Journal: Vaccines

Article Title: A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

doi: 10.3390/vaccines9050431

Figure Lengend Snippet: Augmented antibody titer, viral neutralization, and lymph node retention of RBD-bann protein vaccine. RBD variants were isolated from the supernatants of mammalian cells via affinity chromatography and analyzed for purity and specificity via SDS-PAGE ( a ) and the binding of recombinant proteins at a serial dilution, or buffer alone (0), to immobilized human ACE2 receptor was measured in an ELISA assay ( b ). Dissociation of RBD and RBD-bann from immobilized hACE2 was measured by SPR ( c ). Mice were immunized with RBD and RBD-bann proteins purified from mammalian cell supernatant with/without squalene-mediated adjuvant. End point titer (EPT) for total IgG against RBD ( d – e ) and against spike protein ( f – g ). Graphs represent the mean EPTs of groups of mice ( n = 6 per group). Each dot represents an individual animal. * p < 0.05; ** p < 0.01. All p values are from the Mann–Whitney test compared to RBD group + AddaVax. Neutralization titer in mice sera was determined using a pseudovirus system. Sera of mice immunized with isolated proteins were diluted 50-fold, and Spike-pseudotyped virus infection of ACE2 and TMPRSS2-transfected HEK293 cells was followed by luminescence. Mean and SEM biological replicates are shown ( b ). * p < 0.05, *** p < 0.001, **** p < 0.0001. All p values are from one-way ANOVA followed by Tukey’s multiple comparisons test compared to the buffer group ( h ). A serum virus neutralization assay was performed by infecting Vero E6 cells with SARS-CoV-2-GFP reporter virus (MOI 1) with prior incubation of inoculum with dilutions of mouse sera (1:50) immunized with indicated isolated proteins. As control cells were left uninfected (mock), GFP signal was measured 36-h post-infection using the Incucyte S3 live-cell imaging system. Each point represents the mean of two technical replicates, and the mean +/− SD from six mice per group is indicated. Indicated p -values apply for comparison between individual conditions and the dilution-matched buffer condition and were calculated using a one-sided Student’s t -test between RBD + Addaax and RBD-bann + Addavax. * p < 0.05 ( i ). Trafficking of labeled isolated proteins into the popliteal lymph nodes (PLN). AF-647 labeled RBD proteins were injected into the foot pad (RBD left, RBD-bann right). The fluorescence signal of AF-647 was determined in the popliteal lymph node, depicting the presence of labeled RBD. Each dot represents the measurement of the region of interest (ROI) of an individual animal PLN. * p < 0.05 derived from two-tailed paired t -test ( j ).

Article Snippet: The normalized GFP intensity was computed using the IncuCyte S3 software (Essen Bioscience; version 2019B Rev2, Sartorius, Göttingen, Germany) as integrated GFP intensity per well divided by the total area of the cells per well.

Techniques: Neutralization, Isolation, Affinity Chromatography, SDS Page, Binding Assay, Recombinant, Serial Dilution, Enzyme-linked Immunosorbent Assay, Purification, Adjuvant, MANN-WHITNEY, Virus, Infection, Transfection, Incubation, Control, Live Cell Imaging, Comparison, Labeling, Injection, Fluorescence, Derivative Assay, Two Tailed Test