Differential MicroRNA Expression Profile between Stimulated PBMCs from HIV-1 Infected Elite Controllers and Viremic Progressors

<div><p>Background</p><p>The emerging relationship between microRNAs (miRNA) and viral-control is a topic of interest in the field of HIV. Host-genome might play an important role in the control of viremia. The aim of this study was to assess the specific miRNA profile that could contribute to the control of HIV replication in Elite Controllers</p><p>Results</p><p>After adequate normalization, expression profile of 286 human miRNAs (hsa-miR) was evaluated in phytohaemagglutinin-stimulated PBMCs from 29 individuals classified in 4 groups: 8 elite controllers (EC; viral load <50 cp/ml without treatment), 8 viremic progressors (VP; VL>5000 cp/ml without treatment), 8 patients under antiretroviral treatment (ART; VL<200 cp/ml) and 5 uninfected individuals (HIV-) through TaqMan Array Human microRNA Cards v3.0. A differential expression pattern consisting of 23 miRNAs became significantly different when comparing EC and VP. Profiling analysis segregated the population in two different blocks: while EC and HIV- clustered together in the same block (EC/HIV-_block 1), VP and ART individuals clustered together in a second block (VP/ART_block 2). Two inversely expressed miRNA patterns were determined within those two blocks: a set of 4 miRNAs (hsa-miR-221, -27a, -27b and -29b) was up-expressed in EC/HIV-_block and down-expressed in VP/ART_block while 19 miRNAs were down-expressed in block 1 and up-expressed in block 2. Differential miRNAs were successfully validated through individual RT-qPCR assays.</p><p>Conclusions</p><p>Profile in EC resembled HIV- and differentially clusters with VP and ART. Therefore, differential clustering does not rely on undetectable viremia.</p></div

Differential miRNAs between Elite Controllers (EC) and Viremic Progressors (VP).

<p>A) Hierarchical clustering of the differentially expressed miRNAs between EC and VP. Patients are ordered on vertical lines and candidate miRNAs on horizontal lines. For each miRNA, green represents under-expressed and red over-expressed values compared to the average value (median), in dark. Dendrograms (tree graph) between patients and between miRNAs are depicted, where the closest branches of the tree represent patients/miRNAs with the most similar expression pattern. Two blocks of patients (Block 1/Block 2) with an inverse expression profile were segregated. Two groups of miRNAs (Group 1/Group 2) with an inverse expression profile were segregated within each block. B) Fold change (log 2) of the 23 differentially expressed miRNAs in EC. Differential levels are normalized to all assesed miRNAs and relative to VP. Bars represent standard error means (SEM).</p

Baseline characteristics of the study participants.

<p>VP, viremic progressors; EC, elite controller; ART, antiretroviral therapy; MSM, men who had sex with men; *; median [interquartile range  =  upper quartile (Q3) - lower quartile (Q1)]; N/A, not applicable.</p><p>Baseline characteristics of the study participants.</p

Fold change (log₂) of the 23 differentially expressed miRNAs in EC and VP.

<p>A) normalized to all assessed miRNAs and relative to ART, B) normalized to all assessed miRNAs and relative to HIV-. Bars represent standard error means (SEM); *, p<0.05; **, p<0.01; ***, p<0.001; VP, viremic progressors; EC, elite controllers; ART, antiretroviral therapy.</p

Analysis of virion morphologies

<p><b>by electron microscopy.</b> (A) Electron micrographic analysis of 293-T cells transfected with pNL4-3. Mature virions (black arrows) show their characteristic core (white arrow heads). Budding particles (black arrow heads) are also present. (B) Electron micrograph showing immature viral particles budding from 293-T cells transfected with pNL4-3/ΔRT (black arrows). (C) Electron micrographic analysis of pNL4-3 purified viruses showing mature, immature and eccentric virion forms. (D) Electron micrograph of pNL4-3/ΔRT purified virions in which only immature virion forms can be observed. (E and F) NL4-3 and NL4-3/ΔRT virion magnifications respectively are shown. (G) Virion form measurements of NL4-3 <i>versus</i> NL4-3/ΔRT. NL4-3/ΔRT virion conformation was mostly immature and no mature virions were observed. On the other hand, NL4-3 virions were mainly mature. Error bars represent standard deviations. Samples were imaged using a Tecnai Spirit Twin electron-microscope.</p

Fold change (log₂) of EC, VP and ART, normalized to endogenous control miRNAs and relative to HIV-.

<p>One-way analysis of variance (ANOVA) tests were performed for global comparisons and Turkey post-hoc tests for pair comparisons. Bars represent standard error means (SEM); *, p<0.05; **, p<0.01; ***, p<0.001 for Tuckey post-hoc tests; VP, viremic progressors; EC, elite controllers; ART, antiretroviral therapy.</p

Immature NL4-3 virions improved the elicited cellular immune response.

<p>(A) Electron microscopy analysis of purified NL4-3 virions treated with AT-2 and APV. Different virion morphologies were observed; most were immature and eccentric. (B) 52 electron-micrographs were analysed for virus morphologies. The APV incubation reduces mature virions but increases immature and eccentric forms. Error bars represent standard deviaations. (C) Twenty-one cryopreserved PBMCs from HIV seropositive individuals were assessed by ELISPOT to detect IFN-γ production. PBMCs were pulsed with 200 ng/ml p24 equivalents in all cases. Magnitude response was significantly lower against WT inactivated virions (WT+AT-2) than against WT+APV+AT-2 (*p<0.05). Averaged values from duplicate wells normalized to SFC/10⁶ PBMCs are shown for the stimulation conditions indicated. Plots represent mean ± standard error of the mean (SEM). Positivity threshold for each construct or antigen was defined as at least 50 SFC/10⁶ PBMC and at least twice that of the control medium.</p

NL4-3/ΔRT plasmid renders virions infectious but non-replicative.

<p>(A) PBMC infection was monitored using NL4-3 (WT) and NL4-3/ΔRT (ΔRT) virions labelled with a Gag fusion protein (Gag-EGFP). Samples were imaged using a Leica SP2 confocal microscope. We observed an accumulation of Gag-EGFP in the cells infected with NL4-3 and NL4-3/ΔRT (arrows) but not in cells infected with Gag-EGFP. Cellular nuclei are stained in blue (DAPI), F-actin in red (Phalloidine) and the fusion protein Gag in green (Gag-EGFP). (B) NL4-3 and NL4-3/ΔRT virion replication measurements by luciferase activity in TZM-bl cells. The NL4-3 virions showed a decrease in luciferase activity directly proportional to the p24 quantity, while for NL4-3/ΔRT virions, luciferase activity was under the background independently of the p24 quantity.</p

NL4-3/ΔRT plasmid graphic scheme.

<p>NL4-3 (WT) plasmid was used to generate the NL4-3/ΔRT plasmid by deleting an 892 bp fragment, which codes for the retrotranscriptase (RT) and is inside the <i>pol</i> gene. These plasmids were used to transfect 293-T cells in order to obtain viruses. Numbers refer to pNL4-3.</p

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

<div><p>Background</p><p>Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.</p> <p>Methods</p><p>We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.</p> <p>Results</p><p>The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “<i>full-responders</i>” (positive response against both viral particles), <i>“partial-responders”</i> (positive response only against NL4-3/ΔRT virions) and “<i>non-responders</i>” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.</p> <p>Conclusions</p><p>In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both <i>in vitro</i> and in vaccine trials, by a feasible, simply, effective and low cost assay.</p> </div