Dynamics of Simian Immunodeficiency Virus Two-Long-Terminal-Repeat Circles in the Presence and Absence of CD8⁺Cells

Abstract

CD8 cells play a key role in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, but their specific mechanism(s) of action in controlling the virus is unclear. Two-long-terminal-repeat (2-LTR) circles are extrachromosomal products generated upon failed integration of HIV/SIV. To understand the specific effects of CD8 cells on infected cells, we analyzed the dynamics of 2-LTR circles in SIVmac251-infected rhesus macaques (RMs) treated with an integrase inhibitor (INT). Twenty RMs underwent CD8 cell depletion and received raltegravir (RAL) monotherapy or a combination of both. Blood, lymph nodes (LNs), and gut biopsy specimens were routinely sampled. Plasma viral loads (pVLs) and 2-LTR circles from peripheral blood mononuclear cells (PBMCs) and LN lymphocytes were measured with quantitative reverse transcription-PCR (qRT-PCR). In the CD8 depletion group, an ∼1-log increase in pVLs and a slow increase in PBMC 2-LTRs occurred following depletion. In the INT group, a strong decline in pVLs upon treatment initiation and no change in 2-LTR levels were observed. In the INT and CD8 cell depletion group, an increase in pVLs following CD8 depletion similar to that in the CD8 depletion group was observed, with a modest decline following INT initiation, and 2-LTR circles significantly increased in PBMCs and LNs. Analyzing the 2-LTR data across all treatment groups with a mathematical model indicates that the data best support an effect of CD8 cells in killing cells prior to viral integration. Sensitivity analyses of these results confirm that effect but also allow for additional effects, which the data do not discriminate well. Overall, we show that INT does not significantly increase the levels of 2-LTR circles. However, CD8 cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT. CD8 T cells play an essential role in controlling HIV and SIV infection, but the specific mechanisms involved remain poorly understood. Due to failed viral infection, HIV and SIV can form 2-LTR extrachromosomal circles that can be quantified. We present novel data on the dynamics of these 2-LTR forms in a SIV-infected macaque model under three different treatment conditions: depletion of CD8 cells, administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles, and a combination of the two treatments. We used a new mathematical model to help interpret the data, and the results suggest that CD8 cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8 cells in SIV infection. Confirmation of our results would be an important step in understanding immune control of HIV.

The dynamics of simian immunodeficiency virus after depletion of CD8+ cells

“…We have developed an experimental protocol similar to this one for CD8+ cell depletion and RAL therapy in NHP. 63 Whether these results are in agreement with the schematic results predicted by the modeling and depicted in Figure 7 is still under investigation.…”

“…Here we focus on studies of CD8+ cell depletion during chronic infection, because some of these experiments have been analyzed in more detail through modeling. As schematically shown in Figure B, administration of a CD8‐depleting antibody during chronic infection leads to a profound depletion of CD8+ cells in the periphery, although depletion at other sites, such as lymph node and mucosa, is usually less pronounced and more variable . At the same time, the viral load increases quickly.…”

“…We have developed an experimental protocol similar to this one for CD8+ cell depletion and RAL therapy in NHP . Preliminary results indicate a difference in the slopes of viral decline with RAL therapy in the presence vs. absence of CD8+ cells (B.…”

CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption

“…Replicate wells were merged and analyzed in the QuantaSoft Analysis Pro Software. As described by Bender and others, a correction for shearing was not applied given low and similar shearing rates across samples, and the frequency of 2-LTR + env + circles was subtracted from the frequency of intact (pol + env + ) proviruses 26,52 . Samples with a DNA shearing index >25 were excluded from analyses (RWs16 at d−29 p.t.…”

Increased Proviral DNA in Circulating Cells Correlates with Plasma Viral Rebound in Simian Immunodeficiency Virus-Infected Rhesus Macaques after Antiretroviral Therapy Interruption

“…All of these are necessary for the process of reverse transcription, proviral integration, transcription, viral protein expression, and virion assembling (17)(18)(19)(20)(21). Throughout the HIV/SIV life cycle, various viral nucleic acids, representing different infectious status and clinical significance (10,11,(22)(23)(24)(25)(26), can be measured in tissues. Given HIV/SIV RNA/DNA forms in the viral life cycle, their existence and abundance might be helpful to estimate reservoir size and latency: 1) HIV/SIV full-length, unspliced RNA expresses gag and gag-pol capsid proteins as well as genomic RNA for viral packaging, thus HIV gag RNA transcripts represent bona fide HIV unspliced RNA involved in viral replication (27,28); 2) Correctly spliced HIV/SIV tat/rev RNA can express functional proteins, which are indispensable for viral replication and production (29); 3) Episomal two-long-terminal-repeat (2-LTR) circles are extrachromosomal bystander products generated upon failed integration of HIV/SIV (26); and; 4) a pool of integrated proviral DNA, only ~11.7% of which represents genetically intact HIV genomes in treated patients (30) and the latter are believed to be responsible for most viral rebound after ART cessation.…”