Silvia Nozza

Escape of monocyte-derived dendritic cells of HIV-1 infected individuals from natural killer cell-mediated lysis

Objective: To verify whether the in vitro sensitivity of immature dendritic cells (iDC) to lysis by autologous natural killer (NK) cells from HIV-infected individuals might be correlated with HIV disease progression. Design: Both dendritic cells (DC) and interlekin (IL)-2 activated NK cells were obtained from 13 HIV-infected individuals early after seroconversion and not receiving highly active antiretroviral therapy (HAART) and from 14 individuals with chronic HIV infection under HAART. The rate of NK cell-mediated killing of autologous iDC was correlated with classical parameters of HIV evolution. Methods: Peripheral blood monocytes obtained from the Ficoll-derived leukocyte fraction after adherence to plastic were stimulated with granulocyte–macrophage colony stimulating factor plus IL-4 to induce their differentiation into iDC to be used as target cells in a standard 4-h cytotoxicity assay. A fraction of autologous leukocytes was stimulated with IL-2 to induce activation of NK cells to be used as effector cells. Results: During early HIV infection the extent of ex vivo lysis of monocyte-derived DC by activated autologous NK cells was inversely and directly correlated with the levels of viraemia and with the percentage of circulating CD4 T cells, respectively. In contrast, the capacity of NK cells to kill iDC was lost independently of the levels of plasma viraemia or the concurrence of HAART in chronically infected individuals. Addition of exogenous HIV Tat during the cytotoxicity assay inhibited NK cell-mediated lysis of DC. Conclusions: NK cell-mediated immune surveillance against infected DC may be effective only during early HIV infection and may not be restored by HAART.

B-cell subset alterations and correlated factors in HIV-1 infection.

Objectives:During HIV-1 infection, the development, phenotype, and functionality of B cells are impaired. Transitional B cells and aberrant B-cell populations arise in blood, whereas a declined percentage of resting memory B cells is detected. Our study aimed at pinpointing the demographic, immunological, and viral factors driving these pathological findings, and the role of antiretroviral therapy in reverting these alterations. Design:B-cell phenotype and correlating factors were evaluated. Methods:Variations in B-cell subsets were evaluated by flow cytometry in HIV-1-infected individuals naive to therapy, elite controllers, and patients treated with antiretroviral drugs (virological control or failure). Multivariable analysis was performed to identify variables independently associated with the B-cell alterations. Results:Significant differences were observed among patients’ groups in relation to all B-cell subsets. Resting memory B cells were preserved in patients naive to therapy and elite controllers, but reduced in treated patients. Individuals naive to therapy and experiencing multidrug failure, as well as elite controllers, had significantly higher levels of activated memory B cells compared to healthy controls. In the multivariate analysis, plasma viral load and nadir CD4+ T cells independently correlated with major B-cell alterations. Coinfection with hepatitis C but not hepatitis B virus also showed an impact on specific B-cell subsets. Successful protracted antiretroviral treatment led to normalization of all B-cell subsets with exception of resting memory B cells. Conclusion:Our results indicate that viremia and nadir CD4+ T cells are important prognostic markers of B-cell perturbations and provide evidence that resting memory B-cell depletion during chronic infection is not reverted upon successful antiretroviral therapy.

Raltegravir, maraviroc, etravirine: an effective protease inhibitor and nucleoside reverse transcriptase inhibitor-sparing regimen for salvage therapy in HIV-infected patients with triple-class experience.

We prospectively evaluated 28 triple-class experienced HIV-1-infected patients harbouring R5 virus, who received maraviroc, raltegravir and etravirine. By on-treatment analysis, 26 (92%) had less than 50 copies HIV-RNA/ml at week 48. The median (interquartile range) 48-week increase in CD4+ cell counts was 267 (136–355) cells/μl. Three serious adverse events occurred: one recurrence of mycobacterial spondylodiscitis, one anal cancer, one Hodgkin lymphoma. Although long-term safety needs further study, this protease inhibitor and nucleoside analogue-sparing regimen showed sustained efficacy.

Efficacy of Low-Dose Intermittent Subcutaneous Interleukin (IL)–2 in Antiviral Drug–Experienced Human Immunodeficiency Virus–Infected Persons with Detectable Virus Load: A Controlled Study of 3 IL-2 Regimens with Antiviral Drug Therapy

To evaluate the safety and efficacy of 3 regimens of intermittent subcutaneous (sc) interleukin (IL)--2 in a phase 2 study, 61 antiviral drug-experienced human immunodeficiency virus (HIV)--positive patients were randomly assigned to one of the following study arms: antiretroviral therapy (ART) plus IL-2 (12 million IU [MIU] by continuous intravenous infusion, followed by 7.5 MIU twice a day, sc, every 8 weeks); ART plus IL-2 (7.5 MIU twice a day, sc, every 8 weeks); ART plus IL-2 (3 MIU twice a day, sc, every 4 weeks); or ART alone. A significant increase of circulating CD4 cells was observed in IL-2--treated subjects, compared with those given ART alone. Low doses of IL-2 were better tolerated. Despite the incomplete suppression of viral replication, IL-2 with ART did not increase either plasma viremia or cell-associated HIV DNA levels. Low doses of intermittent sc IL-2 induced a stable increase of peripheral CD4 cells that was indistinguishable from those associated with higher, less well-tolerated doses of IL-2.

Maraviroc is a substrate for OATP1B1 in vitro and maraviroc plasma concentrations are influenced by SLCO1B1 521 T>C polymorphism

Background Organic anion transporting polypeptides (OATPs) are emerging as major determinants of pharmacokinetics for numerous drugs, with the 1B1 isoform-mediating hepatic uptake. The 521 T>C polymorphism has been correlated earlier with higher plasma concentrations of several drugs and the aim of this study was to determine whether this polymorphism influences trough concentrations of maraviroc. Methods The uptake of maraviroc by OATP1B1 was assessed using a heterologous Xenopus laevis oocyte expression system and quantified using a novel liquid chromatography-mass spectrometry method. Regression analyses were conducted to identify factors associated with maraviroc Ctrough in 59 patients treated with maraviroc at 150, 300, or 600 mg twice daily. Results Maraviroc was identified as a substrate for OATP1B1 with a Km of 33.9 &mgr;mol/l. A dose of 600 mg of etravirine or efavirenz [odds ratio (OR)=0.22, 95% confidence interval (95% CI): 0.06–0.76; P=0.016] and SLCO1B1 521 heterozygosity were both associated with maraviroc Ctrough, above the suggested target concentration of 50 ng/ml (OR=20.3, 95% CI: 2.2–182; P=0.007). Conclusion These findings show the importance of OATP1B1 for variability in maraviroc pharmacokinetics. Furthermore, the SLCO1B1 521 T>C polymorphism maybe useful in predicting higher plasma concentrations but these data should be confirmed before prospective clinical studies to define the clinical usefulness.

Induction of protective antibody response by MF59-adjuvanted 2009 pandemic A/H1N1v influenza vaccine in HIV-1-infected individuals

Objective:To determine the immunogenicity of the monovalent vaccine against 2009 pandemic influenza A/H1N1 in HIV-1-infected individuals. Design:A total of 192 participants, including 44 HIV-1-positive individuals and 148 HIV-1-negative healthy controls were enrolled to receive a single dose of MF59-adjuvanted 2009 A/H1N1v vaccine formulated to contain 7.5 μg of haemagglutin antigen. Methods:Standard haemagglutination inhibition (HAI) assay was performed to evaluate seroconversion and seroprotecsion rates against the pandemic virus in serum samples collected at baseline (T0) and 3–5-week postvaccination (T28). Seroconversion to vaccination was defined by either prevaccination HAI titer less than 1: 10 with a postvaccination titer higher than 1: 40, or a prevaccination titer higher than 1: 10 and increase of at least four-fold or more after vaccination. Seroprotection was defined by HAI titers higher than 1: 40. Results:The vaccine induced specific antibody titers in HIV-1-positive individuals similar to those of HIV-1-negative controls [215.3, 95% confidence interval (CI) 150.4–308.1 vs. 275.9, 95% CI 232.6–327.3] with postvaccination seroprotection rates higher than 97%. In contrast, the seroconversion rate was lower in the HIV-1-positive individuals as compared with the HIV-1-negative controls (36.4 vs. 79.0%, P < 0.0001), likely as a consequence of their high HAI baseline titers. Multivariable logistic regression analysis showed that seroconversion was less likely in HIV-1-positive individuals [odds ratio (OR) = 0.237, 95% CI 0.104–0.539, P = 0.0006) and with increasing age (OR = 0.805, 95% CI 0.684–0.947, P = 0.009). Conclusions:A single dose of MF59-adjuvanted 2009 influenza H1N1 vaccine induced an immune response against pandemic H1N1 virus in HIV-1-positive individuals reaching titers similar to those of HIV-1-negative individuals. The seroconversion rate was negatively associated with HIV infection and increasing age.

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT.

BackgroundT-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-γ-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level.ResultsThe cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-γ producing T-cells were also producing MIP-1β whereas T-cells characterized by the sole production of IFN-γ were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-γ+ MIP-1β+ T-cells was equivalent to the measurement of the total IFN-γ+ T-cells, we adopted the IFN-γ+ MIP-1β+ data analysis system to evaluate IFN-γ-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-γ+ MIP-1β+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay.ConclusionThe IFN-γ+ MIP-1β+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study

BackgroundTat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination.FindingsHere we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization.ConclusionsAnti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy.

Resistance to amprenavir before and after treatment with lopinavir/ritonavir in highly protease inhibitor-experienced HIV patients

Anti-hepatitis C virus (HCV) treatment combining IFN-alpha and ribavirin (IFN+R) has still poorly defined effects on immune responses. Frequencies of T helper (Th) type 1 cells specific for HCV, cytomegalovirus (CMV) and HIV were measured in chronically HCV-infected patients with or without HIV co-infection during IFN+R treatment. Although scarce, peripheral blood HCV-specific Th1 cells did not change significantly, while frequencies of HIV and CMV-specific Th1 cells decreased in co-infected patients independently of CD4 cell count changes. This suggests that IFN+R therapy might compromise virus-specific immune defenses in immunosuppressed patients.

Simplification to atazanavir/ritonavir monotherapy for HIV-1 treated individuals on virological suppression: 48-week efficacy and safety results.

Objectives:The objective of this study was to assess the 48-week virological efficacy of atazanavir/ritonavir (ATV/r) monotherapy vs. ATV/r along with two nucleoside reverse transcriptase (NRTIs) in HIV-1 treated individuals with HIV-RNA less than 50 copies/ml. Methods:A multicentre, randomized, open-label, noninferiority trial. HIV-1 treated individuals on ATV/r 300/100 mg along with two NRTIs were randomized to receive ATV/r monotherapy or to maintain their antiretroviral regimen. The primary endpoint was the confirmed viral rebound (CVR: two consecutive HIV-RNA >50 copies/ml) or treatment discontinuation for any reason. Individuals who experienced CVR on ATV/r monotherapy reintroduced NRTIs and discontinued the study if HIV-RNA was more than 50 copies/ml after 12 weeks since reintensification. Results:One hundred and three patients enrolled. By week 48, 11 patients in ATV/r arm and two in ATV/r along with two NRTIs experienced CVR; four (8%) patients in ATV/r and eight (15%) in ATV/r along with two NRTIs discontinued. At the 48-week primary efficacy analysis (re-intensification = failure), treatment success was 73% in ATV/r arm and 85% in ATV/r along with two NRTIs [difference −12.1%, 95% confidence interval (95% CI) −27.8 to 2.1]. According to the analysis considering re-intensification is equal to success, treatment success was 92% in ATV/r arm and 85% in the ATV/r along with two NRTIs arm (difference 7.5%, 95% CI −4.7 to 19.8). At CVR, no mutation was observed in ATV/r arm and reintensification with NRTIs was effective in all individuals. Overall, Grade 3–4 (P = 0.003) and grade 3–4 drug-related (P = 0.027) adverse events were less frequent in ATV/r arm. A significant increase in total and low-density lipoprotein (LDL)-cholesterol was observed as well as a significant improvement in high-density lipoprotein (HDL)-cholesterol, fasting glucose, liver fibrosis and alkaline phosphatase was observed in ATV/r monotherapy in comparison with ATV/r along with two NRTIs. Conclusion:ATV/r monotherapy treatment simplification showed lower virological efficacy in comparison with maintaining triple therapy; NRTIs reintroduction was effective in all the individuals.