Persistence of viral reservoirs in multiple tissues after antiretroviral therapy suppression in a macaque RT-SHIV model

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. © 2013 Kline et al

Antifibrotic Therapy in Simian Immunodeficiency Virus Infection Preserves CD4\u3csup\u3e+\u3c/sup\u3e T-Cell Populations and Improves Immune Reconstitution With Antiretroviral Therapy

Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have CD4+ T cells/μL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival.We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution

Plasma viremia was measured in all twelve macaques by qRT-PCR of RT-SHIV <i>gag</i> RNA.

<p>Animals were infected at week 0 and were (A) untreated or (B) treated with 3 or 4 antiretroviral drugs. Animals treated with 3 drugs (TFV, FTC, EFV) are denoted by closed symbols and treatment was initiated at week 10, denoted by the solid arrow. The animals treated with 4 drugs (TFV, FTC, EFV, and L-870812) are denoted by open symbols and treatment was initiated at week 13 (GV08 and GN19) or week 14 (GG45 and GV40), denoted by the open arrow. Treatment was continued daily until necropsy (week 30 or 31). The limit of detection of the assay was 30 vRNA copies/ml plasma.</p

The ratio of RT-SHIV <i>gag</i> RNA copies per 10⁶ macaque CD4 RNA copies were measured in PBMC isolated at week 1 or 2 and 28 or 30 post-infection from each of the untreated and treated macaques (black bars).

<p>The amount of RT-SHIV gag RNA in the plasma is also plotted for each animal at each time point (white bars). Asterisks (*) denote plasma viremia levels below the limit of detection (<30 copies/ml).</p

Lymphoid tissue viral DNA at necropsy is correlated with week 1 plasma viremia levels.

<p>(A) The ratio of <i>gag</i> copies per 10⁶ CCR5 copies for each tissue of the untreated RT-SHIV-infected macaques. The average of each qPCR reaction was used for the graph. In addition, the week 1 plasma viral load was included for each animal. Asterisks (*) denote samples that were not collected or in which no significant CCR5 DNA were measured. (B) The amount of <i>gag</i> vDNA detected in each of the lymphoid tissues for each animal was plotted against the week 1 plasma viremia level. Statistics determined a Spearman rank-order correlation of 0.996 with p value of < 0.0001.</p

Cytotoxic capacity of SIV-specific CD8(+) T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques.

Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated

SIV-specific CD8⁺ T cells from LTNP/EC mediate greater lysis of SIV-infected CD4⁺ T-cell targets compared with progressors.

<p>GrB target cell activity (<b>A</b>) and infected CD4 elimination (ICE) (<b>B</b>) are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). Horizontal bars represent the median values. <b>C.</b> Correlation between ICE and GrB target cell activity (n = 22) was determined by the Spearman rank method. Red, blue and cyan dots represent LTNP/EC, progressors and one SIV-uninfected animal, respectively.</p

Infected CD4 Elimination (ICE) inversely correlates with plasma SIV RNA levels in SIV-infected rhesus macaques.

<p>Red dots represent LTNP/EC (n = 11). Blue dots represent progressors (n = 11). Statistical analysis was performed by the Spearman rank method.</p