Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model

Abstract

Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood.

Enhanced Antiretroviral Therapy in Rhesus Macaques Improves RT-SHIV Viral Decay Kinetics

“…Their regimen consisted of TFV, (Ϫ)-FTC, and raltegravir, initially for 1.5 months and then intensified with the protease inhibitor darunavir (pharmacokinetically enhanced by ritonavir) for 80 days and, lastly, reinforced with the addition of the CCR5 antagonist maraviroc (42). Likewise, Kline et al monitored RT-SHIV mne in rhesus macaques over 20 weeks and found persistence of viral reservoirs in lymphoid tissues, despite undetectable plasma viremia at the time of necropsy (43). Unfortunately, eradication was not achieved in any of these studies.…”

Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase

“…RNA was washed with isopropanol and ethanol and samples were suspended in 10 mM Tris-HCl (pH 8.0). Tissue vRNA isolation was performed as previously described (54).…”

“…Quantitative PCR was performed on cDNA using SsoFast probes SuperMix (Bio-Rad), SIVgag-F (5=-GTCTGCGTCATCTGGTGCATTC-3=) and SIVgag-R primers, and the SIVgag-probe (5=-FAM-CTTCCTCAGTGTGTTTCACTTTCTCTTCT-GCG-3= 6-carboxytetramethylrhodamine [TAMRA]). CCR5 primers and probe were previously described (54). Reaction conditions were 1 cycle of 95°C for 2 min followed by 45 cycles of 95°C for 15 s and 60°C for 1 min.…”

Glioma initiating cells contribute to malignant transformation of host glial cells during tumor tissue remodeling via PDGF signaling

“…PCR was performed on cDNA corresponding to 50 ng RNA. Reaction condition: 5 min 94°C; 30 cycles of 90 s at specified annealing temperature, 90 s 72°C, 30 s 94°C; 5 min at specified annealing temperature and 10 min 72°C [14,15]. Primer sequences for analytical RT-PCR experiments, length of amplicons and annealing temperatures were as follows: PDFGR-α (5′-GACGAGACCATCGAGGACAT-3′, 5′-GCCTCGGGAACTTTCTCTCT-3′, 207 bp, 55°C); PDGF-B (5′-CTGAGCTGGAC TTGAACATGAC-3′, 5′-CGCACAATCTCAATCTTTCTCA-3′, 307 bp, 60°C); h-Actin (5′-ACATCCGCAAAGACCTGTAC-3′, 5′-GCCATGCCAATCTCATCTTG-3′, 346 bp, 58°C); m-Actin (5′-CTTTGCAGCT-CCTTCGTTG-3′, 5′-TGGTAAACAATGCCATGTTCA-3′, 274 bp, 58°C).…”