TABLE 7.
Manufacturing process for a vaccine containing autologous DCs loaded with ApB of inactivated endogenous HIV-1
| Step | Comment |
|---|---|
| Find HIV-1+ subjects not on ART therapy | |
| Isolate endogenous virus from the subject's PBMCs | p24 titer of CD4+ CD8− T-cell supernatants of ≥25 ng/ml |
| Freeze virus-positive supernatants | |
| Proceed with leukapheresis of the subject | |
| Separate and recover monocytes and T cells by using the Elutra system | |
| Separate CD4+ T cells by negative selection on immunobeads (CliniMACS) and cryopreserve CD4+ T cells | |
| Generate iDCs in the Aastrom Replicell closed system | Yield, 2 × 108 DCs |
| Characterize iDCs, aliquot the iDCs, and cryopreserve the iDCs for vaccine formulation pending superinfection of autologous CD4+ T cells (4 to 10 days) | |
| Superinfect autologous CD4+ CD8− T cells with the viral supernatants | 10 to 20 ml per 5 × 107 T cells |
| Inactivate virus in CD4+ T cells with UVB light and psoralen (30 min) | |
| Coincubate thawed iDCs with ApBs of T cells for 12 to 24 h | Ratio, 2 DCs to 1 CD4+ CD8− T cell or 4 × 107 DCs to 2 × 107 T cells |
| Determine sterility, p24 titer, DC phenotype, and viability | Sterile, endotoxin and mycoplasma negative, >70% purity, >80% viability |
| Establish vaccine release criteria |