Synergistic inhibition of HIV-1 in activated and resting peripheral blood mononuclear cells, monocyte-derived macrophages, and selected drug-resistant isolates with nucleoside analogues combined with a natural product, resveratrol

Resveratrol (trans-3,5,4;-trihydroxystilbene) is a phytoalexin present in grapes, wine, and certain plants, which has recently been reported to possess properties that may protect against atherosclerosis, certain cancers, and inflammation. We now report that resveratrol (RV) synergistically enhances the anti-HIV-1 activity of the nucleoside analogues zidovudine (AZT), zalcitabine (ddC), and didanosine (ddI). RV at 10 microM was not toxic to cells, and by itself reduced viral replication by 20% to 30%. In phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs) infected with HTLV-IIIB, 10 microM RV reduced the 90 % inhibitory concentration (IC90) of AZT, ddC, and ddI by 3.5-, 5.5-, and 17.8-fold, respectively. Similar antiviral activity was demonstrated when ddI was combined with 5 or 10 mM RV in PBMCs infected with clinical isolates of HIV-1. The addition of RV resulted in a >10-fold augmentation of ddI-antiviral activity in infected monocyte-derived macrophages (MDMs). In a resting cell model of T lymphocytes which were infected with HTLV-IIIB, RV plus ddI in combination, but not individually, suppressed establishment of a productive viral infection. In addition, RV plus ddI markedly inhibited the replication of four ddI-resistant viral isolates, three of which presented mutations in the RT gene conferring RT-multidrug resistance. Finally, when compared with hydroxyurea (HU), both 100 mM HU and 10 mM RV showed similar enhancement of ddI-antiviral suppressive activity. However, RV was shown to have less of a cellular antiproliferative effect than HU.     

Effective highly active antiretroviral therapy in patients with primary HIV-1 infection prevents the evolution of the avidity of HIV-1-specific antibodies

OBJECTIVE: To evaluate if the administration of highly active antiretroviral therapy (HAART) during primary HIV infection (PHI) may affect the antibody avidity evolution. METHODS: In 13 subjects with symptomatic PHI, of whom 8 initiated HAART at diagnosis, the Avidity Index (AI) and Western blot evolution patterns were analyzed on serial serum/plasma samples for 1 year. In 4 patients, who subsequently interrupted HAART, additional specimens were analyzed. RESULTS: At diagnosis, the range of HIV viremia was 0.003 to 38 x 10(6) copies/mL. In untreated patients, viremia reached the set point in 4 to 6 months, whereas in treated patients, early suppression of viremia was observed, remaining undetectable during therapy. At diagnosis, the median AI was low in untreated (0.42, range: 0.33 to 0.43) and treated (0.44, range: 0.40 to 0.72) patients. At 3, 6, and 12 months, the AI progressively increased in untreated patients, whereas it remained <0.80 in all treated patients. In the 4 patients interrupting HAART, the AI increased after therapy interruption to greater than 0.80 in < or = 6 months. The Western blot pattern transiently/partially reversed during HAART in 2 patients. CONCLUSIONS: Antibody avidity maturation takes place only in the presence of ongoing viral replication. These results may have relevant implications in understanding the complex mechanism of maturation of the immune response to HIV.     

High-performance liquid chromatographic method for the determination of HIV-1 non-nucleoside reverse transcriptase inhibitor efavirenz in plasma of patients during highly active antiretroviral therapy

A new high-performance liquid chromatographic method for the determination of efavirenz in human plasma is described. Quantitative recovery following liquid-liquid extraction with diethylether from 200 microl of human plasma was achieved. Subsequently, the assay was performed with 67 mM potassium dihydrogen phosphate-acetonitrile as a mobile phase, a XTerraRP 18 column protected with a Phenomenex C18 column and UV detection at 246 nm. Linear standard curves were obtained for concentrations ranging from 25 to 15,000 ng/ml. The calculated intra- and inter-day coefficients of variation were below 10%.     

Induction of HIV-1-specific T cell responses by administration of cytokines in late-stage patients receiving highly active anti-retroviral therapy.

Highly active anti-retroviral therapy (HAART) is associated with reduction in the morbidity and mortality of patients with advanced HIV-1 disease. The ability of such treatment to improve immune responses against HIV-1 and opportunistic pathogens is variable and limited. Addition of cytokine immunotherapy to this treatment may improve immune responses. IL-2 with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered to HIV-1+ individuals receiving HAART with undetectable viral loads, and CD4 counts < 100 cells/microl. In one patient presenting with Mycobacterium avium complex (MAC) infection, we evaluated the effect of cytokine immunotherapy on lymphocyte phenotype; plasma viral load; proliferative responses to mitogens, recall and HIV-1 antigens; cytokine production and message in response to non-specific and specific stimuli; and natural killer (NK) cell activity. Proliferation assays were performed in two similar patients. Before cytokine immunotherapy the predominant CD8+ population was mainly CD28-. No proliferation or IL-2 production was seen in response to mitogens, recall or HIV-1 antigens; and no HIV-1 peptide-specific interferon-gamma (IFN-gamma)-secreting cells were present. Low levels of IL-4 were detected in response to antigens to which patients had been exposed, associated with up-regulated expression of costimulatory molecules influenced by IL-4. Following IL-2 administration, loss of IL-4 was associated with increased NK cell activity and HIV-1 peptide-specific and non-specific IFN-gamma-producing cells. Proliferative responses associated with IL-2 production and responsiveness were only seen after subsequent concomitant administration of GM-CSF with IL-2. These changes mirrored clinical improvement. An imbalance of lymphocyte subsets may account for immune unresponsiveness when receiving HAART. Restoration of responses following immunotherapy suggests a shift towards a lymphocyte profile with anti-pathogen activity.     

Effects of potent antiretroviral therapy on the immune activation marker soluble CD27 in patients infected with HIV-1 subtypes A-D

HIV-1 genetic subtypes might have a different impact on disease progression and response to antiretroviral therapy (ART). Few data are available on the immune activation profile in patients with different HIV-1 subtypes. We have tested by ELISA the plasma levels of an immune activation marker, soluble CD27 (sCD27), in a cohort of 64 patients infected with HIV-1 subtypes A-D, at baseline and after 1 year of virologically successful ART. Plasma sCD27 was significantly higher in the whole HIV-1-infected population as compared to healthy subjects [522 U/ml (188-1,307) vs. 285 U/ml (174-397), P < 0.001]. Among the four different HIV-1 subtypes, patients with subtype C virus had significantly higher plasma sCD27 [684 U/ml, (188-1228)] as compared to patients with subtype A [428 U/ml (247-1307), P < 0.05] and B [454 (211-925), P < 0.05]. After 1 year of ART, plasma sCD27 significantly decreased in all groups but patients with subtype C viruses had the largest reduction of sCD27 from baseline. The data indicate that a similar immune activation profile is present in patients infected with HIV-1 subtypes A, B, and D and that in presence of successful ART these subtypes respond similarly in terms of immune activation. Intriguingly, subtype C infection seems to be associated with higher levels of plasma sCD27, suggesting that HIV-1 genetic subtype C may have a different impact on disease outcome and response to therapy.     

In vivo emergence of drug-resistant mutations at less than 50 HIV-1 RNA copies/mL that are maintained at viral rebound in longitudinal plasma samples from human immunodeficiency virus type-1-infected patients on highly active antiretroviral therapy

OBJECTIVE: Emergence of human immunodeficiency virus type-1 (HIV-1) genotypic drug resistance is generally attributed to noncompliance, poorly absorbed drugs, or drug-to-drug interaction. Attempts to determine emerging genotypic drug resistance from study subjects on highly active antiretroviral therapy (HAART) relied on insensitive polymerase chain reaction (PCR) techniques, revealing wild type HIV-1 or precursor resistant genotypes from few plasma samples successfully amplified with <50 copies/mL. STUDY DESIGN/METHODS: In this analysis, using Applied Biosystems' ViroSeq HIV-1 Genotyping Systems, Version 2.0 (Applied Biosystems, Foster City, CA, USA) and the supplemental, for research use only, nested PCR primers, genotypic drug resistance was determined in longitudinal plasma samples from 11 study subjects on HAART. RESULTS: In 4 of 11 study subjects, newly emerging genotypic primary resistant mutations were detected in plasma samples with <50 copies/mL. Most of these primary drug-resistant mutations were detected in subsequent longitudinal samples with detectable viral load (viral breakthrough). CONCLUSIONS: This analysis suggests sufficient viral replication <50 copies/mL to generate genotypic drug resistance in study subjects on suppressive HAART.     

Rapid and durable antiretroviral effect of the HIV-1 Integrase inhibitor raltegravir as part of combination therapy in treatment-naive patients with HIV-1 infection: results of a 48-week controlled study

BACKGROUND: Raltegravir is an HIV-1 integrase strand-transfer inhibitor with potent in vitro activity. This study explored the antiretroviral activity and safety of raltegravir in treatment-naive patients with plasma HIV-1 RNA levels > or = 5000 copies/mL and CD4 T-cell counts > or = 100 cells/mm. METHODS: Multicenter, double-blind, randomized, controlled study of raltegravir at doses of 100, 200, 400, and 600 mg twice daily versus efavirenz at a dose of 600 mg/d, all in combination with tenofovir at a dose of 300 mg/d and lamivudine at a dose of 300 mg/d (clinicaltrials.gov identifier: NCT00100048). RESULTS: In the 198 patients treated (160 on raltegravir and 38 on efavirenz), the mean HIV-1 RNA level ranged from 4.6 to 4.8 log10 copies/mL at baseline. At weeks 2, 4, and 8, the proportion of patients achieving an HIV-1 RNA level <50 copies/mL was greater in each of the raltegravir treatment groups than in the efavirenz group. By week 24, all treatment groups appeared similar, with plasma HIV-1 RNA levels <400 copies/mL in 85% to 98% of patients and <50 copies/mL in 85% to 95% of patients. These reductions were maintained through week 48 in 85% to 98% of patients and in 83% to 88% of patients, respectively. Five (3%) patients on raltegravir and 1 (3%) on efavirenz experienced virologic failure before week 48. Drug-related clinical adverse events were less common with raltegravir than with efavirenz. After 24 and 48 weeks of treatment, raltegravir did not result in increased serum levels of total cholesterol, low-density lipoprotein cholesterol, or triglycerides. CONCLUSIONS: Raltegravir at all doses studied was generally well tolerated in combination with tenofovir and lamivudine. Raltegravir exhibited potent and durable antiretroviral activity similar to that of efavirenz at 24 and 48 weeks but achieved HIV-1 RNA levels below detection at a more rapid rate.     

Sensitivity of the Multispot HIV-1/HIV-2 rapid test using samples from human immunodeficiency virus type 1-positive individuals with various levels of exposure to highly active antiretroviral therapy

The Multispot HIV-1/HIV-2 rapid test detects human immunodeficiency virus type 1 (HIV-1) gp41 antibodies, which can wane over time in some HIV-1-infected populations, resulting in false-negative screening results. Multispot sensitivity was 100% using 248 sera from one such population, and it correctly identified serostatus in individuals who previously tested false negative with rapid testing.     

High-performance liquid chromatographic method for the simultaneous determination of HIV-1 protease inhibitors indinavir, saquinavir and ritonavir in plasma of patients during highly active antiretroviral therapy

A new high-performance liquid chromatographic method for the simultaneous determination of indinavir, saquinavir and ritonavir in human plasma is described. Quantitative recovery following liquid-liquid extraction with diethyl ether from 500 microl of human plasma was achieved. Subsequently, the assay was performed with a linear gradient starting at 67 mM potassium dihydrogenphosphate-acetonitrile (65:35 to 40:60, v/v) as a mobile phase, a Phenomenex C18 column and UV detection at 240 and 258 nm, respectively. Linear standard curves were obtained for concentrations ranging from 75 to 20,000 ng/ml for indinavir, from 10 to 6000 ng/ml for saquinavir, and from 45 to 30,000 ng/ml for ritonavir. The calculated intra- and inter-day coefficients of variation were below 6%.