Rob A. Gruters

Functional and phenotypic evidence for a selective loss of memory T cells in asymptomatic human immunodeficiency virus-infected men.

In addition to a well-documented depletion of CD4+ T helper cells in later stages of human immunodeficiency virus (HIV) infection, evidence has been provided for a specific unresponsiveness to triggering either by specific antigen in the context of autologous major histocompatibility molecules (self + X) or anti-CD3 monoclonal antibodies (MAb) in both CD4 and CD8 cells from asymptomatic HIV-infected individuals. In the present study we analyzed this unresponsiveness using mitogenic antibodies to distinct T cell membrane receptors. T cells from HIV-infected men who had normal numbers of CD4+ T cells responded poorly to activation signals via the CD3 membrane antigen in both accessory cell-dependent as well as accessory cell-independent culture systems. A similar low response was observed in an anti-CD2-driven system. In contrast, proliferation induced by anti-CD3, anti-CD2, or the phorbol ester Phorbol myristate acetate could be normally enhanced by anti-CD28 MAb. We demonstrated that this unresponsiveness is not due to a failure to induce early events required for activation, such as increased intracellular concentration of free calcium and activation of protein kinase C, but is caused by an imbalance between naive and memory T cells. In HIV-infected asymptomatic men, CD29+ memory T cells are selectively depleted which results in a poor responsiveness to self + X. These findings provide new insights that may have implications for our understanding of the immunopathogenesis of AIDS.

Vaccination with Rev and tat against AIDS

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.

Biomedical application of MALDI mass spectrometry for small-molecule analysis

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.

Current and future applications of dried blood spots in viral disease management.

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.

Vaccine-induced enhancement of viral infections.

Abstract 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.

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes

Recent studies indicate that the time required for virus‐infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV‐1‐specific CTL, we measured virus production in cultures containing CD4+ T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev‐specific CTL prevented at least 2 log10 more HIV‐1 production, in 10 days, than similar numbers of RT‐specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL‐target cell interactions during successive infection cycles. The results show that substantially higher CTL‐mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT‐specific CTL were at least as effective as the Rev‐specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

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.

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.

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

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.

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

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.