The use of photo-labelled non-nucleoside reverse transcriptase inhibitors of HIV-1 to inactivate viral replication capacity
S. Colby-Germinario1, A. Rios2, J. Quesada2, D. Anderson2, A. Goldstein3, T. Fossum4, M.A. Wainberg1
1Jewish General Hospital, McGill AIDS Center, Lady Davis Institute,, Montreal, Canada, 2PhotoImmune Biotechnology Inc,, Houston, United States, 3George Washington University School of Medicine and Health Sciences, Washington, United States, 4Texas A &M University,, College Station, United States
Background: We describe a new methodology for the potential development of an inactivated HIV vaccine using targeted photo-inactivation of HIV reverse transcriptase (RT) with preservation of conformational integrity of viral surface proteins.
Methods: An azido (-N3) group was introduced into each of a nevirapine analog (NVPa), and a dapivirine analog (DPVa), creating photo-active NNRTI analogs PA-NVPa and PA-DPVa. HIV-1 NL4-3 wild-type virus was attached to surfaces of 96-well plates using solid phase immobilization. Plates coated with poly-l-lysine, were incubated 1hr with NL4-3 and washed. The photoactive analogs PA-NVPa or PA-DPVa were then incubated with HIV-1 ± 2hrs and exposed to ultraviolet (UV) light, irreversibly causing cross-linking to the HIV-1 RT. Plates were then washed to remove unbound compound. MT-2 or PM-1 human leukemia cells were added to the wells in 10% RPMI and cultured for 3 to 10 days. Supernatant samples were collected at various times for p24 and RT assays.
Results: IC50 for NVP, PA-NVPa, DPV, and PA-DPVa were determined in MT-2 and PM-1 cells infected with HIV-1 NL4-3 wild type. Both the unmodified and modified compounds inhibited replication in a similar concentration range indicating that addition of the photo-active moiety did not alter the antiviral activity. Although the PA drugs were present only during pre-treatment of the virus they did reduce replication compared to control (ANOVA p< 0.0001). The PA-NVPa compound significantly reduced viral infectiousness (» 75%) and lowered viral RT activity in situ by >90%. The PA-DPVa compound completely inhibited both viral RT activity as well as viral infectiousness in susceptible target cells.
Conclusions: These findings illustrate that treatment of HIV-1 with PA-modified NNRTIs, in particular PA-DVPa, together with UV irradiation, can inactivate HIV-1.
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