Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Immunofluorescent assay for colocalization of AXL with B19V virions. CD36 + EPCs cells were infected with B19V at an MOI of ~5000 vgc per cell for 1.5 hours at 4°C. The cells were then washed with ice-cold PBS, then cytospun onto slides, fixed, permeabilized, and stained with an anti-B19V capsid antibody (clone MAB860-55D). Confocal images were taken under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. ( B to D ) Kinetics of the in vitro interaction between AXL and VP1u using Ni-NTA biosensors. The binding kinetics depicts the associations and dissociations of 4 μM rAXL EC with GST-VP1u at the indicated concentrations (B). Binding kinetics of 4 μM AXL His with 4 and 8 μM GST was used as a control (C). The binding parameters of AXL and GST-VP1u are shown (D). Equilibrium dissociation constant K D value represents the ratio of dissociation [ K d (1/s)] and association [ K a (1/Ms)] computed from the real-time binding curves of rAXL EC to GST-VP1u. The values are shown with means ± SDs based on at least three repeated experiments.

Article Snippet: The fixed cells were then incubated with an anti-B19V capsid antibody [#MAB8292/clone 521-5D ( ) or #MAb860-55D ( , )] to detect intact B19V virions or costained with anti-AXL antibody (#AF154, R&D Systems).

Techniques: Infection, Staining, Microscopy, In Vitro, Binding Assay, Control

Journal: Vaccines

Article Title: Rational Design of a Chimpanzee Adenoviral-Vector Vaccine Against Yellow Fever Through the Modification of Antigen Transmembrane Domains

doi: 10.3390/vaccines14030273

Figure Lengend Snippet: Vaccine design and antigen expression: ( a ) A schematic representation of the yellow fever antigen designs on the vaccines used. The yellow box represents the tPa signal sequence, the black box the transmembrane domain, and the blue box the anchored-membrane envelope glycoprotein. Based on the YF polyprotein strain 17D; prME is amino acids 121 to 778; PrMEΔTM is amino acids 121 to 731; E is amino acids 286 to 778; EΔTM is amino acids 286 to 731). ( b ) YFV envelope antigen expression in HEK-293A cells according to Western blots. The antigen was detected using an anti-YFV-E polyclonal antibody in both cell lysates and immunoprecipitated supernatants. Red arrows corresponds to YFV envelope protein. Black arrow corresponds to DBP, a viral protein, used as a ChAdOx1 infection control. Representative of three independent experiments. From left to right: M, marker; prME, ChAdOx1 prME; prMEΔTM, ChAdOx1 prMEΔTM; E, ChAdOx1 E; EΔTM, ChAdOx1 EΔTM; GFP, ChAdOx1 GFP; NI, not infected. The full blot is included in , and the density reading and intensity ratios are in . ( c ) Confocal immunofluorescence microscopy of infected T-Rex™-293 cells expressing the four YFV-E antigens to assess the cellular distribution ( top ). These are representative confocal images where the fluorescence signal of the envelop E proteins is shown in green, cis Golgi (GM130) in red, and the nucleus (DAPI) is shown in blue. The colocalization of the YFV envelope protein with Golgi ( bottom ) is shown in white. The scale bar represents 5 μm. The graph represents the fraction of viral protein signal associated with the Golgi, calculated as the integrated viral protein fluorescence intensity within the Golgi ROI divided by the total cellular viral protein intensity. Bars indicate mean ± SD (n = 4 images per condition in which 3 to 5 ROIs were analyzed). Data were analyzed with an ordinary one-way ANOVA with Tukey’s multiple-comparison test (ChAdOx1-YFprMEΔTM vs. ChAdOx1-YFE mean difference= 0.597, 95%CI [0.1812, 0.8569], ** p = 0.0031; ChAdOx1-YFE vs. ChAdOx1-YFEΔTM mean difference = −0.3385, 95%CI [−0.6757, −0.001387], * p = 0.0490).

Article Snippet: Blots were blocked with Pierce Protein Free T20 (Thermo Fisher Scientific, UK) Blocking Buffer and incubated with either the Yellow Fever Virus Envelope Protein (strain 17D vaccine) Recombinant Rabbit Monoclonal Antibody (Invitrogen, UK) or the Rabbit anti-Human adenovirus C serotype 5 (HAdV-5) (Human adenovirus 5) DBP Polyclonal antibody (Cusabio, Houston, TX, USA), followed by incubation with an anti-rabbit HRP-conjugated antibody (Agiletn technologies, Cheshire, UK).

Techniques: Expressing, Vaccines, Sequencing, Membrane, Western Blot, Immunoprecipitation, Infection, Control, Marker, Immunofluorescence, Microscopy, Fluorescence, Comparison