Closing the manufacturing process of dendritic cell vaccines transduced with adenovirus vectors

Differential Effects of Viral Vectors on Migratory Afferent Lymph Dendritic CellsIn VitroPredict Enhanced ImmunogenicityIn Vivo

“…However, these models have limitations, especially in terms of relevance in vaccination protocols which rely on the delivery of antigen parentally. Another drawback for using MoDC is that these are poorly transduced by one of the most promising recombinant vaccine vectors, human replication-deficient recombinant adenovirus 5 (rhuAdV5) (6,23,39).…”

Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use

“…The infection process by Adv for hematopoietic cells is dependent upon 3 parameters: time, VP number and concentration, and to an extent, DC concentration [14]. Prior studies to optimize infection parameters revealed that DCs were optimally transduced using a concentration of 22,500:1 VP:DC and DCs cultured at 5 × 10 6 cells/ ml [12]. Following a 3-hour co-culture, the infected cells were washed and concentrated using an Eppendorf 5810R centrifuge (Eppendorf, Westbury, NY) and/or a Cobe 2991 (Gambro BCT, Lakewood, CO), depending upon volume.…”

“…The concentrated cells were then transferred to 72 ml bags (2PF-0072, Afc) and centrifuged at 18°C for 10 min at 290 ×g. As previously described, a Fenwal Plasma Extractor (Fenwal, Inc., Lake Zurich, IL) was used to remove the supernatant [12]. Reflux of expressed supernatant was closely monitored and the 2PF-0072 bag drained of as much media and virus as possible.…”

“…Following informed consent, healthy donors (males or females, ages 19-70) underwent mononuclear cell leukapheresis on a Cobe Spectra (Gambro BCT, Lakewood, CO) apheresis cell separator. PB monocytes were enriched directly from the leukapheresis products using the Elutra® (CaridianBCT, Lakewood, CO) cell separation system semi-automated method [12,13]. All manufacturing procedures were performed in a class-10,000 clean room.…”

“…All manufacturing procedures were performed in a class-10,000 clean room. DCs were differentiated from monocytes using a closed system with CellGenix DC media (CellGenix, Antioch, IL) with recombinant human cytokines (IL-4, CellGenix, Antioch, IL) and GM-CSF (Leukine, Berlex, Seattle, WA) in VueLife® cell culture bags, (American Fluoroseal Corporation, Afc, Gaithersburg, MD) for use as a therapeutic TC-DC product [12].…”