Restriction Factors expression decreases in HIV-1 patients after cART.

Activation of interferon (IFN) mediated responses and the consequent expression of restriction factors (RFs) represent an early line of defense against HIV-1 infection. The levels of viral replication and the antiviral are among the determinants influencing RFs' expression pattern. A deeper understanding of the molecular mechanisms regulating RFs activity and their relationship with viral replication factors might lead to new therapeutic strategies based on the enhancement of immune response against the virus. The aim of this study is to perform a longitudinal evaluation of the variations in the levels of a group of selected RFs (APOBEC3G, BST2, TRIM5α, MX2, SAMHD1, SERINC3/5, IFI16 and STING) to determine the impact of cART on their expression in HIV-1 positive patients. Together with RFs expression, immunological and virological parameters (plasma HIV1-RNA load and total HIV1-DNA) were longitudinally evaluated in a cohorts fourteen HIV-1 cART na ve patients, who were evaluated at diagnosis (T0) and followed at 4 (T1) and 8 (T2) months after starting cART. Fourteen long-term treated patients who achieved sustained undetectable viremia for at least 2 years were also included in the study as a reference group. We observed a restoration of immunological conditions during cART, together with a progressive decrease of HIV1-RNA load, which became undetectable at 8 months after starting treatment. On the other hand, despite showing a trend towards decrease, total HIV1-DNA remained detectable after reaching viral suppression, similarly to what observed in long term treated patients. The expression of APOBEC3G, SAMHD1, BST2, IFI16, SERINC3, and SERINC5 was higher at the time of diagnosis and decreased significantly during therapy, reaching levels similar to the ones observed in virally suppressed patients. On the other hand, MX2 and TRIM5a high expression values up to T0, reaching lower levels immediately after the initiation of cART treatment. Correlation analysis showed a positive association between the expression levels of APOBEC3G, IFI16, MX2, SAMHD1, SERINC3 and TRIM5α with the HIV-1 viral load. On the contrary, no significant association was observed for BST2, SERINC5 and STING, even BST2 expression showed a tendency to correlate with viral load. We observed a tendency for a positive association of MX2, SAMHD1 and SERINC5 with the size of viral reservoir and a trend for a negative association for STING. STING appeared also as the only one factor whose expression correlates with the CD4 count and the CD4/CD8 ratio. Our data confirm the correlation between viral replication and expression of RFs, with, the levels of cellular defense proteins decreasing in parallel to the reduction of viral replication.

Development of C-TILDA: A modified TILDA method for reservoir quantification in long term treated patients infected with subtype C HIV-1.

A better characterization of the HIV reservoir is pivotal for the development of effective eradication strategies. Accurate quantification of the latent reservoir remains challenging. Starting from a regular blood draw, the Tat/Rev induced limiting dilution assay (TILDA) combines serial dilution of CD4+ T cells with a PCR-based detection of HIV-1 spliced mRNA produced upon cell stimulation. Here we adapted the original protocol for HIV-1 subtype B to detect tat/rev mRNAs transcribed from reactivated latently infected cells in long term suppressed patients infected with HIV-1 subtype C. Given the heterogeneity of global HIV epidemiology, it is pivotal to develop assays with optimal performances also in patients infected with non-B subtypes. We observed that, in these patients infected with subtype C virus, the HIV reservoir quantified by TILDA correlates with both the time of virological suppression and CD4/CD8 ratio.

Host Restriction Factors and Human Immunodeficiency Virus (HIV-1): A Dynamic Interplay Involving All Phases of the Viral Life Cycle.

Mammalian cells have evolved several mechanisms to prevent or block lentiviral infection and spread. Among the innate immune mechanisms, the signaling cascade triggered by type I interferon (IFN) plays a pivotal role in limiting the burden of HIV-1. In the presence of IFN, human cells upregulate the expression of a number of genes, referred to as IFN-stimulated genes (ISGs), many of them acting as antiviral restriction factors (RFs). RFs are dominant proteins that target different essential steps of the viral cycle, thereby providing an early line of defense against the virus. The identification and characterization of RFs have provided unique insights into the molecular biology of HIV-1, further revealing the complex host-pathogen interplay that characterizes the infection. The presence of RFs drove viral evolution, forcing the virus to develop specific proteins to counteract their activity. The knowledge of the mechanisms that prevent viral infection and their viral counterparts may offer new insights to improve current antiviral strategies. This review provides an overview of the RFs targeting HIV-1 replication and the mechanisms that regulate their expression as well as their impact on viral replication and the clinical course of the disease.

Comparison of the Aptima HIV-1 Quant Dx assay with the COBAS AmpliPrep/COBAS TaqMan HIV-1 v2.0 Test for HIV-1 viral load quantification in plasma samples from HIV-1-infected patients.

BACKGROUND AND AIMS: HIV-1 RNA viral load (VL) in plasma samples of HIV-1-positive patients is used to assess the level of viral replication, the risk of disease progression, and the response and efficacy to antiretroviral treatment. Knowing the performance of different tests for HIV-1 RNA detection is, therefore, important for clinical care. This study compared the performance of the recently introduced Aptima HIV-1 Quant Dx assay (Hologic, Inc) and the standard COBAS AmpliPrep/COBAS TaqMan HIV-1 v2.0 Test (CAP/CTM2) (Roche Molecular System, Inc) for HIV-1 RNA quantitation. METHODS: Assay performance was assessed using 335 clinical samples, a standard HIV-1 low VL panel, and 2 diluted samples from well-characterized patients infected with different HIV-1 subtypes tested in 5 replicates over 3 days. All samples were tested on both assays to evaluate inter-assay agreement, both qualitatively and quantitively. Altogether, we evaluated assay sensitivity, linearity, accuracy, precision, repeatability, and reproducibility. RESULTS: Assay agreement for qualitative results in 335 clinical samples was fair (80.6%). Correlation of quantitative assay results (n = 164) was excellent (R 2 = 0.97), with 96.3% of the results within the 95% limit of assay agreement (-0.42 to +0.86 log), and 98.8% within 1 log of each other. Aptima-HIV-1 yielded results, on average, 0.22 log higher than CAP/CTM2. Both assays accurately quantitated the HIV-1 standard at low VL (R 2 ≥ 0.94), with all samples within 0.5 log of the target. CONCLUSION: Aptima-HIV-1 assay demonstrated sensitivity, accuracy, reproducibility, and precision for the detection and quantitation of HIV-1 RNA across a wide dynamic range of VLs. Its performance, together with full automation and high throughput, suggests that Aptima-HIV-1 could be a suitable assay for reliable monitoring of HIV-1 VL in patients undergoing treatment.

Impact of Different Antiretroviral Strategies on Total HIV-DNA Level in Virologically Suppressed HIV-1 Infected Patients.

BACKGROUND: Total HIV-DNA load in peripheral blood cell (PBMCs) reflects the global viral reservoir that seems not to be affected by antiretroviral treatment. However, some studies reported a different permeability of different drugs in cellular compartments. OBJECTIVE: To investigate the relation between the amount of total HIV-1 DNA and different treatment strategies. METHODS: Total HIV-1 DNA was quantified by real time PCR in PBMCs collected from 161 patients with long-term undetectable HIV-RNA receiving different therapy schedules (3-drug regimens or 2-drug regimen containing Raltegravir as integrase inhibitor). RESULTS: Overall, HIV patients who started therapy with a median pre-ART CD4+ cell count >400 cells/mm3 and HIV viral load of 3 log10 copies/ml, achieved a lower amount of HIV total DNA. No significant correlation was found in DNA size when patients were stratified on the basis of different therapeutic protocols. However, HIV DNA load analysis, when only performed in HIV patients with a median pre-ART CD4+ cell count >200 cells/mm3 and HIV viral load < 3 log10 copies/ml, showed a significative DNA decrease in Raltegravir treated group with respect to the NNRTIs-treated group. CONCLUSION: The data emphasize that HIV-DNA level represents a predictive factor in long-term suppressive therapy patients. In addition, the diminished reservoir, only observed in patients treated with the NRTI-sparing regimen RAL plus PI/r before immunological and virological derangement, suggests that latest generation drugs, such as integrase inhibitors, might represent an optimal chance in the management of HIV infection.

HIV and kidney: a dangerous liaison.

Kidney disease represents an important health concern among HIV-infected individuals, with an estimated prevalence ranging between 2.4 and 17%. The widespread use of antiretroviral drugs has changed the epidemiology of kidney disease in the HIV positive population, drastically reducing the percentage of patients affected by HIV-associated nephropathy (HIVAN), a complication characterized by apoptosis and de-differentiation of renal epithelial cells and podocytes. However, impaired kidney function remains an important issue among HIV-infected patients because of their long-term exposure to antiretroviral drugs and the growing burden of traditional risk factors associated with chronic renal disease. Furthermore, since HIV infects renal epithelial cells, kidney is a potential reservoir site that needs to be considered in future eradication studies. This review summarizes the main risk factors associated with chronic kidney disease in HIV-infected patients and discusses the contribution of viral infection and antiretroviral therapy to the pathogenesis of renal damage, emphasizing the need to monitor kidney status during the follow-up of HIV-infected patients.

A SPRY2 mutation leading to MAPK/ERK pathway inhibition is associated with an autosomal dominant form of IgA nephropathy.

IgA nephropathy (IgAN) represents the most common primary glomerulonephritis worldwide with a prevalence of 25-50% among patients with primary glomerulopathies. In ~5-10% of the patients the disease segregates with an autosomal dominant (AD) pattern. Association studies identified loci on chromosomes 1q32, 6p21, 8p23, 17p13, 22q12, whereas classical linkage studies on AD families identified loci on chromosomes 2q36, 4q26-31, 6q22, 17q12-22. We have studied a large Sicilian family where IgAN segregates with an AD transmission. To identify the causal gene, the exomes of two affected and one unaffected individual have been sequenced. From the bioinformatics analysis a p.(Arg119Trp) variant in the SPRY2 gene was identified as the probable disease-causing mutation. Moreover, functional characterization of this variant showed that it is responsible for the inhibition of the MAPK/ERK1/2 pathway. The same effect was observed in two sporadic IgAN patients carriers of wild-type SPRY2, suggesting that downregulation of the MAPK/ERK1/2 pathway represents a common mechanism leading to IgAN.