Production of a dendritic cell-based vaccine containing inactivated autologous virus for therapy of patients with chronic human immunodeficiency virus type 1 infection

Abstract

In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8(+) and CD4(+) T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4(+) T cells with the virus; (iii) inactivation of the virus in CD4(+) T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4(+) T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4(+) T cells. CD4(+) T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID(50); which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4(+) T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID(50) of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 microg/ml) and UVB irradiation (312 nm) reduced the TCID(50) of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4(+) T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).

Figures

FIG. 1.

Inactivation of cell-associated HIV IIIb by UVB (312-nm) irradiation in the presence of psoralen (20 μg/ml). Infected CD4+ T cells were treated with UVB light and psoralen for various periods of time. Following inactivation, the infectivity of the virus was determined in TCID50 assays.

FIG. 2.

Effects of psoralen and UVB light treatment on CD4+ T-cell viability: 30 min of treatment is sufficient to induce apoptosis and necrosis (annexin V and 7-AAD positivity) in >75% of the cells. Annexin V and 7-AAD staining were performed 4 h after completing irradiation.

FIG. 3.

DCs loaded with endogenous virus-infected, inactivated CD4+ T cells. (A) Confocal microscopy image illustrating the uptake of DiOC6-labeled ApBs by autologous iDCs surface stained with fluorescein isothiocyanate-labeled anti-CD11c Ab; (B) flow cytometry analysis of the uptake of DiOC6-labeled ApBs after 12 h of their coculture with iDCs.