Roberta Amici

Microbial translocation is associated with residual viral replication in HAART-treated HIV+ subjects with <50 copies/ml HIV-1 RNA

BACKGROUND Recent data have shown that plasma levels of lipopolysaccharide (LPS) are a quantitative indicator of microbial translocation in HIV infected individuals. OBJECTIVES To assess the impact of residual viral replication on plasma LPS in HAART-treated HIV+ subjects with <50copies/ml HIV-1 RNA and to evaluate LPS changes during repeated HAART interruptions not exceeding 2-month duration. STUDY DESIGN LPS was measured in 44 HIV+ subjects at T0 (during HAART) and at day 15 of the first and fourth HAART interruption. Ten uninfected, healthy donors were studied as well. Residual plasma HIV-1 RNA was measured at T0 by an ultra-ultrasensitive method with limit of detection of 2.5copies HIV-1 RNA/ml. Subjects with less than 2.5copies/ml (fully suppressed - FS) were compared to those with 2.5-50copies/ml (partially suppressed - PS). RESULTS At T0, plasma LPS levels were comparable in FS and uninfected subjects, whereas in PS they were higher than in uninfected subjects (p=0.049). After 4 HAART interruptions, they did not change significantly. However, LPS values were lower in FS than in PS (p=0.020). An inverse correlation was found between CD4 and LPS levels (p=0.044) in PS group only. CONCLUSIONS A reduced degree of microbial translocation was seen in subjects with a more complete suppression of viral replication. Repeated HAART interruptions had no significant impact on plasma LPS levels.

Novel Quinolinonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, and Biological Activities

Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as an anti-IN agent highly potent against both the 3′-processing and ST steps. Compound 6d was the most potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN−DNA acceptor site without affecting the DNA donor site.

Maternal antiretroviral therapy for the prevention of mother-to-child transmission of HIV in Malawi: maternal and infant outcomes two years after delivery.

Background Optimized preventive strategies are needed to reach the objective of eliminating pediatric AIDS. This study aimed to define the determinants of residual HIV transmission in the context of maternal antiretroviral therapy (ART) administration to pregnant women, to assess infant safety of this strategy, and to evaluate its impact on maternal disease. Methodology/Principal Findings A total of 311 HIV-infected pregnant women were enrolled in Malawi in an observational study and received a nevirapine-based regimen from week 25 of gestation until 6 months after delivery (end of breastfeeding period) if their CD4+ count was > 350/mm3 at baseline (n = 147), or indefinitely if they met the criteria for treatment (n. 164). Mother/child pairs were followed until 2 years after delivery. The Kaplan-Meier method was used to estimate HIV transmission, maternal disease progression, and survival at 24 months. The rate of HIV infant infection was 3.2% [95% confidence intervals (CI) 1.0-5.4]. Six of the 8 transmissions occurred among mothers with baseline CD4+ count > 350/mm3. HIV-free survival of children was 85.8% (95% CI 81.4-90.1). Children born to mothers with baseline CD4+ count < 350/mm3 were at increased risk of death (hazard ratio 2.6, 95% CI 1.1-6.1). Among women who had stopped treatment the risk of progression to CD4+ count < 350/mm3 was 20.6% (95% CI 9.2-31.9) by 18 months of drug discontinuation. Conclusions HIV transmission in this cohort was rare however, it occurred in a significative proportion among women with high CD4+ counts. Strategies to improve treatment adherence should be implemented to further reduce HIV transmission. Mortality in the uninfected exposed children was the major determinant of HIV-free survival and was associated to maternal disease stage. Given the considerable proportion of women reaching the criteria for treatment within 18 months of drug discontinuation, life-long ART administration to HIV-infected women should be considered.

Risk factors and occurrence of rash in HIV-positive patients not receiving nonnucleoside reverse transcriptase inhibitor: data from a randomized study evaluating use of protease inhibitors in nucleoside-experienced patients with very low CD4 levels (<50 cells/microL).

Most of the studies evaluating rash in HIV‐positive patients have focused on nonnucleoside reverse transcriptase inhibitors (NNRTI), particularly nevirapine, and little is known about the occurrence of rash and the risk factors for its development in patients receiving regimens not based on NNRTI.

Development of a human immunodeficiency virus vector-based, single-cycle assay for evaluation of anti-integrase compounds.

ABSTRACT Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.

High Prevalence of M184 Mutation among Patients with Viroimmunologic Discordant Responses to Highly Active Antiretroviral Therapy and Outcomes after Change of Therapy Guided by Genotypic Analysis

ABSTRACT Whether highly active antiretroviral therapy (HAART) should be modified in patients with persistent increases in CD4+ T cells despite detectable viral loads is an unresolved question. Forty-three heavily pretreated human immunodeficiency virus (HIV)-infected patients with virologic failure during HAART were studied before a change of therapy guided by genotypic analysis and during follow-up. Patients with an increase in CD4+ cell count (>100 cells/ml) over pre-HAART values were considered to be discordant patients (20 individuals), whereas patients with a lower increase or no increase in CD4+ cell count were considered failing patients (23 individuals). Based on univariate analysis, a high CD4+ cell count before antiretroviral treatment, homosexual behavior as a risk factor for HIV infection, reduced drug exposure to nonnucleoside reverse transcriptase inhibitors, low replicative capacity of HIV isolates, and more frequent detection of HIV isolates with a non-B subtype, an R5 biological phenotype, and M184V and T215Y/F mutations were factors associated with a discordant response to HAART. Based on multivariate analysis, only the M184V mutation remained significantly associated with a viroimmunologic discordant response (odds ratio, 25.48; 95% confidence interval, 1.43 to 453.93). No difference in lamivudine exposure was found between discordant (95%) and failing (91%) patients. Twelve months after the genotypic analysis-guided change of therapy, 3 discordant (15%) and 6 failing patients (26%) achieved undetectable viral loads (<50 copies/ml), whereas in patients with HIV RNA loads of >500 copies/ml, discordant responses were observed in 5 out of 15 discordant patients and in 4 out of 16 failing patients. A relationship between the M184V mutation and a viroimmunologic discordant response to HAART was found. After the genotypic analysis-driven change of therapy, similar rates of virologic suppression were detected in the two groups.

Quantification of HIV-RNA from dried blood spots using the Siemens VERSANT® HIV-1 RNA (kPCR) assay

OBJECTIVES Simplified methods for virological monitoring in resource-limited settings are increasingly needed. We evaluated the performance of the VERSANT(®) HIV-1 RNA (kPCR) assay for the determination of HIV-1 viral load from dried blood spots (DBS). Assay sensitivity and correlation with plasma quantification values were assessed. METHODS A total of 98 DBS were prepared from fresh blood samples of HIV-infected patients. DBS were kept at room temperature for 6 weeks or 7 months before processing while the corresponding plasma samples were stored at -80°C. DBS were first pre-treated in a special DBS buffer. The DBS extracts and the plasma samples were then purified and amplified using the VERSANT assay reagents. RESULTS In the first series of tests, performed after 6 weeks of storage, there was good correlation between quantification of viral load in plasma and in DBS (r = 0.95, P < 0.001). The detection rate in DBS was 100% when plasma levels were >1000 copies/mL. The sensitivity and specificity of the DBS assay were 88.2% [95% confidence interval (CI) 79.4-93.6] and 69.2% (95% CI 42.0-87.4), respectively. Using the 5000 copies/mL threshold (defining virological failure in resource-limited settings), both positive and negative predictive values were high (95.2% and 87.5%, respectively). After 7 months of storage there was a modest decrease in the detection rate and less significant correlations for samples with HIV-RNA <5000 copies/mL. CONCLUSIONS Quantification of HIV-RNA from DBS by the VERSANT automated sample preparation and detection method can be used to diagnose virological failure in HIV-positive patients.

Emergence of lamivudine resistance hepatitis B virus mutations in pregnant women infected with HBV and HIV receiving antiretroviral prophylaxis for the prevention of mother‐to‐infant transmission in Malawi

HIV/HBV co‐infection is highly prevalent in sub‐Saharan Africa. The aim of this study was to determine if the use of triple combination lamivudine‐containing prophylaxis for the prevention of mother‐to‐infant HIV transmission was associated with the emergence of lamivudine HBV mutations. The study included 21 pregnant co‐infected women in Malawi who received either zidovudine or stavudine plus lamivudine and nevirapine from week 25 of gestation until 6 months after delivery or indefinitely if they met the criteria for treatment (CD4+ <350/mm3). HBV‐DNA was determined using the Roche COBAS assay. Resistance mutations were assessed by the Trugene assay (Siemens Diagnostics). At baseline 33% of the women were HBeAg positive and had HBV‐DNA > 104 IU/ml. Median CD4 count was 237 cells/mm3 and median HIV‐RNA was 3.8 log10 copies/ml. After a median of 259 days of treatment, HBV‐DNA was detectable in 9 out of 21 patients (42.8%). In three cases the HBV‐DNA level was >104 IU/ml. Resistance mutations (M204I in five cases and L180M + M204I/V in one case) were present in 6 (28.6%) patients. Women with a resistant virus had significantly higher baseline HBV‐DNA levels than those not developing resistance (1.1 × 107 IU/ml vs. 20.8 IU/ml, P = 0.022). Levels of ALT and AST were higher in women with resistant viruses compared to those retaining a wild‐type virus. A high rate of lamivudine resistance was seen in this cohort of pregnant women. Follow‐up of these patients will clarify if the presence of resistance has a significant impact on liver disease. J. Med. Virol. 84:1553–1557, 2012.

Evaluation of HIV-1 integrase inhibitors on human primary macrophages using a luciferase-based single-cycle phenotypic assay.

Macrophages represent an important site for productive infection of HIV-1 and the evaluation of integrase (IN) inhibitors on this cell subset is of fundamental importance. In this report, preclinical evaluation of IN inhibitors on primary human macrophages was attempted successfully using a 96-well microtiter phenotypic assay developed recently for the evaluation of IN inhibitors in a cell-based system by taking advantage of HIV-derived lentiviral vectors expressing luciferase. IN inhibitors were also tested using a lentiviral vector containing an IN with introduced T66I/S153Y mutations, known to affect the activity of azido-group-containing diketo acid (DKA) IN inhibitors. Utilizing different classes of HIV integrase inhibitors against the wild-type IN and the mutant mentioned above, some of the IN inhibitors used were also active on this particular mutant, suggesting that should HIV-1 develop additional or different mutations to become resistant to such anti-IN drugs, new drugs can be developed with a better resistance profile. This assay provides a standardized method for the preclinical evaluation of the efficacy of IN inhibitors on wild-type and mutated IN that can be adapted easily for the evaluation of anti-IN activity on IN sequences derived from patients.

The mutational archive in proviral DNA does not change during 24 months of continuous or intermittent highly active antiretroviral therapy

Objectives The aim of the study was to determine the modifications of the mutational archive in proviral HIV‐1 DNA occurring during 24 months of intermittent or continuous highly active antiretroviral therapy (HAART).