Utility of Systematic Isolation of immune cell subsets from HIV-infected individuals for miRNA profiling.

INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune cell subsets by immunomagnetic separation and the RNA integrity after isolated cells from samples of HIV-infected patients. METHODS: PBMC from Healthy Controls (HC, n=15), Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=15) and HIV-infected patients on therapy (ART, n=15) were isolated by Ficoll-Paque density gradient centrifugation. Subsets were separated with monoclonal antibodies (CD56, CD14, CD4, and CD8) conjugated to microbeads. We evaluated the yield and purity of each subset isolated from PBMCs under resting and activated conditions; LPS, anti-CD3/CD28 and anti-CD16 were used to activate monocytes, PBMC, T cells and NK cells, respectively. The quality of extracted RNA was tested by 2100 Bioanalyzer. RESULTS: In resting conditions, the average yield of CD14+ (monocytes) was decreased (p=0.021) in HIV+ patients compared with healthy controls. CD56+ (Natural Killer-NKs; p=0.03) and CD8+ (Cytotoxic T lymphocytes-CTL p=0.001) cells were increased in HIV+ patients after 72h of activation. The purity assay detected significant differences in CD14+ (p≤0.001) and CD8+ (p=0.034) subpopulations when comparing PBMC isolated either from healthy controls or HIV+ patients. The number of activated cells in HIV+ presented differences in CD8 subset (p=0.003). Finally, similar quantities of high quality RNA were extracted from immune cells subsets obtained by our method. Specifically, we show that Bioanalyzer electrophenograms reveal optimal RIN values in HIV positive and negative patients in resting condition (EC:8;HC:6.5;VC:8.80;VP:8;HAART:7.5) and activated condition (EC:9;HC:6.7;VC:8.2;VP:7.2;HAART:8.6). CONCLUSION: This method allowed us to obtain a sufficient quantity of different isolated immune cell subsets from HIV-infected individuals at different disease stages. Moreover, the assessed qualities of nucleic acids allow us to perform subsequent molecular studies, such as microRNA profiling.

Preclinical evaluation of an mRNA HIV vaccine combining rationally selected antigenic sequences and adjuvant signals (HTI-TriMix).

BACKGROUND: The development of a prophylactic vaccine against HIV-1 has so far not been successful. Therefore, attention has shifted more and more toward the development of novel therapeutic vaccines. Here, we evaluated a new mRNA-based therapeutic vaccine against HIV-1-encoding activation signals (TriMix: CD40L + CD70 + caTLR4) combined with rationally selected antigenic sequences [HIVACAT T-cell immunogen (HTI)] sequence: comprises 16 joined fragments from Gag, Pol, Vif, and Nef). METHODS: For this purpose, peripheral blood mononuclear cells from HIV-1-infected individuals on cART, lymph node explants from noninfected humans, and splenocytes from immunized mice were collected and several immune functions were measured. RESULTS: Electroporation of immature monocyte-derived dendritic cells from HIV-infected patients with mRNA encoding HTI + TriMix potently activated dendritic cells which resulted in upregulation of maturation markers and cytokine production and T-cell stimulation, as evidenced by enhanced proliferation and cytokine secretion (IFN-γ). Responses were HIV specific and were predominantly targeted against the sequences included in HTI. These findings were confirmed in human lymph node explants exposed to HTI + TriMix mRNA. Intranodal immunizations with HTI mRNA in a mouse model increased antigen-specific cytotoxic T-lymphocyte responses. The addition of TriMix further enhanced cytotoxic responses. CONCLUSION: Our results suggest that uptake of mRNA, encoding strong activation signals and a potent HIV antigen, confers a T-cell stimulatory capacity to dendritic cells and enhances their ability to stimulate antigen-specific immunity. These findings may pave the way for therapeutic HIV vaccine strategies based on antigen-encoding RNA to specifically target antigen-presenting cells.

MicroRNA Profile in CD8+ T-Lymphocytes from HIV-Infected Individuals: Relationship with Antiviral Immune Response and Disease Progression.

BACKGROUND: The relationship between host microRNAs (miRNA), viral control and immune response has not yet been elucidated in the field of HIV. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of viral replication control and immune response. METHODS: miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n = 11), Elite Controllers (EC, n = 15), Viremic Controllers (VC, n = 15), Viremic Progressors (VP, n = 13) and HIV-infected patients on therapy (ART, n = 14) was assessed using Affymetrix miRNA 3.1 arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatic algorithms. RESULTS: A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV-. A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to VP, being hsa-miR-4492 the most downregulated. Although a preferential miRNA downregulation was observed when stimulated samples were compared to the respective resting samples, VP presented a differential miRNA expression pattern. In fact, hsa-miR-155 and hsa-miR-181a were downregulated in VP whereas in the other groups, either an upregulation or no differences were observed after stimulation, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in signal transduction pathways, metabolic regulation, apoptosis, and immune response. CONCLUSIONS: Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non-infected individuals. Moreover, a specific miRNA pattern is present in stimulated CD8+ T-cells from VP which could reflect a detrimental pattern in terms of CD8+ T-cell immune response.

Detection of HIV-1-specific T-cell immune responses in highly HIV-exposed uninfected individuals by in-vitro dendritic cell co-culture.

BACKGROUND: Although virus-specific responses are rarely detected by conventional approaches, we report here the detection of T-cell responses in HIV-exposed seronegative (HESN) patients by two distinct assays. METHODS: HIV-specific T-cell responses were analyzed by enzyme-linked immunospot in peripheral blood mononuclear cells from HESN patients after a 48-h co-culture with boosted dendritic cells. Additionally, a boosted flow cytometry approach was used to capture antiviral T-cell responses. Host genetic factors and T-cell activation were also analyzed to assess their implication on HIV exposure. RESULTS: Of the 45 HESN individuals tested, up to 11 (24.4%) showed at least one response to peptide pools covering HIV Gag and Nef. A positive correlation was observed between the intensity (P = 0.0022) and magnitude (P = 0.0174) of the response detected in the HESN, and the viral load of the HIV-positive partner. Moreover, the result from the boosted flow and cytomix analyses showed a dominant Th1-like response pattern against HIV antigens, especially in CD8 T-cell populations. CONCLUSIONS: The combined use of our boosted dendritic cell technique with a boosted flow cytometric approach allows us both to detect specific HIV-positive responses in a higher percentage of HESN patients and to define specific effector function profiles. This study contributes to a better understanding of resistance to HIV infection.