sad b19 g  (Addgene inc)

Journal: Cell Reports

Article Title: Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups

doi: 10.1016/j.celrep.2020.107920

Figure Lengend Snippet: Structure-Based Design of Chimeric Lyssavirus Glycoproteins (A–C) Structural models of lyssavirus glycoprotein (G) ectodomain monomers. (A) Representative structural model of a lyssavirus G with proposed structural domains highlighted (clip in yellow, core in orange, flap in red). Structural model of Chimeric G 1 (B) and Chimeric G 2 (C) clip domains highlighted in white, and core and flap domains highlighted in blue or red, corresponding to patterns in (E) and (F). (D–F) Linear schematics of a representative lyssavirus G monomer (D) and the RABV G/MOKV G chimeric Gs named Chimeric G 1 (E) and Chimeric G 2 (F). In (D), proposed structural domains of the ectodomain are noted (clip in yellow, core in orange, flap in red), as are the antigenic regions as they are known to exist on the RABV G: site I (residues 224–229), site II (34–42 and 198–200), site III (330–338), site IV (263–264), and minor site “a” (342–343). R333E, attenuating mutation at RABV G residue 333; TM, transmembrane domain. See also and .

Article Snippet: To make the rRABV vector, we inserted a human codon-optimized RABV G (SAD B19 strain with R333E mutation, synthesized by Genscript USA) into the BNSPΔG vector ( ) using the BsiWI and NheI restriction sites.

Techniques: Mutagenesis

Journal: Cell Reports

Article Title: Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups

doi: 10.1016/j.celrep.2020.107920

Figure Lengend Snippet: Construction and Recovery of a Chimeric Lyssavirus G Vaccine (A) Viral genome schematics. BNSP333 is the parent vaccine vector based on RABV strain SAD B19. Its G is located in the native fourth position and contains the attenuating R333E mutation. BNSPΔG is based on BNSP333 but lacks the native G. All of the following experimental constructs are based on BNSPΔG: rRABV contains a human codon-optimized (c.o.) RABV G with the attenuating mutation R333E at the second position; rMOKV contains human c.o. MOKV G at the second position; rChimera1 (LyssaVax) contains Chimeric G 1, with the attenuating R333E mutation, at the second position; and rChimera2 contains Chimeric G 2 at the second position. (B) Infection immunofluorescence. VERO cells infected with either LyssaVax (left column), rMOKV (second column), rRABV (third column), or uninfected (right column) were fixed and stained with a DyLight 488-conjugated human anti-RABV G mAb 4C12 (green) and mouse anti-MOKV G sera (red). Nuclei labeled in blue by DAPI. Scale bars represent 50 μm. (C) Analysis of purified virions. 3-μg viral particles denatured and resolved by SDS-PAGE, then total protein stained with SYPRO Ruby. Viral proteins are indicated.

Article Snippet: To make the rRABV vector, we inserted a human codon-optimized RABV G (SAD B19 strain with R333E mutation, synthesized by Genscript USA) into the BNSPΔG vector ( ) using the BsiWI and NheI restriction sites.

Techniques: Plasmid Preparation, Mutagenesis, Construct, Infection, Immunofluorescence, Staining, Labeling, Purification, SDS Page

Journal: Cell Reports

Article Title: Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups

doi: 10.1016/j.celrep.2020.107920

Figure Lengend Snippet: Humoral Response to LyssaVax (A) Schematic timeline of immunization (green syringe), sera collection (red drop), and challenge (orange bolt) through necropsy (NEC). (B and C) Development of antibodies over time in groups of mice (n = 10 mice per group, analyzed in triplicate, mean ± SD) immunized three times with either LyssaVax (purple), rRABV (blue), or rMOKV (red). Graphs compare half-maximal responses (EC 50 s) between sera from immune mice probed against RABV G (B) or MOKV G (C) antigens in ELISA format. Day 0 samples did not seroconvert, so EC 50 values were not calculated. Analysis within time points by Kruskal-Wallis test and Dunn’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for adjusted p values; n.s. is not significant). See also Figure S5 .

Article Snippet: To make the rRABV vector, we inserted a human codon-optimized RABV G (SAD B19 strain with R333E mutation, synthesized by Genscript USA) into the BNSPΔG vector ( ) using the BsiWI and NheI restriction sites.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell Reports

Article Title: Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups

doi: 10.1016/j.celrep.2020.107920

Figure Lengend Snippet:

Article Snippet: To make the rRABV vector, we inserted a human codon-optimized RABV G (SAD B19 strain with R333E mutation, synthesized by Genscript USA) into the BNSPΔG vector ( ) using the BsiWI and NheI restriction sites.

Techniques: Recombinant, Staining, Plasmid Preparation, Cell Culture, Lysis, BIA-KA, Luciferase, Software, Clone Assay, Transfection