anti wnv e protein  (Novus Biologicals)

Journal: PLOS Pathogens

Article Title: B Cell Receptor’s function in virus entry: Anti-SARS-CoV-2 B cell receptors can mediate viral entry in an ACE2-independent mechanism

doi: 10.1371/journal.ppat.1013946

Figure Lengend Snippet: (A) Ramos cells were transduced with lentiviral vectors expressing the single-chain antibody (ScFv) form of the BCRs. Ramos cells, along with Ramos cells expressing control BCR (anti-HIV-1 3BNC117) , ZKA190 BCR, and ZKA230 BCR, were incubated with fluorescently labeled ZIKV (DiD ZIKV) for one hour at room temperature. Virus binding was then analyzed by flow cytometry. N = 3 and averages are shown with SD. Significance was calculated using a two-sample two-sided unpaired student t-test (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). This experiment was repeated twice in singlicate and once in triplicate, and representative flow cytometry histograms are shown. Infection of B) Ramos or C) CD79 Sp2/0 cells with or without ectopic expression of control or anti-ZIKV BCR with replication-competent ZIKV. Both cell types were transduced with lentiviral vectors expressing the ScFv form of the BCRs. The ScFv versions of the BCRs feature N-terminal Flag tags to facilitate the specific detection of lentiviral-mediated expression on B cells; therefore, ScFv expression can be confirmed via flow cytometry following staining with fluorescently labeled anti-Flag antibodies. The cells were infected with replication-competent ZIKV at a multiplicity of infection (MOI) of 10. The viral titer was determined by TCID 50 assay using Vero E6 cells. At 48 hours post-infection, ZIKV infection was quantified via flow cytometry following intracellular staining of the ZIKV E protein using an Alexa 647-conjugated anti-ZIKV E protein antibody (4G2). N = 3 and averages are shown with SD (error bar). This experiment was repeated twice in singlicate and once in triplicate, and the results of the triplicated experiments are shown. (D) Supernatants from ZIKV-infected cells (shown in panels B and C) were harvested at 48 hours post-infection and used to inoculate Vero E6 cells. After an additional 48 hours, ZIKV infection levels in Vero E6 cells were quantified by flow cytometry following intracellular staining for the ZIKV E protein using an Alexa 647-conjugated 4G2 antibody. Data represent the mean ± SD of three independent experiments (n = 3). This experiment was repeated once in singlicate and once in triplicate, and the results of the triplicated experiments are shown. (E) Comparison of expression levels of endogenous IgG1 BCR and lentivirally transduced ScFv BCR on human primary B cells. The cells were stained with human IgG1 antibody conjugated with APC. Black: Unstained whole B cell; Red: Staining of endogenous IgG1 BCR in untransduced population; Blue: Staining of exogenous IgG1 BCR in IgD and FLAG + population; Green: Staining of endogenous IgG1 BCR in untransduced IgD+ population. (F) Infection of primary human B cells from two donors with or without ectopic expression of control or anti-ZIKV BCR with ZIKV replicon. The cells were infected with a ZIKV replicon expressing EGFP at a multiplicity of infection (MOI) of 10 with the replicon titer determined by flow cytometry using Vero E6 cells. At 48 hours post-infection, ZIKV infection, defined by EGFP expression, was quantified via flow cytometry in untransduced CD19-positive B cells (None) and B cells expressing either the control or anti-ZIKV BCR (Flag tag-positive). N = 3 and averages are shown with SD (error bar). We performed within-donor pairwise analyses using Welch’s two-sample t-tests with pre-specified contrasts versus None: Donor 1: None vs Control p = 0.57, ZKA190 significantly increased infection relative to None, p = 0.034. ZKA230 significantly increased infection relative to None, p = 0.05. Donor 2: None vs Control, p = 0.45. ZKA190 also significantly increased infection relative to none, p = 0.004. ZKA230 significantly increased infection relative to None, p = 0.005. Pooled analysis (Experiments 1 + 2): When data were combined across both experiments, ZKA190 and ZKA230 remained highly significantly different from none, p < 1 × 10 ⁻ 3 . Pooled None vs Control was not significant, p = 0.32.

Article Snippet: The cells were then fixed by 4% paraformaldehyde, followed by staining with Alexa 647-conjugated Flavivirus group antigen antibody (4G2) (Novus Biologicals, Littleton, CO) in cell permeabilization buffer (Thermo Fisher Scientific).

Techniques: Transduction, Expressing, Control, Incubation, Labeling, Virus, Binding Assay, Flow Cytometry, Infection, Staining, Comparison, FLAG-tag