jev signal peptide sequence  (Moderna)

Journal: Viruses

Article Title: mRNA Vaccine Against Japanese Encephalitis Virus Genotype IV Protects Against Lethal Infection

doi: 10.3390/v18020171

Figure Lengend Snippet: mRNA vaccine construction. ( a ) Schematics illustrating design of the JEV NSW2022 GIV vaccine mRNA constructs. Two mRNA vaccine constructs were generated, one with the Native signal peptide sequence, and one with a Shorter peptide sequence, which is based on a Moderna ZIKV mRNA vaccine. UTR HBB—untranslated regions of human beta globin. (For full sequence details see ). ( b ) Final purified mRNA species for the two vaccines analyzed by capillary electrophoresis. Sizes by nucleotide (nt) length illustrated by electropherograms and simulated gel images. ( c ) HEK293 cells were transfected with the vaccine mRNA species and after 24 h cells and supernatants were harvested. Cells and proteins precipitated from supernatants were loaded onto 3 parallel SDS-PAGE gels (35 µg protein per lane). Left—Western blotting using anti-envelope (E) monoclonal antibody, 4G2; blue dotted line—lining up markers (KDa) with E. Middle—SDS-PAGE gels stained with Coomassie blue (red marker is 70 KDa). Right—Western blotting using anti-GAPDH (housekeeping protein, expected molecular weight ≈ 37 KDa). Text labeling of the lanes is color coded to facilitate comparisons. ( d ) Final LNP vaccines with encapsulated mRNA or empty (Control). The mean diameter (Z-average) and polydispersity index (PdI) are provided for each vaccine.

Article Snippet: Herein, we evaluated two signal peptide sequences, the full-length 23 amino acid (a.a.) Native signal peptide sequence from JEV NSW2022 , and a Shorter 16 a.a. sequence from JEV NSW2022, with the latter based on a JEV signal peptide sequence used for a ZIKV mRNA vaccine (Moderna, mRNA-1893) [ , ] ( a and ).

Techniques: Construct, Generated, Sequencing, Purification, Vaccines, Electrophoresis, Transfection, SDS Page, Western Blot, Staining, Marker, Molecular Weight, Labeling, Control

Journal: Viruses

Article Title: mRNA Vaccine Against Japanese Encephalitis Virus Genotype IV Protects Against Lethal Infection

doi: 10.3390/v18020171

Figure Lengend Snippet: Mouse experiments. ( a ) Time line of vaccination, challenge, weight change, disease monitoring, and sample collection. ( b ) End point ELISA titers after the first vaccination (serum collected week 3) and after the second vaccination (serum collected week 8), with the indicated mRNA doses. Limit of detection 1 in 30 dilution; ND—not detected. Statistics by Mann–Whitney U tests. ( c ) Reciprocal 50% neutralization titers for the same serum samples as those described in ( b ), plus Imojev included as a positive control. Neutralization of JEV NSW2022 (left), with neutralization of JEV Nakayama (GIII isolate) (right) also shown for serum samples from mice receiving two doses of 5 µg. Limit of detection 1 in 20 (left) or 1 in 10 (right) serum dilution; ND—not detected. Statistics by Kolmogorov–Smirnov exact tests. ( d ) Percent body-weight change post-challenge for the mice described in c. Lines represent mean body weigh change, except for three mice which were plotted individually ( n = 1). These were the one out of five mice that survived from the 1 µg Shorter group (blue line) and the 1 µg Native group (red line); and the one mouse that did not survive in the 5 µg Shorter group (purple line). † indicates mice reached the clinically defined end point for euthanasia. ( e ) Kaplan–Meier plot of survival for the mice shown in d. Statistics by log rank statistic; compared with LNP control p < 0.023 for the 1 µg groups, and p = 0.005 for the 5 µg groups and Imojev. ( f ) Mean viremia post-challenge for the mice shown in ( e ) (legend as in ( e )). For the LNP Control group on day 3, n = 2. Dotted line—limit of detection for individual mice ≈ 2 log 10 CCID 50 /mL; ND—not detected. Statistics by Kolmogorov–Smirnov exact tests for day 2 relative to LNP control; for all groups p = 0.008.

Article Snippet: Herein, we evaluated two signal peptide sequences, the full-length 23 amino acid (a.a.) Native signal peptide sequence from JEV NSW2022 , and a Shorter 16 a.a. sequence from JEV NSW2022, with the latter based on a JEV signal peptide sequence used for a ZIKV mRNA vaccine (Moderna, mRNA-1893) [ , ] ( a and ).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Neutralization, Positive Control, Control