Authors:
Vladimir Lukashov, M Quinones-Mateu, G McGarrity, T Rebello, and L Humeau
Summary:
Background: In a phase II open-label clinical trial, 40 HIV-infected subjects were infused with their own CD4 T cells previously transduced ex vivo with VRX496, a lentiviral vector encoding for a 937 nt antisense against the env gene. VRX496 molecular pressures on the diversity and the fitness of the HIV-1 quasispecies pre- and post-infusions were investigated.
Methods: Plasma samples were collected at different time-points during the course of the study. Samples from 9 subjects were randomly included in this study. HIV-1 env sequences were amplified by real-time polymerase chain reaction (RT-PCR), cloned into expression vectors, sequenced for phylogenetic studies (5 of 9) and viral replicative fitness analyzed (6 of 9).
Results: Cloning and sequencing showed the presence of wild type HIV-1 virions with deleted envelopes (3 of 5) in the area targeted by the antisense. No deletion was found in any of the pre-dose samples. Quasispecies diversities were dramatically reduced post-infusion, by as much as 70%, and nucleotide changes in the antisense targeted area were from G to A (hypermutation) instead of the usual A to G in the non antisense targeted areas of the envelope. Growth competition experiments showed an overall reduction in viral replicative fitness in 5 of 6 subjects studied. Of interest, 1 subject exhibited 2 distinct strains of HIV-1 at time of enrollment. At 1 month post-infusion, no envelope containing HIV-1 virions were detected while standard viral load assay detected gag sequences. At 2 month post-infusion, viral load level were below limits of detection, and only 1 HIV-1 strain re-emerged, with significantly reduced diversity, major amounts of envelope deletions, and impaired replicative fitness with decreased ability to infect and propagate in cell culture.
Conclusions: This is the first in vivo demonstration of anti-HIV antisense activity in cells delivered by a lentiviral vector. Antisense pressures resulted in env deleted mutants, massive quasispecies reductions and impaired replicative fitness. The parameters studied indicate that VRX496 exerts high pressure on wild type HIV, which were not reflected by the conventional read out methods to monitor HIV infection.
Source:
15th Conference on Retroviruses and Opportunistic Infections, Session 124, Tues. 1-4 pm, Hall A