Fig. 2.

Both pLACK and sLACK formulated MPER/liposome vaccines efficiently deliver LACK peptides to antigen presenting cells in vivo. (A) Schematic representation of various incorporation methods for LACK into MPER/liposome: (1) “post-extrusion”, sLACK added in extruded MPER/liposome solution, (2) “pLACK”, surface associated palmitoylated LACK to MPER/liposome, (3) “sLACK”, free and encapsulated sLACK in MPER/liposome solution utilized to test the delivery efficiency to APCs following immunization. (B) In vivo quantification of LACK:I-A^d complexes on the DCs by 2C44 monoclonal antibody 1 day post-immunization. Gating strategy (left) and the frequency of LACK⁺ DCs (CD19⁻ CD11c⁺) in draining iLN was determined by flow cytometry analysis using 2C44 (right) to compare the efficacy of different LACK formulation methods (1–3 above; green, blue and red, respectively) on the LACK delivery to DCs. (C and D) Upregulation of MHCII (C) and CD86 (D) was quantified from the geometric mean fluorescent intensity (gMFI) of surface expression (representative histograms in left panels) for LACK⁺ DCs and LACK⁻ DCs as determined in (B) and graphed in the right panels (open bars and filled inlay, respectively). (E) IL-2 secretion by LACK-specific T cell hybridomas upon incubation with B cells (left) or the DC containing non-B cell fraction (right) in post-immunization ex vivo cultures. Purified B cells and the remaining non-B cells from draining iLN of 1 day immunized mice were cultured with LACK-specific T cell hybridoma cells overnight, and supernatant IL-2 was quantified by ELISA. Points represent mean with error bars denoting SEM. Results are representative of two independent experiments, each with at least four mice per group. (ns) = not significant by the unpaired student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)