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. 2020 Mar 12;12(5):e10938. doi: 10.15252/emmm.201910938

Figure EV1. Serum antibody responses against the AAV vector capsid and PR8‐specific IgA.

Figure EV1

  1. Total IgG ELISA titer induction over vaccination period against AAV9‐vector capsids in pooled sera (AAV‐HA, ‐cHA, ‐GFP, WIV n = 18; AAV‐NP n = 11; AAV‐HL, ‐mHL1, ‐mHL2 n = 7 mice per group) at indicated time points.
  2. AAV9‐vector‐neutralizing antibody titers in pooled pre‐challenge sera (AAV‐HA, ‐cHA, ‐GFP, WIV n = 18; AAV‐NP n = 11; AAV‐HL, ‐mHL1, ‐mHL2 n = 7 mice per group, technical duplicates). Dotted line indicates the limit of detection (LOD) at a dilution of 1:100. Mean ± SD.
  3. Regression analysis of total AAV9‐vector IgG ELISA titers and MN50 titers. Coefficient of correlation (r 2) and P‐value are shown.
  4. Total IgG ELISA titers expressed as AUC against homologous Cal/7/9 virus in pre‐challenge serum pools (AAV‐HA, ‐cHA, ‐GFP, WIV n = 18; AAV‐NP n = 11; AAV‐HL, ‐mHL1, ‐mHL2 n = 7 mice per group, technical duplicates) of the indicated vaccine groups. Mean ± SD.
  5. Immunofluorescence microscopy staining of MDCKII cells 48 h after transfection with pAAV plasmids expressing the indicated constructs. Cells were fixed, permeabilized, and stained with the indicated mouse pre‐challenge serum pools or with anti‐V5‐tag antibody groups (n = 2, technical duplicates) (bar, 50 μm).
  6. Immunoblot analysis with lysates obtained from 293T 48 h after transfection with pAAV plasmids expressing the indicated constructs. Immunoblot was performed with the indicated pre‐challenge mouse serum pools (top, AAV‐HA n = 18, AAV‐HL, ‐mHL1, ‐mHL2 n = 7 mice per group). Hereafter, membranes were stripped and stained with anti‐V5‐tag antibody (bottom). Position of detected protein bands is indicated to the right (bottom).
  7. Immunofluorescence microscopy staining of MCDKII cells 24 h after transfection with wild‐type Cal/7/9 NP or HA and mHL1 plasmids with pre‐challenge serum from AAV‐NP or AAV‐mHL1 + NP immunization groups (n = 2, technical duplicates) (bar, 50 μm).
  8. IgA ELISA titers against Cal/7/9 or PR8 virus in pooled pre‐challenge sera (AAV‐HA, ‐cHA, ‐GFP, WIV n = 18; AAV‐NP n = 11 mice per group, technical duplicates). Mean ± SD.
  9. IgA ELISA titers in post‐challenge lung homogenates of individual mice of the Cal/7/9 and PR8 low‐dose challenge groups of the indicated vaccine groups against PR8. Statistical significance between vaccine groups was determined using Kruskal–Wallis test with Dunn's multiple comparison testing (**P < 0.01, ***P < 0.001). Lines indicate mean. ELISAs were done in technical duplicates.

Source data are available online for this figure.