Novel antigen panel for modern broad-spectrum recombinant rotavirus A vaccine

Figures

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  Fig. 1. (A) Cryo-electron microscopy reconstruction of rotavirus particle and high-resolution structure of outer and middle layers (Protein Data Bank [PDB]: 4V7Q [32333435]). (B) High-resolution structures of VP4 and VP7 trimers showing the location of fragments ΔVP8* (65–223 amino acid [aa]), ΔVP5* (247–478 aa) and location of epitopes ep8, ep5, ep7 (PDB: 4V7Q [323435]). (C) Linear diagrams of VP4 and VP7 showing color-coded fragments and epitopes (not to scale), and linear diagram of recombinant antigen ep-875 (not to scale). VP4 fragment (232–247 aa) removed during VP4 in vivo proteolysis is highlighted in white.

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  Fig. 2. Characterization of rotavirus A (RVA) recombinant antigens. Lanes: VP8*-[P8] (1), VP8*-[P6] (2), VP8*-[P4] (3), ep-875 (4), VP5* (5). (A) Electrophoresis analysis, 8%–20% SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis), staining with Coomassie G-250. (B) Western blot membrane stained by Ponceau S. (C) Western blot analysis with commercial polyclonal antisera to 5 RVA strains: Wa-G1P[8], Ds-1 G2P[4], Ito-G3P[8], HOCHI-G4P[8], 69M-G8P[10] (Cat# MBS316568; MyBioSource Inc., San Diego, CA, USA) as primary antibodies and secondary anti-species antibodies conjugated with horseradish peroxidase. Positions of molecular weights (kDa) markers are indicated in the right.

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  Fig. 3. Phylogenetic tree based on the amino acid sequences of the VP8* protein fragment (residues 65–223) of human rotavirus A isolates representing genotypes P[4], P[6], and P[8]. Synthetic consensus sequences are shown in bold. The tree was reconstructed using the PhyML 3.0 version of maximum likelihood algorithm [28]. Bootstrap values (>50%, 1,000 replicates) are displayed next to the corresponding nodes. GenBank accession numbers of the previously characterized isolates are indicated on the right of the tree with the exception of Hochi (BAF80174), Ito (BAA77545), Wa (AGJ72856), Rotarix (JN849113), RotaTeq (GU565044), YO (AB008279), L26 (M58292), RV5 (M32559), DS1 (AEG25325), AU19 (AB770153), M37 (L20877), ST3 (L33895), 1076 (M88480), 69M (AAA47336), and AU1 (D10970). The scale bar indicates the number of substitutions per residue.