A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption.

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses.

Comprehensive analysis of the intracellular metabolism of antiretroviral nucleosides and nucleotides using liquid chromatography-tandem mass spectrometry and method improvement by using ultra performance liquid chromatography.

Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1 × 10(6) cells. The platform consists of two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC-MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC-MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1 nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%.

Ultra-fast analysis of plasma and intracellular levels of HIV protease inhibitors in children: a clinical application of MALDI mass spectrometry.

HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 microL plasma and 1x10(6) PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots.

Kaletra (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)-ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.

A mass spectrometry based imaging method developed for the intracellular detection of HIV protease inhibitors.

Mass spectrometry imaging is a promising technique for measuring drugs and drug metabolites in cells and tissues. In this manuscript we describe a method for the imaging of HIV protease inhibitors. As a model system we used Mono Mac 6 cells cultured with the HIV protease inhibitors saquinavir and nelfinavir deposited on glass slides using a cytocentrifuge. A sublimation/deposition device for homogeneous matrix deposition was constructed which allows imaging of these HIV protease inhibitors at clinically relevant concentrations. Using this matrix sublimation/deposition method, glass slides containing the cytocentrifuged cells can be measured and analyzed by two types of mass spectrometry techniques, viz. matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and MALDI Fourier transform ion cyclotron resonance (FTICR), and this makes it possible to perform imaging rapidly (MALDI-TOF) and with a very high selectivity (MALDI-FTICR).

Quantitative analysis of HIV-1 protease inhibitors in cell lysates using MALDI-FTICR mass spectrometry.

In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and poor spot-to-spot reproducibilities. We found that the quantitative precisions improved significantly when DMSO (dimethylsulfoxide) was added to the matrix solution. For lopinavir and ritonavir, currently the most frequently prescribed HIV-1 protease inhibitors, the signal-to-noise ratios improved significantly when potassium iodide was added to the matrix solution. The mean quantitative precisions, expressed as % relative standard deviation, were 6.4% for saquinavir, 7.3% for lopinavir, 8.5% for ritonavir, 11.1% for indinavir, and 7.2% for nelfinavir. The mean quantitative accuracies, expressed as % deviation, were 4.5% for saquinavir, 6.0% for lopinavir, 5.9% for ritonavir, 6.6% for indinavir, and 8.0% for nelfinavir. The concentrations measured for the individual quality control samples were all within 85-117% of the theoretical concentrations. The lower limits of quantification in cell lysates were 4 fmol/microL for saquinavir, 16 fmol/microL for lopinavir, 31 fmol/microL for ritonavir, and 100 fmol/microL for indinavir and nelfinavir. The mean mass accuracies for the protease inhibitors were 0.28 ppm using external calibration. Our results show that MALDI-FTICR mass spectrometry can be successfully used for precise, accurate, and selective quantitative analyses of HIV-1 protease inhibitors in cell lysates. In addition, the lower limits of quantification obtained allow clinical applications of the technique.

Fluorescent antigen-transfected target cell cytotoxic T lymphocyte assay for ex vivo detection of antigen-specific cell-mediated cytotoxicity.

Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigentransfected target cellCTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigengreen fluorescent protein (GFP) fusion proteins. After coculture at various effector : target (E : T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with human immunodeficiency virus (HIV) and influenza virusspecific CTL clones and revealed cytotoxicity at lower E : T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated immunity.