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.

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.

Safety and immunogenicity of a modified pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02).

BACKGROUND: To investigate the safety and immunogenicity of a modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed. METHODS: 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1×10(8)pfu/dose) of MVA-B (n=24) or placebo (n=6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity. RESULTS: A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers. CONCLUSIONS: MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497.

Phenotypic and functional characteristics of HIV-specific CD8 T cells and gag sequence variability after autologous dendritic cells based therapeutic vaccine.

A decrease in HIV-1 specific CD8 T-cell responses associated with a partial control of viral replication occurred in 12 HIV-1-infected patients during autologous dendritic cells vaccination. HIV CD8 T cells were detected in 6/10 patients during immunizations, increasing after HAART discontinuation in 3 of them. Tet+ CD8 cells mainly had an effector phenotype (CD45RA-/+ CCR7- and CD28- and Perf+/-) and maintained IFN-gamma release throughout follow-up. By contrast, patients with CD45RA-/+ CCR7+ Perf+ HIV-specific cells showed a decrease in peptide-specific IFN-gamma production during vaccinations while levels were recovered when off HAART. No major mutations in either Gag p24 and p17 immunodominant epitopes were observed that might have explained the impaired CD8+ T-cell responses. Taken together, heterogeneity in the maturation status of HIV-specific CD8 T cells may be partially involved in the drop of peptide-specific IFN-gamma production during immunizations.

Dynamics of T cells subsets and lymphoproliferative responses during structured treatment interruption cycles and after definitive interruption of HAART in early chronic HIV type-1-infected patients.

Little is known about the consequences of short cycles of structured treatment interruption or definitive interruption of HAART for both T cell subset dynamics and T lymphoproliferative responses (LPR). Immunological follow-up was performed in 45 early chronical HIV-1-infected patients during short STI cycles during the first 12 weeks after the definitive interruption of HAART (DTI) and, thereafter, until VL reached a plateau. During STI cycles, CD8(+), CD8(+), CD28(+), activation markers and naive CD4(+) T cells increased significantly (p < 0.0001), while both naive CD8(+) and memory CD4(+) T cells decreased. During DTI, CD8(+) CD28(+) T cells fell and CD4(+) naive T cells stabilized and the rest of the T cell subsets presented changes similar to those during STI cycles. Despite a transient increase in LPR to recall antigens and HIV proteins during STI cycles, LPR to polyclonal stimuli and pathogens decreased over the study. Differences in T cell subset dynamics and LPR observed throughout the study suggest that multiple exposures to low levels of antigen could improve the immune system, mainly by driving T cell maturation. Conversely, higher and longer viral replication after cessation of HAART overwhelms the immune system. These data may help to guide future immune-based therapies.

Immune restoration in HIV-positive, antiretroviral-naive patients after 1 year of zidovudine/lamivudine plus nelfinavir or nevirapine.

OBJECTIVES: To evaluate the immunological response in HIV-1-infected, antiretroviral-naive patients receiving highly active antiretroviral therapy regimen of two nucleosides plus a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor. DESIGN AND METHODS: Of 142 patients included in a randomized, open, multicentre trial comparing zidovudine/lamivudine plus nelfinavir (NFV) or nevirapine (NVP), 36 patients (16 NFV, 20 NVP) were enrolled in an immunological substudy. Mean baseline CD4 T-cell counts was 360/mm3 (range: 11-679) and mean baseline plasma viral load >50000 copies/ml (range: 2240-1468210). Viral load (VL), T-cell subsets and T-cell functions were analysed at baseline and after 1 year of treatment. RESULTS: After 12 months of follow-up, plasma viral load was reduced similarly in both groups, with 78% (NFV) and 83% (NVP) of patients achieving a VL <200 copies/ml. A significant increase in CD4 T cells was observed in both groups (mean: +182 cells, P=0.001). Both regimens were similarly effective in reducing activated T cells (CD38 and DR). A significant increase of both CD4 and CD8 CD28 T cells occurred in both arms of treatment. Patients of both regimens showed a significant decrease of activated memory (CD45RA-CD45RO+) CD8 T cells and a clear increase of naive (CD45RA+CD45RO-) CD8 T cells. Peripheral blood mononuclear cell proliferative responses to polyclonal stimuli (CD3 and CD3 +CD28) as well as to ubiquitous cytomegalovirus antigen increased significantly in both groups after 12 months of follow-up. Nevertheless, neither at baseline nor after 1 year of treatment, these patients showed any significant T-cell responsiveness to HIV-1 recombinant proteins gp160 or p24. CONCLUSIONS: Our data indicate that immune restoration achieved after 1 year of therapy with either NFV or NVP was similar. This reinforces the role of NVP-containing regimens as a valid option for initiating antiretroviral therapy. Nevertheless, additional therapeutic approaches should be envisaged to restore HIV-1-specific T-cell responses.

Immunologic reconstitution after 1 year of highly active antiretroviral therapy, with or without protease inhibitors.

OBJECTIVES: To assess the effectiveness of two triple antiretroviral combinations (2 nucleoside reverse transcriptase inhibitors [NRTIs] + 1 protease inhibitors [PI] vs. 2 NRTIs + 1 nonnucleoside reverse transcriptase inhibitor [NNRTI]) to correct T-cell subsets abnormalities and to restore immune functions in asymptomatic antiretroviral-naive HIV-1-infected patients with a baseline CD4 T-cell counts >500/mm3 and plasma viral load >5000 copies/mL. DESIGN AND METHODS: Twenty randomized patients from 2 cohort studies receiving either stavudine (d4T) + lamivudine (3TC) + indinavir (n = 9), or d4T + didanosine (ddI) + nevirapine (NVP) (n = 11) were studied. Viral load, T-cell subsets and T-cell functions were analyzed at baseline and after 1 year of treatment. RESULTS: After 1 year of follow-up, the PI regimen was significantly more effective in reducing plasma and lymphoid tissue VL to undetectable levels. A significant increase in CD4+ T cells was observed in patients treated with PI (p =.0007) compared with those treated with NVP. Percentages of CD8+ T-cells and of activated CD8+ T-cells (CD38+ and DR+ as well as memory CD45RO+) decreased in all patients. An increase of the CD28+ subset of CD8+ T-cells also occurred in both groups of treatment. Naive T cells were maintained in the CD4+ subset and augmented in the CD8+ subset in all patients. In both PI and NVP groups, memory CD4+ T-cells increased significantly (p =.03). Peripheral blood mononuclear cell responsiveness to polyclonal stimuli and to tetanus toxoid and cytomegalovirus (CMV) antigen was similar in both groups of treatment. HIV-infected patients treated for 1 year with both triple combinations lacked significant T-cell responsiveness to HIV-1 proteins. CONCLUSIONS: These data suggest that immune reconstitution achieved after 1 year of therapy with PI-containing or PI-sparing regimens is similar, despite the higher effectiveness of PI-containing regimens in reducing viral load. Additional therapeutic approaches should be designed to restore HIV-1-specific responses.