CD8⁺ T cell immunogenicity of Nanopatch-delivered viral vector vaccines in prime boost schedules.

<p>Mice (n = 5/6) were primed with 5×10⁷ PFU MVA.PbCSP (no TH or SC) (A) or 5×10⁹ VP ChAd63.ME-TRAP +10% ^w/_v TH+SC (B), either by coated Nanopatch or ID injection. Two weeks post-MVA.PbCSP priming, a boost immunisation of MVA.PbCSP was given and 8 weeks after ChAd63.METRAP a boost immunisation of MVA.ME-TRAP (no TH or SC) was given (dose 5×10⁷ PFU, given either ID or by Nanopatch). One week post-MVA (A) or 3 weeks post-ChAd63 (B) or 2 weeks post-boost (C+D), blood was taken for analysis of Pb9-specific IFN-γ secreting cells. SFC = spot forming cells. PBMC = peripheral blood mononuclear cells.</p

Imaging of skin penetration by Nanopatch microprojections.

<p>(A) The size of a single Nanopatch relative to a forefinger. (B+C) SEM images of microprojection morphology after dry-etch fabrication. (D) Representative cryo-SEM images of the mouse ear skin surface following application of a single Nanopatch. D(i) shows a far field view of the corner of the patched area, with micro-channel openings characteristic of microprojection penetration, adjacent to unbroken skin. D(ii) shows perforated area in higher magnification, with a single micro-channel next to a hair follicle inset. Scale bar inset = 10 µm. (E) Representative micrographs of ear tissue sections following delivery of Nanopatch coated with a fluorescent dye. BF: brightfield image, F: fluorescence image, BF+F: both brightfield and fluorescent images overlaid. SC = <i>stratum corneum</i>; VE = viable epidermis; D = dermis.</p

Viral viability throughout formulation and during Nanopatch dry-coating.

<p>(A+B) ChAd63.ME-TRAP (1×10⁹ VP) and MVA.GFP (1×10⁷ PFU) were mixed with combinations of MC and PS20, with or or without the disaccharides TH+SC (10% ^w/_v each sugar). Formulations were added to DF-1 cell monolayers (A; MVA, n = 4) or HEK-293A cells (B; ChAd63, n = 5) to evaluate viral titre, which was compared to unformulated virus. (C+D) Formulations containing ChAd63.ME-TRAP (C; 1×10⁹ VP) and MVA.GFP (D; 1×10⁷ PFU) were coated onto Nanopatch and immediately eluted into D-MEM. Eluates (n = 4/5) were added to cell monolayers in infectivity assays as before. Eluted viral titres were compared against unformulated, liquid virus. Negative control wells contained D-MEM only. NS = not significant. nd = no data. IFU = infectious units, PFU = plaque forming units.</p