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