Neil, Stuart John Douglas; (2001) Lentiviral-mediated gene delivery to human antigen presenting cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Viral vectors based on lentiviruses exhibit distinct advantages over other retroviral vectors because of their ability to transduce non-dividing cells. The use of lentiviral vectors for gene delivery of antigens and immuno-modulatory factors to human antigen presenting cells will allow initiation and modulation of clinically relevant immune responses. To this end we have explored the use of vectors based on the human immunodeficiency virus type 1 (HIV-1) to transduce human macrophages and dendritic cells. Human peripheral blood monocytes were susceptible to HIV vector transduction only after maturation into macrophages following 5 days culture. This maturation-dependence of infection was observed with vectors carrying HIV-1 accessory proteins and with transgene expression driven by 4 different promoters. Analysis of reverse transcription in freshly isolated monocytes and differentiated macrophages infected with HIV-based vectors showed that levels of viral DNA synthesis were equivalent. However nuclear viral DNA could only be detected in differentiated macrophages. Moreover, wild-type HIV-1 virions carrying the envelope of VSV-G also exhibited this pattern of differentiation-dependent transduction. Taken together these results demonstrate a differentiation-regulated restriction to HIV-1 in primary human monocytes. However, monocytes differentiated into dendritic cells in the presence of the cytokines IL-4 and GMCSF were susceptible to transduction at all stages of culture. Infection in freshly isolated monocytes could be rescued by subsequent dendritic cell differentiation, an effect that was shown to be due to culture of the cells in foetal calf serum. These results are of importance in the design of protocols for the infection of human antigen presenting cells by lentiviral vectors. They also provide evidence for a post-entry block to HIV-1 infection in freshly isolated human monocytes.

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