Autologous dendritic cells (DCs) are generated from isolated monocytes. DC maturation is induced and cells are exposed to autologous HIV-1 antigen RNAs with CD40L RNA via electroporation.
RNA is extracted and amplified from pre-antiretroviral therapy (ART) plasma samples to generate HIV Gag, Rev, Vpr and Nef antigens. CD40L RNA is transcribed in vitro from a CD40L-encoding plasmid using a co-transcriptional capping method. The purified, sterile, endotoxin-free HIV antigen RNAs and polyadenylated CD40L RNA are formulated at a ratio of 0.25:1:1:1:4 μg for Nef, Gag, Rev, Vpr and CD40L in vitro transcribed RNAs, respectively.
Peripheral blood mononuclear cells (PBMCs) are obtained from each subject by standard leukapheresis. PBMCs are isolated using Ficoll density gradient centrifugation. Monocytes are isolated from PBMCs, through adherence to tissue culture plastic, and cultured for six days with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to generate immature DCs. On day 6, DC maturation is initiated by overnight culture with TNF-α, IFN-γ, and PGE2.
CD40L RNA and autologous HIV-1 antigen RNA encoding Nef, Vpr, Gag, and Rev are electroporated into autologous DCs on day 7.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
