Polypropylene Sulfide Nanoparticle p24 Vaccine Promotes Dendritic Cell-Mediated Specific Immune Responses against HIV-1  Stephan M. Caucheteux, John P. Mitchell, Matthew O. Ivory, Sachiko Hirosue, Svetlana Hakobyan, Garry Dolton, Kristin Ladell, Kelly Miners, David A. Price, June Kan-Mitchell, Andrew K. Sewell, Frank Nestle, Arnaud Moris, Richard O. Karoo, James C. Birchall, Melody A. Swartz, Jeffrey A. Hubbel, Fabien P. Blanchet, Vincent Piguet  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1172-1181 (June 2016) DOI: 10.1016/j.jid.2016.01.033 Copyright © 2016 The Authors Terms and Conditions

Figure 1 p24NP rapid uptake by dendritic cells (DC) does not induce early maturation. (a) Monocyte-derived dendritic cells (MDDC) were incubated with 0.1 or 1 μg/ml p24NP for 24 hours and collected at the indicated time points up to 24 hours. Uptake of p24NP was measured by anti-p24 intracellular staining. MDDCs were incubated overnight with various concentrations of p24NP. CD83 and tumor necrosis factor (TNF)-α were stained and analyzed by flow cytometry. Unconjugated nanoparticles (NP) and 1 μg/ml lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Statistical analysis was performed with (b) MDDC and (d) dermal-like DC. Differences were not statistically significant. (c) Representative flow cytometric analysis of TNF-α staining with 0.5 μg/ml NP or p24NP, and 1 μg/ml LPS used. MFI, mean fluorescence intensity. Journal of Investigative Dermatology 2016 136, 1172-1181DOI: (10.1016/j.jid.2016.01.033) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Anti-p24 CD8-specific response to p24NP-loaded dendritic cells. p24 and p24NP-loaded autologous (a, c) monocyte-derived dendritic cells (MDDC) or (b, c) dermal-like dendritic cells were co-cultured overnight with TW10-specific CD8+ T-cell clones. IFN-γ production was measured by intracellular staining on CD3+ cells and analyzed by flow cytometry. NP, nanoparticle; PMA, phorbol myristyl acetate. *P < 0.05; **P < 0.01; ***P < 0.0001. Journal of Investigative Dermatology 2016 136, 1172-1181DOI: (10.1016/j.jid.2016.01.033) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Anti-p24 CD4-specific response to p24NP-loaded dendritic cells. (a, c) Monocyte-derived dendritic cells or (b, d) dermal-like dendritic cells were incubated with various concentrations of p24 or p24NP, and co-cultured with Ox97 CD4+ T-cell clones overnight. pOx97-specific peptide is used a positive control. IFN-γ production in CD3+ cells was measured by intracellular staining and analyzed by flow cytometry. NP, nanoparticle. Journal of Investigative Dermatology 2016 136, 1172-1181DOI: (10.1016/j.jid.2016.01.033) Copyright © 2016 The Authors Terms and Conditions

Figure 4 p24NP priming induces CD4 T-cell response to HIV-1 infection. CD4+ T cells were stimulated with p24/ p24NP-loaded dendritic cells for two rounds. (a, b) At the end of the second round, IFN-γ production was measured by flow cytometry after restimulation. (a) Statistical analysis. (b) Representative experiment. CD4+ T cells were immunized as in panel a with whole blood dendritic cells, then co-cultured with monocyte-derived Langerhans cells (MDLC) or monocyte-derived dendritic cells (MDDC) infected with HIV-1 pR9 for 6 days. (c) IFN-γ and (d) IL-17 production was tested after restimulation with phorbol myristyl acetate/ionomycin. *P < 0.05. Journal of Investigative Dermatology 2016 136, 1172-1181DOI: (10.1016/j.jid.2016.01.033) Copyright © 2016 The Authors Terms and Conditions

Figure 5 p24NP induces a strong antibody response. (a) CD4+ T cells were immunized as in Figure 4. and co-cultured with autologous B cells. After 12–14 days, supernatants were tested for antibody production by ELISA. Intradermal microneedles were coated with yellow fluorescent–coated nanobeads and inserted into skin explants for 60 seconds. Frozen sections were stained with CD207 anti-langerin antibody (red) and Hoechst 33342 for nucleus staining (blue). (b, c) Two representative images. Bar = 100 μm. Journal of Investigative Dermatology 2016 136, 1172-1181DOI: (10.1016/j.jid.2016.01.033) Copyright © 2016 The Authors Terms and Conditions

Figure 6 p24NP delivered into skin-resident dendritic cells stimulates CD4+ T cells. (a) Epidermal and dermal dendritic cells were purified from skin explants and treated with nanoparticles (NP), p24NP, or lipopolysaccharide (LPS) for 24 hours, and then tumor necrosis factor (TNF)-α secretion was measured by intracellular staining. (b) N12 CD4 T cells were then co-cultured with dermal or epidermal dendritic cells overnight in the presence of brefeldin A and stained for IFN-γ. (c) Corresponding data for the irrelevant peptide and N12-positive peptide control. Journal of Investigative Dermatology 2016 136, 1172-1181DOI: (10.1016/j.jid.2016.01.033) Copyright © 2016 The Authors Terms and Conditions