Dynamics of Simian Immunodeﬁciency Virus Two-Long-Terminal-Repeat Circles in the Presence and Absence of CD8 (cid:2) Cells

CD8 (cid:2) 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 (cid:2) 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 (cid:2) 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 (cid:3) 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 (cid:2) 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 (cid:2) 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 (cid:2) cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT. IMPORTANCE CD8 (cid:2) 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 (cid:2) cells, administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles, and a combination of the two treat-ments. We used a new mathematical model to help interpret the data, and the results suggest that CD8 (cid:2) cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8 (cid:2) cells in SIV infection. Conﬁrmation of our results would be an important step in understanding immune control of HIV. ultiple lines of demonstrate that are in immunodeﬁciency (HIV)/simian immunodeﬁciency virus infection: (i) a strong association exists between speciﬁc host major histocompatibility complex class I (MHC-I) alleles and HIV/SIV disease progression (1); (ii) a temporal association can be established between the postpeak decline in plasma viremia and the increase in virus-speciﬁc CD8 (cid:2) cytotoxic T lymphocyte (CTL) responses (2, 3); (iii) virus escape mutations consistently arise in the face of host cytotoxic responses during all stages of infection, starting from the time when cellular immune responses are generated (4–8); (iv) CD8 (cid:2) cell depletion in vivo results in a rapid and sustained increase in plasma viremia, which then returns to predepletion levels following the rebound of CD8 (cid:2) cells (9–21). Furthermore, CD8 (cid:2) T cells suppress HIV infection in vitro (22, 23). However, the speciﬁc mechanism(s) of action these cells take to control viral replication is poorly understood. To gain some insight, we assessed the role of CD8 (cid:2) cells in controlling two-long-terminal-repeat-positive (2-LTR (cid:2) ) cells, which we used as a surrogate for HIV-infected cells preintegration. A recent study showed that cells containing virus prior to integration can represent a sizeable fraction of the total population of infected cells (24). since cells are still de novo infected during therapy, thus allowing better detection of the effects of CD8 (cid:2) cells. We then compared our data to an extended model of viral dynamics to explore the effect of CD8 (cid:2) cells on 2-LTR circle dynamics. We report that the levels of 2-LTR circles modestly increase following CD8 (cid:2) cell depletion, are unchanged after RAL monotherapy, and dramatically increase following the combined CD8 (cid:2) cell depletion and RAL monotherapy. Using the model of viral dynamics that includes 2-LTR circles, we found that model predictions are most similar to the observed data when CD8 (cid:2) cell killing of infected cells occurs prior to viral integration. We present all results in blood as the number of 2-LTR circles/ml, which we calculated using the absolute count of PBMCs determined from the TruCounts. It is important to note that the number of 2-LTR circles/ml is a more faithful measure of the dynamics of 2-LTR circles than this number normalized by a million cells, because the latter also depends on the dynamics of cells without 2-LTR circles, which change with treatment. For tissue samples (such as LN biopsy specimens), we present the results as the number of 2-LTR circles/million CCR5 (cid:2) cells, because it is difﬁcult to calculate absolute numbers of 2-LTR (cid:2) cells. Due to low cell counts in the jejunal biopsy specimens, we were unable to quantify the levels of 2-LTR circles in these samples. CA). However, we also tested the sensitivity , by repeating the described below with and/or 10 and p (cid:5) 10 4 virions/cell/day. The value of p had no effect on the ﬁts or the conclusions presented On the other hand, (cid:4) inﬂuenced the quantitative values of the parameters but did not change the overall conclusions, although the predicted proﬁles of 2-LTR (cid:2) cells/ml were not as good. We then performed ﬁts of the model to the changes in 2-LTR/ml to estimate (cid:5) and the effect of CD8 (cid:2) cell depletion. In these ﬁts, we searched for the best set of parameters for all the data together, i.e., the 2-LTRs in the 20 RMs, normalized at the initial time and assuming ﬁve different effects of CD8 (cid:2) cell depletion (and their combinations two by two): (i) on the death of infected cells before integration, by reducing (cid:3) 1 in the model; (ii) on the death of productively infected cells by reducing (cid:3) 2 ; (iii) on the viral production, by increasing p ; (iv) on the infectivity of virus, by increasing (cid:4) ; and (iv) on the integration rate, by increasing k . The ﬁrst three effects have been referred to in the literature of CD8 (cid:2) cell depletion studies as putative targets for the action of CD8 (cid:2) T cells (15, 16, 20, 42), and we include the last two for completeness. However, our interest is not in the speciﬁc values of the parameters but rather in the general behavior of the model and how it compares with our observed 2-LTR dynamics under different treatment conditions. In addition, in the current manuscript, we are studying only the 2-LTR dynamics and did not ﬁt virus or T-cell dynamics. The model was simulated using R (https://cran.r-project.org/), and we used the DEoptim package of R for ﬁtting. We compared models and selected the best one based on the corrected Akaike information criteria (AICc) (56). The best ﬁts resulted systematically in (cid:5) (cid:5) 0/day or very near 0, indicating that R 2 (cid:5) 0 cells/ml. So, we also performed analyses imposing a lower bound of (cid:5) (cid:5) 0.1/day. Although these ﬁts were not as good, the model may be more physiological.