Antibody]) were added simultaneously to the upper chamber of transwell inserts to a monolayer of HPMEC or HPMEC/ACE2 and TEER was measured 24 hours post-treatment (hpt). Data are from n=2 biological replicates. In all panels, the dotted line is the normalized TEER value of the untreated control condition. All data are plotted as mean +/− SD. For all panels, values are compared to untreated controls by ANOVA with multiple comparisons with *p<0.05, **p<0.01, ***p<0.001, and n.s. p>0.05.

Article Snippet: Purified proteins were formulated at ~1 mg/mL in PBS and stored in aliquots at −80° C. Recombinant DENV serotype 2 NS1 was purchased from the Native Antigen Company and characterized previously (Dengue virus serotype 2 NS1 [accession # P29990.1, Thailand/16681/84]) ( ).

Techniques: Positive Control, Inhibition

Journal: bioRxiv

Article Title: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

doi: 10.1101/2021.12.10.472112

Figure Lengend Snippet: (A) A representative mouse back from a dermal leak experiment. The dorsal dermises of mice were injected intradermally with the treatments and doses indicated. Mice then received a dextran-680 tracer molecule intravenously. Mouse dermises were collected 2 h post-treatment and quantification of local dermal leak was assessed by a fluorescent scanner. (B) Quantification of A from mice treated with PBS (n=27), DENV2 NS1 (15 μg; n=5), S (10 μg; n=25), and S (25 μg; n=5). (C) Representative lung images from a SARS-CoV-2 S systemic vascular leak assay. Mice were administered 50 μg of SARS-CoV-2 S or ovalbumin intranasally as indicated, and 22 hpt were administered a dextran-680 tracer intravenously as in A. Organs of mice were collected 2 hours post dextran-680 administration (24 hours post-S treatment), and accumulation of dextran-680 was measured with a fluorescent scanner. (D) Quantification of C from n=6 mice. (E) Same as C except representative images of spleens. (F) Quantification of E from n=6 mice. (G) Same as C except representative images of small intestine. (H) Quantification of G from n=5 mice. MFI is mean fluorescence intensity. All data are plotted as mean +/− SEM with *p<0.05, **p<0.01, and ***p<0.001 by unpaired t-test.

Article Snippet: Purified proteins were formulated at ~1 mg/mL in PBS and stored in aliquots at −80° C. Recombinant DENV serotype 2 NS1 was purchased from the Native Antigen Company and characterized previously (Dengue virus serotype 2 NS1 [accession # P29990.1, Thailand/16681/84]) ( ).

Techniques: Injection, Fluorescence

Journal: Immuno

Article Title: A Serological Multiplexed Immunoassay (MIA) Detects Antibody Reactivity to SARS-CoV-2 and Other Viral Pathogens in Liberia and Is Configurable as a Multiplexed Inhibition Test (MINT)

doi: 10.3390/immuno4010007

Figure Lengend Snippet: The multiplexed immunoassay (MIA) differentiates SARS-CoV-2 serological status. ( A ) Graphical representation of MIA process created with BioRender.com and Adobe Illustrator. ( B ) MIA of samples collected in Hawaii. One-way ANOVA multiple comparison analysis. Grey lines indicate BSA cut-offs, and black lines indicate antigen cut-offs. ( C ) Principal component analysis of samples collected in Hawaii. Calculations completed in GraphPad Prism, Principal component analysis. * < 0.5, ** < 0.01, **** < 0.0001. 2°Ab-PE = phycoerythrin-coupled secondary antibody, NC = negative controls (n = 10), N = naïve (n = 7), C = convalescent (n = 7), PD1 = post-dose 1, PD2 = post-dose 2. Wu-1 Trimer = Wuhan Hu1 strain spike trimer, Alpha-S1 = SARS-CoV-2 Alpha strain spike domain 1, Beta S1 + S2 = SARS-CoV-2 Beta strain spike domains 1 + 2, Wu-1 NP = Wuhan Hu1 strain nucleocapsid protein, NL63 S1 = hCoV-NL63 spike domain 1, DENV-2E = Dengue virus serotype 2 envelope protein, DENV-2 NS1 = Dengue virus serotype 2 nonstructural protein 1.

Article Snippet: The coupling of individually addressable microspheres with Wu-1 Trimer, Wu-1 S1 (SinoBiological 40591-V08H), Alpha S1 (SinoBiological 40591-V08H7), Beta S1 + S2 (SinoBiological 40589-v08B16), Wu-1 NP, NL63 S1 (SinoBiological 40600-V08H), DENV-2 E (SinoBiological 40471-V08Y1), DENV-2 NS1 (Native Antigen Company, Oxford, UK), and CHIKV E2 proteins, was conducted as described previously [ , , ].

Techniques: Comparison, Virus

Journal: Immuno

Article Title: A Serological Multiplexed Immunoassay (MIA) Detects Antibody Reactivity to SARS-CoV-2 and Other Viral Pathogens in Liberia and Is Configurable as a Multiplexed Inhibition Test (MINT)

doi: 10.3390/immuno4010007

Figure Lengend Snippet: The multiplexed inhibition test (MINT) reveals antibody functionality. ( A ) Graphical representation of the MINT assay created with BioRender.com and Adobe Illustrator. ( B ) MINT curves from Hawaii samples were performed on baseline and post-dose 2 samples for the Wuhan Hu-1 trimer antigen. ( C ) MINT curves for Liberia samples were performed on Wuhan Hu-1 trimer, Alpha-S1, and Beta-S1 + S2 antigens. ( D ) Correlations of MINT 50 values and interpolated IgG concentrations for samples collected in Hawaii or Liberia. Pearson r and p values were calculated using GraphPad Prism. ***< 0.001.

Article Snippet: The coupling of individually addressable microspheres with Wu-1 Trimer, Wu-1 S1 (SinoBiological 40591-V08H), Alpha S1 (SinoBiological 40591-V08H7), Beta S1 + S2 (SinoBiological 40589-v08B16), Wu-1 NP, NL63 S1 (SinoBiological 40600-V08H), DENV-2 E (SinoBiological 40471-V08Y1), DENV-2 NS1 (Native Antigen Company, Oxford, UK), and CHIKV E2 proteins, was conducted as described previously [ , , ].

Techniques: Inhibition