Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Vaccinated antisera blocks RBD-ACE-2 interaction. (A) 3-fold serial diluted antisera was added to micro-titer plates coated with recombinant RBD protein. After 30 min incubation, FLAG-tagged ACE-2 protein were added and detected with anti-FLAG antibody. Pooled pre-immune serum of each group was used as a control and theamount of bound ACE-2 determined. Each colored line represents serum of an indvidual mouse with pooled pre-immune serum in grey. (B) Reciprocal Inhibitory Dose 50 (IC50) was calculated for sera of each animals. Comparison of IC50 values between the two adjuvants is shown. (*) indicates p < 0.05 in unpaired T-test.

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Recombinant, Incubation

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Vaccination-induced neutralizing antibodies (nAb) protects from SARS-CoV-2 infection. Pre-immune or post-vaccinated mice sera were evaluated for their ability to neutralize SARS-CoV-2 pseudoparticles (CoV2pp) (A) or infectious SARs-CoV-2 virions (B). (A) Neutralization CoV2pp was performed using pre- and post- vaccination sera (1:50) in 293 T ACE-2 cells. The group means (in triplicate) with SEM were plotted and virus/particle entry normalized to entry of CoV2pp in the presence of PBS as 100%. (B) Neutralization titer (N100) of vaccinated mouse sera was determined in Vero E6 cells using infectious SARS-CoV-2. Serially diluted sera were pre-incubated with 100 PFU of SARS-CoV-2 for 1 h at 37 °C followed by addition to Vero E6 cells. Three days post-infection, cells were formaldehyde fixed and stained with crystal violet. Neutralization titer was determined as the minimal dilution of each mouse serum required to prevent CPE. A representative of two independent experiments (performed in duplicate) is shown. (*) indicates p < 0.05 in Tukey’s multiple comparisons test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Infection, Neutralization, Incubation, Staining

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Antisera exhibits similar neutralization activity against SARS-CoV-2 pp containing variant mutations N501Y/E484K/K417N. Neutralization activity of mouse sera were tested at 1:50 to 1:109,350 in 3-fold dilution against lentivirus particles pseudotyped with either spike of SARS-CoV-2(WT) or S encoding triple mutations N501Y/E484K/K417N. Pre- or Post-vaccination sera were pre-incubated with pseudoparticles (PP) encoding different surface proteins for 1 h followed by addition to 293 T ACE-2 cells. 48 h post-transduction, entry of PP were quantitated by luciferase activity andresults (in duplicate) normalized to entry of PP in the presence of PBS. IC50 of neutralization activity was calculated and compared between groups. (A) Mean and standard error of IC50 values is shown. (*) indicates p < 0.05 in Tukey’s multiple comparisons test. (B) Change in neutralization (IC50) between WT and variant (501/484/417) PP. A representative of two independent experiments (performed in duplicate) is shown with fold change of the mean IC50 value compared to WT.

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Neutralization, Activity Assay, Variant Assay, Incubation, Transduction, Luciferase

Journal: Vaccine

Article Title: SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1

doi: 10.1016/j.vaccine.2021.08.081

Figure Lengend Snippet: Antisera from RBD SARS-CoV-2 vaccinated mice exhibits cross-neutralization against SARS-CoV-1. RBD is either formulated with Alum + MPLA (left) or with AddaS03 (right). Neutralization activity of mouse sera were tested at 1:100 against pseudotyped virus particles (PP) expressing either SARS-CoV-2 spike (CoV-2), SARS-CoV-1 spike (CoV-1) or the glycoprotein of VSV (VSV). Pre- or post-vaccination sera were pre-incubated with PP and added to 293 T ACE-2 cells and assessed for luciferase activity according to the Materials and Methods. Triplicate samples were normalized to entry of Pre-immune sera and the mean with SEM plotted. (*) indicates p < 0.05 in Tukey's multiple comparison test. A representative of two independent experiments done in triplicates is shown.

Article Snippet: Full length human ACE-2-MycDDK in pCMV-6 entry vector (Origene, cat #RC208442) was expressed in Expi293™ cells.

Techniques: Neutralization, Activity Assay, Expressing, Incubation, Luciferase