Figure 3.

Capsid display of DogCatcher-NANP18 ligand shields the particles from anti-vector neutralizing antibodies and elicits potent ligand-specific humoral immunity

(A) SDS-PAGE and Coomassie staining analysis of Ad virions displaying DogTag at HVR5, (1E+10 viral particles) incubated with DogCatcher-NANP9 (5 μM) or DogCatcher-NANP18 (5 μM) at 4°C for 16 h. (B) Vector infectivity (GFP focus assay) performed in 293A cells on the samples from (A). Data show mean +SD of triplicate wells. (C) Vector neutralization assay using anti-hexon mAb 9C12. Ad particles encoding a GFP transgene with or without a capsid ligand were added to 293A cells in the presence of a varying concentration of neutralizing mAb. Vector transduction was measured via fluorescence of encoded GFP expressed in the cells. (D) Vector neutralization assay using anti-Ad5 serum. Ad particles encoding a GFP transgene with or without a capsid ligand were added to 293A cells in the presence of a varying concentration of serum from mice immunized with Ad5. (C) and (D) show mean + range of duplicate wells. (E) BALB/c mice (five per group) were immunized intramuscularly as described (vector-encoded antigens in brackets). Note that Ad(C-NANP18) has an unmodified hexon (no DogTag). The DogCatcher-NANP18 protein dose in group 2 was calculated to be < 0.05 μg per mouse. (F) Serum IgG antibody responses to NANP18 in groups 2–5 measured by endpoint ELISA 14 days post immunization. (G) CD8⁺ T cell responses in the spleen to encoded epitope EGFP_200-208 were measured in groups 1 and 2 by overnight ex vivo IFNγ-ELISpot 14 days post immunization. SFC, spot forming cells. (H) Serum IgG antibody responses to encoded GFP in groups 1 and 2 were measured by endpoint ELISA 14 days post immunization. In (F)–(H), bars show median responses. Dashed line represents limit of detection.