Immunomonitoring technologies for the evaluation of Modified Vaccinia Virus Ankara expressing HIV-1 nef as a vaccine against AIDS

The WHO/UNAIDS “Global summary of the AIDS epidemic” released in December 2009, estimates that 33 million of people are living with HIV, 2.7 million were newly infected and 2.0 million of people died of AIDS in the last year. After more than two decades of research an effective preventive or therapeutic vaccine against HIV remains elusive and immunological correlates of protection remain unknown. T-cell mediated immunity is considered to play an important role in controlling HIV infection and progression to AIDS. Several candidate vaccines against HIV aiming to stimulate cellular immune responses are investigated in phase I to phase III clinical trials and assays enabling for a reliable, informative and sensitive measurement of CD4 and CD8 T-cell need to be implemented. Among the most promising vaccine candidates is recombinant modified vaccinia virus Ankara (MVA), a live viral vector system for the delivery of HIV-derived antigens. Several vaccination trials have made use of the modified vaccinia virus Ankara (MVA) as delivery vector. At present, the IFN-γ-based ELISPOT assay is considered as a gold standard and preferred primary assay in vaccine trials. However, despite its high sensitivity the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. Polychromatic flow-cytometry-based assays as intracellular cytokine staining (ICS) offer the possibility to detect several markers on the same cell. Several findings from cross-sectional and longitudinal studies investigating different grades of HIV control highlight the importance of developing assays able to simultaneously measure several parameters on a single-cell level and strongly suggest the use of flow cytometry to monitor immune responses. In this work polychromatic flow-cytometry based assays were developed, optimized, and standardized for T-cell immunomonitoring purposes. In addition, these ICS based methods were compared with ELISPOT assays performed in two different experienced laboratories. The comparative study provided evidence that by the use of a special analysis system, the sensitivity of the ICS could be increased up to levels comparable to the sensitivity of the ELISPOT assay. The established polychromatic ICS together with a polychromatic CFSE-based proliferation assay were applied to a re-evaluation study of a vaccination trial using recombinant MVA expressing HIV-1-Nef (MVA-nef) in HIV-1 infected HAART treated individuals. In this study, the impact of the immunologic intervention with MVA-nef on the specific anti-Nef T-cell immune response was investigated in regard to cytokine production (IFN-γ and IL-2), chemokine production (MIP-1β), activation and differentiation marker expression (CD154 and CD45RA, respectively) and proliferative potential. Vaccine-induced polyfunctionality and proliferative capacity, which were associated with nonprogressive HIV-1 infection in several studies, were detectable by combining the two immune assays. By means of short-term ICS, a significant increase of polyfunctional Nef-specific CD4 T cells expressing IFN-γ, IL-2 and CD154 was observed following vaccination, whereas changes in the quality of the CD8 T-cell response could not be observed. Only the additional use of a long-term polychromatic CFSE-based proliferation assay revealed vaccine-induced Nef-specific CD8 as well as CD4 T cells with proliferative capacity. The correlation between the vaccine-induced IL-2 production by CD4 T cells and the increase of proliferating Nef-specific CD8 T cells suggests a causal link between these two functions. The insight gathered in this reevaluation study exceeded by far the information obtained in the original work using a simple IFN-γ-based immune assay. These results highlight the importance of combining sophisticated immunomonitoring tools to unravel concealed effects of immunologic interventions and support the use of the poxvirus-derived MVA vector to stimulate effective HIV-specific T-cell responses. From a technical point of view, these findings are important to guide the choice for suitable immune assays able to characterize the phenotype and function of specific T-cells in a highly sensitive way

Modeling cellular deformations using the level set formalism

BACKGROUND: Many cellular processes involve substantial shape changes. Traditional simulations of these cell shape changes require that grids and boundaries be moved as the cell's shape evolves. Here we demonstrate that accurate cell shape changes can be recreated using level set methods (LSM), in which the cellular shape is defined implicitly, thereby eschewing the need for updating boundaries. RESULTS: We obtain a viscoelastic model of Dictyostelium cells using micropipette aspiration and show how this viscoelastic model can be incorporated into LSM simulations to recreate the observed protrusion of cells into the micropipette faithfully. We also demonstrate the use of our techniques by simulating the cell shape changes elicited by the chemotactic response to an external chemoattractant gradient. CONCLUSION: Our results provide a simple but effective means of incorporating cellular deformations into mathematical simulations of cell signaling. Such methods will be useful for simulating important cellular events such as chemotaxis and cytokinesis

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT

<p>Abstract</p> <p>Background</p> <p>T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-γ-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level.</p> <p>Results</p> <p>The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-γ producing T-cells were also producing MIP-1β whereas T-cells characterized by the sole production of IFN-γ were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-γ+ MIP-1β+ T-cells was equivalent to the measurement of the total IFN-γ+ T-cells, we adopted the IFN-γ+ MIP-1β+ data analysis system to evaluate IFN-γ-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-γ+ MIP-1β+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay.</p> <p>Conclusion</p> <p>The IFN-γ+ MIP-1β+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.</p

Nef-specific CD45RA+ CD8+ T cells secreting MIP-1β but not IFN-γ are associated with nonprogressive HIV-1 infection

<p>Abstract</p> <p>Background</p> <p>Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease.</p> <p>Results</p> <p>We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gamma^negMIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gamma^negMIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease.</p> <p>Conclusion</p> <p>The novel antigen-specific CD45RA+ IFN-gamma^negMIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.</p

The potential of statistical matching for the analysis of wider benefits of learning in later life

It is challenging to investigate wider benefits of adult learning, especially in later life, due to limited data on educational activities and non-monetary returns in large, longitudinal surveys. Statistical matching provides an approach to exploit the potential of existing data by combining data sources with complementary features based on shared information. The paper describes the matching of two data sources (German Ageing Survey and Study of Educational Attainment and Interests of Older People) in order to examine the effects of educational participation on well-being in later life. We emphasize the matching procedure and how to identify the best-matched dataset. Based on matched data, effects of educational activities on life satisfaction are examined in later life. The discussion focuses on future demands on data and methods for investigating wider benefits of adult learning in quantitative research. (DIPF/Orig.

Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells

Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR-3 are localised primarily to vasculature in human primary solid cancers

Purpose: Vascular endothelial growth factor (VEGF) signaling is key to tumor angiogenesis and is an important target in the development of anticancer drugs. However, VEGF receptor (VEGFR) expression in human cancers, particularly the relative expression of VEGFR-2 and VEGFR-3 in tumor vasculature versus tumor cells, is poorly defined. Experimental Design: VEGFR-2– and VEGFR-3–specific antibodies were identified and used in the immunohistochemical analysis of human primary cancers and normal tissue. The relative vascular localization of both receptors in colorectal and breast cancers was determined by coimmunofluorescence with vascular markers. Results: VEGFR-2 and VEGFR-3 were expressed on vascular endothelium but not on malignant cells in 13 common human solid tumor types (n > 400, bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid). The signal intensity of both receptors was significantly greater in vessels associated with malignant colorectal, lung, and breast than adjacent nontumor tissue. In colorectal cancers, VEGFR-2 was expressed on both intratumoral blood and lymphatic vessels, whereas VEGFR-3 was found predominantly on lymphatic vessels. In breast cancers, both receptors were localized to and upregulated on blood vessels. Conclusions: VEGFR-2 and VEGFR-3 are primarily localized to, and significantly upregulated on, tumor vasculature (blood and/or lymphatic) supporting the majority of solid cancers. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumor vessels rather than tumor cells. The upregulation of VEGFR-3 on tumor blood vessels indicates a potential additional antiangiogenic effect for dual VEGFR-2/VEGFR-3–targeted therapy