Gruters, R.A.

Induction of humoral and cellular immune responses by antigen-expressing immunostimulatory liposomes (Article)

2012-12-28T00:00:00Z

Recently we have shown that liposomes can be used as artificial microbes for the production and delivery of DNA-encoded antigens. These so-called antigen-expressing immunostimulatory liposomes (AnExILs) were superior in inducing antigen-specific antibodies compared to conventional liposomal protein or DNA vaccines when tested in mice after i.m. immunization. In this study, we investigated the capacity of AnExILs to induce T-cell responses. By using a plasmid vector encoding a model antigen under control of both the prokaryotic T7 and the eukaryotic CMV promoter we hypothesized that antigen production could lead to CTL activation via two distinct routes: i. production of antigens inside the AnExILs with subsequent cross-presentation after processing by APCs and ii. endogenous production of antigens after AnExIL-mediated transfection of the pDNA. Although we were not able to demonstrate transfection-mediated expression of luc-NP in mice, i.m. injection of AnExILs producing luc-NP resulted in T-cell responses against the encoded NP epitope, as determined by tetramer staining. T-cell responses were comparable to the responses obtained after i.m. injection of naked pDNA. In order to find out whether CTL activation was caused by cross-presentation of the exogenous antigens produced inside AnExILs or by endogenous antigen production from transfection with the same pDNA source a second study was initiated in which the contribution of each of these effects could be separately determined. These results demonstrate that the observed T-cell responses were not exclusively caused by cross-presentation of the AnExIL-produced antigens alone, but were rather a combination of dose-dependent antigen cross-presentation and low levels of endogenous antigen production.

Dried matrix on paper disks: The next generation DBS microsampling technique for managing the hematocrit effect in DBS analysis (Article)

2012-08-01T00:00:00Z

Background: The hematocrit effect is a hurdle for successful introduction of the dried blood spot (DBS) in a regulated environment. Recently, attempts were taken to overcome the hematocrit effect by whole-cut DBS analysis. This paper presents the next-generation whole-cut DBS; dried matrix on paper disks (DMPD). Results: DMPD eliminated the hematocrit effect and demonstrated better accuracy and precision than regular DBS with partial punching. Observed accuracy and precision were 6.0 and 2.3% for DMPD, respectively, and-10.4 and 17.1%, for DBS, respectively. Conclusion: The DMPD technique performed better than regular DBS by eliminating the hematocrit effect related blood volume bias. Although this effect was not observed with DMPD, a systematic error of 6.0% was detected and further technical development of DMPD could improve the performance.

Efficient Nef-mediated downmodulation of TCR-CD3 and CD28 Is associated with high CD4+ T cell counts in viremic HIV-2 infection (Article)

2012-05-01T00:00:00Z

The role of the multifunctional accessory Nef protein in the immunopathogenesis of HIV-2 infection is currently poorly understood.Here, we performed comprehensive functional analyses of 50 nef genes from 21 viremic (plasma viral load,>500 copies/ml) and 16 nonviremic (<500) HIV-2-infected individuals. On average, nef alleles from both groups were equally active in modulatingCD4, TCR-CD3, CD28, MHC-I, and Ii cell surface expression and in enhancing virion infectivity. Thus, many HIV-2-infected individuals efficiently control the virus in spite of efficient Nef function. However, the potency of nef alleles indownmodulating TCR-CD3 and CD28 to suppress the activation and apoptosis of T cells correlated with high numbers of CD4+T cells in viremic patients. No such correlations were observed in HIV-2-infected individuals with undetectable viral load. Furtherfunctional analyses showed that the Nef-mediated downmodulation of TCR-CD3 suppressed the induction of Fas, Fas-L, PD-1, and CTLA-4 cell surface expression as well as the secretion of gamma interferon (IFN-γ) by primary CD4+T cells. Moreover, we identified a single naturally occurring amino acid variation (I132T) in the core domain of HIV-2 Nef that selectivelydisrupts its ability to downmodulate TCR-CD3 and results in functional properties highly reminiscent of HIV-1 Nef proteins. Taken together, our data suggest that the efficient Nef-mediated downmodulation of TCR-CD3 and CD28 help viremic HIV-2-infected individuals to maintain normal CD4+T cell homeostasis by preventing T cell activation and by suppressing the inductionof death receptors that may affect the functionality and survival of both virally infected and uninfected bystander cells.

Sequence evolution and escape from specific immune pressure of an HIV-1 Rev epitope with extensive sequence similarity to human nucleolar protein 6 (Article)

2012-03-01T00:00:00Z

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8+T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8+T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4+T-cell count and viral load remained stable despite escape from T-cell recognition.

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

2012-03-01T00:00:00Z

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.

Current and future applications of dried blood spots in viral disease management (Article)

2012-03-01T00:00:00Z

Almost five decades after their first application in diagnostics, dried blood spot (DBS) cards remain to be of key interest in many research areas and clinical applications. The advantages of sample stability during transport and storage, can now be combined with the high sensitivity of novel diagnostic techniques for the measurement and analysis of nucleic acids, proteins and small molecules which may overcome the limitations of the small samples sizes in DBS cards. Here we present a survey of the literature on the use of DBS cards for diagnosis, monitoring and epidemiological studies of virus infections other than HIV, including CMV, HBV, HCV, HAV, HEV, HTLV, EBV, HSV, measles-, rubella- and dengue-virus. The minimal invasiveness of sampling and the relative ease of handling and storing DBS cards is expected to offer additional opportunities to measure and analyze biomarkers of viral disease in resource poor settings or when limited amount of blood can be obtained. Large retrospective studies of virus infections in newborns using stored DBS cards have already been undertaken for screening of congenital infections. In addition, DBS cards have been used prospectively for prevalence studies, outbreak surveillance, mass screening for viral infections, follow-up of chronic infection and its treatment in resource-limited areas. We do not expect that current wet sampling techniques of plasma or serum will be replaced by DBS sampling but it allows extension of sampling in persons and settings that are currently difficult to access or that lack suitable storage facilities. In conclusion, DBS card sampling and storage will aid adequate outbreak management of existing and emerging viral diseases.

Incurred sample reanalysis comparison of dried blood spots and plasma samples on the measurement of lopinavir in clinical samples (Article)

2012-02-01T00:00:00Z

Background: Undoubtedly, incurred sample reanalysis (ISR) will become an integral part of regulated bioanalysis of dried blood spot (DBS) samples. In this article, we report results from an ISR study on DBS specimen and their corresponding plasma samples. Incurred samples were reanalyzed on their concentration of the antiretroviral drug lopinavir (LPV). Results: Bland-Altman comparison plots showed a high degree of agreement between the measurements; 94.7% of observed LPV concentrations were within bias ±2 SD. Moreover, 73.7% of obtained LPV concentrations from DBS ISR were in good compliance with general acceptance criteria (4-6-20 rule) on ISR testing, while plasma ISR failed on these acceptance criteria due to the low compliance of 10.5%. Conclusion: It was demonstrated that plasma ISR testing failed on acceptance criteria while corresponding incurred DBS specimens passed. Furthermore, the current article demonstrates that the stability of the antiretroviral drug LPV was significantly different in both biological matrices.

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 (Article)

2011-09-15T00:00:00Z

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. 1×106cells. 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 1nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%.

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate-a validated assay for the clinic (Article)

2011-05-03T00:00:00Z

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5-1,500 ng/mL for oseltamivir and 20-1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®.

Determination of the antiretroviral drug tenofovir in plasma from HIV-infected adults by ultrafast isotope dilution MALDI-triple quadrupole tandem mass spectrometry (Article)

2011-03-01T00:00:00Z

A new and reliable mass spectrometric method using an isotope dilution method in combination with matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (ID-MALDI-QqQ-MS/MS) has been developed and validated for the determination of concentrations of the antiretroviral drug tenofovir (TNV) in plasma from HIV-infected adults. The advantage of this new method is that (1) the method is ultrafast and(2) can be applied for high-throughput measurement of TNV in plasma. The method is based on a simple plasma deproteinization step in combination with the use of [adenine-13C5]-TNV as the internal standard. TNV and [adenine-13C5]-TNV were monitored by multiple reaction monitoring using the transition m/z 288.0 → 176.2 and m/z 293.2 → 181.2 for TNV and [adenine-13C5]-TNV, respectively. The method was validated according to the most recent FDA guidelines for the development and validation of (new) bio-analytical assays. Validated method parameters were: linearity, accuracy, precision and stability of the method. The lowest limit of quantification was 0.10 μmol/l, whereas the limit of detection determined at a signal-to-noise ratio (S/N = 3 : 1) in pooled drug free human control plasma was 0.04 μmol/l. The validated method was successfully applied and tested for its clinical feasibility by the analysis of plasma samples from selected HIV-infected adults receiving the prodrug tenofovir disoproxil fumarate. Observed plasma TNV concentrations ranged between 0.11 and 0.76 μmol/l and measured plasma TNV concentrations were within the therapeutically relevant concentration range. Copyright

Biomedical application of MALDI mass spectrometry for small-molecule analysis (Article)

2011-01-01T00:00:00Z

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is an emerging analytical tool for the analysis of molecules with molar masses below 1,000 Da; that is, small molecules. This technique offers rapid analysis, high sensitivity, low sample consumption, a relative high tolerance towards salts and buffers, and the possibility to store sample on the target plate. The successful application of the technique is, however, hampered by low molecular weight (LMW) matrix-derived interference signals and by poor reproducibility of signal intensities during quantitative analyses. In this review, we focus on the biomedical application of MALDI-MS for the analysis of small molecules and discuss its favorable properties and its challenges as well as strategies to improve the performance of the technique. Furthermore, practical aspects and applications are presented.

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

2010-09-01T00:00:00Z

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.

Ultra-fast analysis of plasma and intracellular levels of HIV protease inhibitors in children: A clinical application of MALDI mass spectrometry (Article)

2010-08-13T00:00:00Z

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 μL plasma and 1 × 106 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 ofthese 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.

P18-03. Dendritic cell-based immune therapy against HIV-1 (Article)

2009-10-22T00:00:00Z

Characterization of recombinant influenza A virus as a vector for HIV-1 p17Gag (Article)

2009-09-25T00:00:00Z

We have generated a recombinant influenza A virus with the HIV-1 p17Gag(rFlu-p17) gene inserted into the influenza virus neuraminidase (NA) gene. Expression of HIV-1 p17 protein was detected by conventional Western blot analysis and also by liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of rFlu-p17 infected cells. The latter method does not depend on protein-specific antibody preparations and proved to be a powerful tool to detect proteins of interest. Next, antigen presentation of p17 expressed after infection of antigen-presenting cells was determined. Cloned p17-specific CD8+ T-cells were co-cultured with rFlu-p17 infected B-cells and produced IFN-γ upon stimulation. Furthermore, we showed that immunization with rFlu-p17 elicited a humoral immune response in mice. This study shows that replication-deficient rFlu-p17 is antigenic in vitro and immunogenic in vivo and warrants further development as a candidate vaccine vector.

A Mass spectrometry based imaging method developed for the intracellular detection of HIV protease inhibitors (Article)

2009-04-30T00:00:00Z

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-flght (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). Copyright

Vaccine-induced enhancement of viral infections (Article)

2009-01-22T00:00:00Z

Examples of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis have been documented for infections by members of different virus families. Several mechanisms, many of which still are poorly understood, are at the basis of this phenomenon. Vaccine development for lentivirus infections in general, and for HIV/AIDS in particular, has been little successful. Certain experimental lentiviral vaccines even proved to be counterproductive: they rendered vaccinated subjects more susceptible to infection rather than protecting them. For vaccine-induced enhanced susceptibility to infection with certain viruses like feline coronavirus, Dengue virus, and feline immunodeficiency virus, it has been shown that antibody-dependent enhancement (ADE) plays an important role. Other mechanisms may, either in the absence of or in combination with ADE, be involved. Consequently, vaccine-induced enhancement has been a major stumble block in the development of certain flavi-, corona-, paramyxo-, and lentivirus vaccines. Also recent failures in the development of a vaccine against HIV may at least in part be attributed to induction of enhanced susceptibility to infection. There may well be a delicate balance between the induction of protective immunity on the one hand and the induction of enhanced susceptibility on the other. The present paper reviews the currently known mechanisms of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis.

FATT-CTL assay for detection of antigen-specific cell-mediated cytotoxicity (Article)

2008-11-01T00:00:00Z

Here we describe a flowcytometric assay that measures the defining function of virusspecific cytotoxic T lymphocytes (CTL), i.e., killing viral protein expressing cells. The fluorescent antigen-transfected target cell (FATT)-CTL assay requires no viruses, recombinant viral vectors, or radioactive isotopes to generate CTL target cells that present naturally processed epitopes. It facilitates developing standardized applications in clinical trial settings. Plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used directly to nucleofect immortalized B cells or peripheral blood mononuclear cells (PBMCs). Elimination of antigen-GFP expressing cells by cloned CTL, in vitro sensitized PBMC, or ex vivo PBMC was quantified following a 4-18-h coculture period by flowcytometry. This technology successfully detected cellmediated cytotoxicity in studies involving human PBMC and various viral antigens, including structural proteins of influenza A virus, and structural and nonstructural HIV proteins. Standardized protocols are currently being developed in the framework of a clinical immunotherapy trial in HIV-infected individuals. The FATT-CTL assay principles facilitate standardized flowcytometric detection of antigenic protein-specific cell-mediated cytotoxicity in many different basic research and clinical trial settings. By measuring their defining function, the FATT-CTL assay contributes to a more complete assessment of antigen-specific CTL responses to infection and vaccination.

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef (Article)

2008-07-04T00:00:00Z

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4+and CD8+cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.

Quantitative analysis of antiretroviral drugs in lysates of peripheral blood mononuclear cells using MALDI-triple quadrupole mass spectrometry (Article)

2008-07-01T00:00:00Z

We report here on the use of a prototype matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometer for quantitative analysis of six anti-retroviral drugs in lysates of peripheral blood mononuclear cells (PBMC). Of the five investigated MALDI matrixes, 2,5-dihydroxybenzoic acid (DHB) and the novel 7-hydroxy-4-(trifluoromethyl) coumarin (HFMC) showed the broadest application ranges for the antiretroviral drugs. For DHB, the mean relative errors ranged from 8.3 (ritonavir) to 4.3% (saquinavir). The mean precisions (CV) ranged from 17.3 (nevirapine) to 10.8% (saquinavir). The obtained lower limits of quantitation (LLOQ) readily allow clinical applications using just 1 million PBMC from HIV-infected patients under therapy. The new matrix HFMC was used for quantitative analysis of the HIV protease inhibitor indinavir using a stainless steel target plate as well as a target plate with a novel, strongly hydrophobic fluoropolymer coating. Using the coated target plate, the mean relative error improved from 10.1 to 4.6%, the mean precision from 33.9 to 9.9% CV, and the LLOQ from 16 to 1 fmol. In addition, the measurement time for one spot went down from 6 to only 2.5 s.

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

2008-05-15T00:00:00Z

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/μL for saquinavir, 16 fmol/μL for lopinavir, 31 fmol/μL for ritonavir, and 100 fmol/μL 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.

AIDS vaccine research: Consider co-infections (Article)

2008-03-07T00:00:00Z

The loss of immunodominant epitopes affects interferon-γ production and lytic activity of the human influenza virus-specific cytotoxic T lymphocyte response in vitro (Article)

2007-05-01T00:00:00Z

In the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)418-426epitope on interferon (IFN)-γ-production and lytic activity of the human cytotoxic T lymphocyte (CTL) response in vitro. Extensive amino acid variation at T cell receptor contact residues of the NP418-426epitope has led to repeated evasion from specific CTL. We generated recombinant influenza viruses with variants of the NP418-426epitope, which were used to stimulate peripheral blood mononuclear cells obtained from six HLA-B*3501-positive study subjects in order to expand virus-specific CTL. Loss of the NP418-426epitope resulted in a significant reduction of IFN-γ-expressing CD8+T cells, similar to that observed previously after the loss of the HLA-B*2705-restricted NP383-391epitope. In addition, the effect of the loss of the NP418-426epitope on the lytic activity of the virus-specific CTL response was assessed. Also this functional property of the virus-specific CTL response was affected significantly by the loss of this and the NP383-391epitope, as determined using the newly developed fluorescent antigen-transfected target cell (FATT)-CTL assay. These findings indicate that the loss of single immunodominant epitopes affects the functionality of the virus-specific CTL response significantly.

Validation of an HIV-1 inactivation protocol that is compatible with intracellular drug analysis by mass spectrometry (Article)

2007-02-15T00:00:00Z

Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this end, we extracted HIV-1 producing cells with methanol or ethanol at 4 °C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6 log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.

CCR5, GPR15, and CXCR6 are major coreceptors of human immunodeficiency virus type 2 variants isolated from individuals with and without plasma viremia. (Article)

2005-02-01T00:00:00Z

Human immunodeficiency virus type 2 (HIV-2) is generally considered capable of using a broad range of coreceptors. Since HIV-2 variants from individuals with nonprogressive infection were not studied previously, the possibility that broad coreceptor usage is a property of variants associated with progressive infection could not be excluded. To test this, we determined the coreceptor usage of 43 HIV-2 variants isolated from six long-term-infected individuals with undetectable plasma viremia. Using GHOST indicator cells, we showed for the first time that the only coreceptors efficiently used by low-pathogenic HIV-2 variants are CCR5, GPR15 (BOB), and CXCR6 (BONZO). Surprisingly, control HIV-2 variants (n = 45) isolated from seven viremic individuals also mainly used these three coreceptors, whereas use of CCR1, CCR2b, or CCR3 was rare. Nearly a quarter of all HIV-2 variants tested could infect the parental GHOST cells, which could be partially explained by CXCR4 usage. Use of CXCR4 was observed only for HIV-2 variants from viremic individuals. Thirty-eight variants from aviremic and viremic HIV-2-infected individuals were additionally tested in U87 cells. All except one were capable of infecting the parental U87 cells, often with high efficiency. When virus production in parental cells was regarded as background in the coreceptor-transduced cell lines, the results in U87 cells were largely in agreement with the findings in GHOST cells. HIV-2 isolates from aviremic individuals commonly use as coreceptors CCR5, GPR15, and CXCR6, as well as an unidentified receptor expressed by U87 cells. Broad coreceptor usage, therefore, does not appear to be associated with pathogenicity of HIV-2.

SIVdrl detection in captive mandrills: are mandrills infected with a third strain of simian immunodeficiency virus? (Article)

2004-11-01T00:00:00Z

A pol-fragment of simian immunodeficiency virus (SIV) that is highly related to SIVdrl-pol from drill monkeys (Mandrillus leucophaeus) was detected in two mandrills (Mandrillus sphinx) from Amsterdam Zoo. These captivity-born mandrills had never been in contact with drill monkeys, and were unlikely to be hybrids. Their mitochondrial haplotype suggested that they descended from founder animals in Cameroon or northern Gabon, close to the habitat of the drill. SIVdrl has once before been found in a wild-caught mandrill from the same region, indicating that mandrills are naturally infected with a SIVdrl-like virus. This suggests that mandrills are the first primate species to be infected with three strains of SIV: SIVmnd1, SIVmnd2, and SIVdrl.

Comparison of the efficacy of early versus late viral proteins in vaccination against SIV. (Article)

2002-07-26T00:00:00Z

The immune response against early regulatory proteins of simian- and human immunodeficiency virus (SIV, HIV) has been associated with a milder course of infection. Here, we directly compared vaccination with Tat/Rev versus Pol/Gag. Challenge infection with SIVmac32H (pJ5) suggested that vaccination with Tat/Rev induced cellular immune responses that enabled cynomolgus macaques to more efficiently control SIV replication than the vaccine-induced immune responses against Pol/Gag. Vaccination with Tat/Rev resulted in reduced plasma SIV loads compared with control (P=0.058) or Pol/Gag-vaccinated (P=0.089) animals, with undetectable plasma viral loads in two of the four Tat/Rev-vaccinated animals. Therefore, the results warrant further investigation of the early regulatory proteins and their potential for vaccination against HIV.

The advantage of early recognition of HIV-infected cells by cytotoxic T lymphocytes. (Article)

2002-05-06T00:00:00Z

Accumulating evidence indicates that cytotoxic T-lymphocytes (CTL) play an important role in the clearing of primary and control of chronic human immunodeficiency virus (HIV) infection. Here, we discuss recent findings that indicate that the timing of target cell recognition critically contributes to CTL effectiveness. In this light several problems that have troubled CTL research are discussed. The use of early proteins like Tat and Rev is proposed for future vaccines design

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef. (Article)

2002-01-01T00:00:00Z

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.

Antibody-mediated enhancement of human immunodeficiency virus type 1 infectivity is determined by the structure of gp120 and depends on modulation of the gp120-CCR5 interaction (Article)

2002-01-01T00:00:00Z

In this study, we characterized the viral determinants of coreceptor usage in relation to susceptibility to antibody-mediated neutralization or enhancement of infectivity by using chimeras of three highly related human immunodeficiency virus type 1 (HIV-1) isolates of different phenotypes. We found that the V3 region was the main determinant of antibody-mediated enhancement and coreceptor specificity but that the overall structure of gp120 was also important for these properties. Constructs susceptible to antibody-mediated enhancement preferentially use CCR5 as a coreceptor, in contrast to constructs that were neutralized or not affected. Using monoclonal antibodies directed against CD4 or CCR5, we were able to show that antibody-mediated enhancement was CD4 dependent. Altogether, our results suggest that the modulation of the interaction of gp120 with CCR5 is the mechanism underlying antibody-mediated enhancement of HIV-1 infectivity.

Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure. (Article)

2001-03-13T00:00:00Z

Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.

Broadening of coreceptor usage by human immunodeficiency virus type 2 does not correlate with increased pathogenicity in an in vivo model. (Article)

2000-01-01T00:00:00Z

The pathogenic properties of four primary human immunodeficiency virus type 2 (HIV-2) isolates and two primary HIV-2 biological clones were studied in an in vivo human-to-mouse chimeric model. The cell-associated viral load and the ability to reduce the severity of the induced graft-versus-host disease symptoms, the CD4/CD8 ratio and the level of repopulation of the mouse tissues by the graft, were determined. All HIV-2 strains, irrespective of their in vitro biological phenotype, replicated to high titres and significantly reduced graft-versus-host disease symptoms as well as the CD4/CD8 ratios. Reduction of graft repopulation caused by infection with the respective HIV-2 strains showed that the in vitro replication rate, syncytium-inducing capacity and ability to infect human macrophages did influence the in vivo pathogenic potential whereas broadening of coreceptor usage did not.

Decline of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in the peripheral blood of long-term nonprogressing macaques infected with SIVmac32H-J5. (Article)

1999-12-01T00:00:00Z

The evolution of simian immunodeficiency virus (SIV)–specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV‐infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction–detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.

Vaccination with Rev and Tat against AIDS. (Article)

1999-06-04T00:00:00Z

Intro: More than 15 years after the discovery of HIV-1 as the causative agent of AIDS, and numerous attempts to develop a vaccine, it has become clear that the efficacy of the currently considered HIV-1 vaccine candidates will generally be limited. This is at least in part due to the relative resistance of so-called primary HIV strains to neutralization by HIV-1 envelope specific antibodies: even the most potent HIV-1 neutralizing antibodies failed to provide protection in in vivo models, at concentrations that can be maintained for longer periods in human vaccinees.

Coreceptor usage of human immunodeficiency virus type 2 primary isolates and biological clones is broad and does not correlate with their syncytium-inducing capacities (Article)

1998-01-01T00:00:00Z

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with alpha- or beta-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8+ cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.

Kinetics of antiviral activity by human immunodeficiency virus type 1-specific cytotoxic T lymphocytes (CTL) and rapid selection of CTL escape virus in vitro (Article)

1998-01-01T00:00:00Z

The antiviral activity of a CD8(+) cytotoxic T-lymphocyte (CTL) clone (TCC108) directed against a newly identified HLA-B14-restricted epitope, human immunodeficiency virus type 1 (HIV-1) Rev(67-75) SAEPVPLQL, was analyzed with respect to its kinetics of target cell lysis and inhibition of HIV-1 production. Addition of TCC108 cells or CD8(+) reverse transcriptase-specific CTLs to HLA-matched CD4(+) T cells at different times after infection with HIV-1 IIIB showed that infected cells became susceptible to CTL-mediated lysis before peak virus production but after the onset of progeny virus release. When either of these CTLs were added to part of the infected cells immediately after infection, p55 expression and virus production were significantly suppressed. These data support a model in which CTLs, apart from exerting cytolytic activity which may prevent continued virus release, can interfere with viral protein expression during the eclipse phase via noncytolytic mechanisms. TCC108-mediated inhibition of virus replication in peripheral blood mononuclear cells caused rapid selection of a virus with a mutation (69E-->K) in the Rev(67-75) CTL epitope which abolished recognition by TCC108 cells. Taken together, these data suggest that both cytolytic and noncytolytic antiviral mechanisms of CTLs can be specifically targeted to HIV-1-infected cells.

Coreceptor usage of human immunodeficiency virus type 2 primary isolates and biological clones is broad and does not correlate with their syncytium-inducing capacities. (Article)

1998-01-01T00:00:00Z

HIV-2 infection in 12 European residents: virus characteristics and disease progression. (Article)

1996-01-01T00:00:00Z

OBJECTIVE: To assess the disease progression rate among 12 HIV-2-infected West European residents (nine of West African descent), compared with the disease progression rate among HIV-1-infected individuals of the same population, and the characteristics of the HIV-2 strains involved. METHODS: HIV-2-infected individuals were identified by commercially available serological assays, their clinical status and CD4+ cell counts were monitored, and HIV-2 was isolated from their peripheral blood mononuclear cells. T-cell-line tropism and syncytium-inducing capacities of the isolated viruses were determined and their phylogenetic relationships were analysed by comparing polymerase chain reaction-amplified nucleotide sequences of reverse transcriptase (RT) gene segments. RESULTS: Eight of the 12 HIV-2-infected individuals presented with progressive disease and one of them progressed from Centers for Disease Control and Prevention group A1 to A3 within 36 months after seroconversion. The ratios of asymptomatic versus symptomatic individuals among residents of the Rotterdam region of West African descent were 2:7 for HIV-2 and 8:9 for HIV-1-infected individuals. HIV-2 was isolated from six of the nine individuals with progressive disease. The time required for virus isolation correlated inversely with the individuals' CD4+ cell counts. Five of the HIV-2 isolates replicated in immortalized T-cell lines, and two isolates from patients with AIDS were syncytium-inducing. Five HIV-2 isolates from patients born in the Cape Verdian Isles grouped together within subtype A. The HIV-2 isolate from a patient of Ghanese origin belonged to subtype B. Mutations were identified in the RT genes from HIV-2 isolates of two zidovudine-treated patients, one of which has also been shown to be involved in zidovudine resistance in HIV-1. CONCLUSION: Disease progression in HIV-2 infection may be as rapid as in HIV-1. HIV-2 isolation and viral phenotype were related to disease status, and mutations identical to those observed in HIV-1 zidovudine resistance were observed in patients treated with zidovudine.