Journal: PLoS Pathogens

Article Title: Human monoclonal antibodies against Ross River virus target epitopes within the E2 protein and protect against disease

doi: 10.1371/journal.ppat.1008517

Figure Lengend Snippet: (A) Pre-attachment and post-attachment neutralization assays were performed for representative mAbs from each competition group, and a focus-forming assay was used to quantify reduction in infection. In the pre-attachment assay, antibody was incubated with virus at 4°C before addition to Vero cells kept at 4°C. For the post-attachment assay, virus was applied to Vero cell monolayer cultures at 4°C before addition of antibody to cells at 4°C. Two independent eperimnents were performed in triplicates for each antibody, and representative curves are shown. (B) A fusion from without (FFWO) assay was used to measure antibody inhibition of virus fusion with the cell membrane under low pH conditions. Virus was adsorbed to Vero cell culture monolayers at 4°C for an hour before addition of antibody dilutions, also at 4°C, after removing excess virus. Cells then were exposed to a pH 5.5 medium or a control medium at neutral pH for two minutes and incubated at 37°C. The acidic pH medium was removed and cells were incubated for an additional 14 h before fixing, permeabilizing, and staining for intracellular virus antigens before flow cytometric analysis. Intracellular virus was quantified by measuring percent PE-positive cells relative to a virus-only control. Three separate experiments were performed in triplicates for each antibody (Kruskal-Wallis one-way ANOVA with Dunn’s post-test, with mean ± S.D. compared to virus-only control. (*p < 0.05, **p < 0.01, ***p < 0.001)). (C) Representative flow cytometry contour plots are shown for the FFWO assay. Mock-infected cells under a low-pH condition and cells with no antibody under a neutral pH condition (to ensure that virus only entered the cell through pH-mediated fusion) are shown as negative controls, and cells with a non-specific mAb (ZIKV-117) under a low pH condition are shown as a positive control. (D) Antibody blocking of RRV binding to mouse Mxra8-Fc fusion protein was determined through competition ELISA. Virus was captured on the plate with a human mAb before addition of RRV mAbs followed by Mxra8-mFc (mouse Fc). A loss of signal indicates competition of RRV mAbs with Mxra8-mFc for binding to virus. Three independent experiments were performed in quadruplicate (Kruskal-Wallis one-way ANOVA with Dunn’s post-test, with mean ± S.D. compared to isotype control (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)). (E) Residues that result in loss of Mxra8 binding to cell-surface-displayed chikungunya proteins are mapped onto the CHIKV E1/E2 trimer of heterodimers (PDB 3J2W) in red, and the alanine footprint of mAbs that block binding of mouse Mxra8-Fc protein to RRV are shown in yellow. Overlapping epitopes of RRV mAbs and Mxra8 contact residues are denoted in orange.

Article Snippet: For staining prior to flow cytometry analysis, cells were incubated sequentially with RRV mouse ascites fluid (1:6000 dilution ATCC Cat. No. VR-1246AF) for 1 h and PE conjugated goat anti-mouse IgG secondary antibody for 1 h (ThermoFisher).

Techniques: Neutralization, Focus Forming Assay, Infection, Incubation, Virus, Inhibition, Membrane, Cell Culture, Control, Staining, Flow Cytometry, Positive Control, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: The Approved Live-Attenuated Chikungunya Virus Vaccine (IXCHIQ ® ) Elicits Cross-Neutralizing Antibody Breadth Extending to Multiple Arthritogenic Alphaviruses Similar to the Antibody Breadth Following Natural Infection

doi: 10.3390/vaccines12080893

Figure Lengend Snippet: Summary of alphavirus neutralizing antibody responses in all participants.

Article Snippet: Alphaviruses were sourced through the Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Manassas, VA, USA): MAYV BeAr505411 (BEI NR-49910), ONNV UgMP30 (BEI NR-51661), and RRV T-48 (BEI NR-51457).

Techniques: Standard Deviation