Cell populations are never truly homogeneous; individual cells exist in biochemical states that define functional differences between them. New technology based on microfluidic arrays combined with ...multiplexed quantitative polymerase chain reactions now enables high-throughput single-cell gene expression measurement, allowing assessment of cellular heterogeneity. However, few analytic tools have been developed specifically for the statistical and analytical challenges of single-cell quantitative polymerase chain reactions data.
We present a statistical framework for the exploration, quality control and analysis of single-cell gene expression data from microfluidic arrays. We assess accuracy and within-sample heterogeneity of single-cell expression and develop quality control criteria to filter unreliable cell measurements. We propose a statistical model accounting for the fact that genes at the single-cell level can be on (and a continuous expression measure is recorded) or dichotomously off (and the recorded expression is zero). Based on this model, we derive a combined likelihood ratio test for differential expression that incorporates both the discrete and continuous components. Using an experiment that examines treatment-specific changes in expression, we show that this combined test is more powerful than either the continuous or dichotomous component in isolation, or a t-test on the zero-inflated data. Although developed for measurements from a specific platform (Fluidigm), these tools are generalizable to other multi-parametric measures over large numbers of events.
All results presented here were obtained using the SingleCellAssay R package available on GitHub (http://github.com/RGLab/SingleCellAssay).
We describe here a method for optimizing the use of polychromatic flow cytometry (with up to 17 fluorochromes simultaneously) in surface and intracellular staining of human T lymphocytes. We will ...highlight and discuss how to procedurally optimize key steps in the experimental process before an intracellular cytokine staining assay protocol is finalized. These include but are not limited to the titration of monoclonal antibodies, use of a dead-cell discriminator and 'dump' channel, selection of a cytokine secretion inhibitor, selection of fixation and permeabilization reagents, and inclusion of compensation controls. Building on this basic protocol, we then establish a polychromatic assay designed to detect five separate functions of T lymphocytes (production of three cytokines and one chemokine, and degranulation) while simultaneously identifying multiple surface markers on the responding cells.
Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single ...cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a “bulk” approach that measures expression patterns from 100 cells at a time in high throughput to define gene signatures, and a single-cell approach to define the coordinate expression patterns of multiple genes and reveal unique subsets of cells.
•Primer qualification method for highly multiplexed, single-cell gene expression•Nonhuman primate cross-reactivity of primers•Conditions for single copy detection; established quantitative scale for system•Limited value of endogenous controls in single-cell gene expression systems•Comparison of bulk (multi-cell, “pooled-cell array”) approach to single-cell assay
Membrane-damaged cells caused by either mechanical trauma or through normal biological processes can produce artifacts in immunophenotyping analysis by flow cytometry. Dead cells can nonspecifically ...bind monoclonal antibody conjugates, potentially leading to erroneous conclusions, particularly when cell frequencies are low. To date, DNA intercalating dyes (Ethidium monoazaide (EMA), Propidium Iodide, 7AAD, etc.) or Annexin V have been commonly used to exclude dead cells; however, each suffer from technical problems. The first issue with such dyes is the dependence on a consistent dead cell source for fluorescence compensation. Another major issue is the stability of the staining; except for EMA, fixation and permeablization used for intracellular staining procedures can cause loss of fluorescence. EMA requires a UV exposure to covalently bond to DNA; while this dye is effective and is not affected by intracellular treatments it is weakly fluorescent. Here we report on the optimization of a new class of viability dyes, the amine reactive viability dyes (ViD) as a dead cell exclusion marker. The inclusion of ViD into the staining panel was found to be simple, reproducible and can have a significant benefit on the accuracy of identifying appropriate cell populations. We show the fluorescence of cells stained with these dyes correlates with traditional dead cell discriminating markers, even after fixation and permeabilization. Amine reactive viability dyes are a powerful tool for fluorescence immunophenotyping experiments.
Induction of HIV-1-specific T-cell responses relevant to diverse subtypes is a major goal of HIV vaccine development. Prime-boost regimens using heterologous gene-based vaccine vectors have induced ...potent, polyfunctional T cell responses in preclinical studies.
The first opportunity to evaluate the immunogenicity of DNA priming followed by recombinant adenovirus serotype 5 (rAd5) boosting was as open-label rollover trials in subjects who had been enrolled in prior studies of HIV-1 specific DNA vaccines. All subjects underwent apheresis before and after rAd5 boosting to characterize in depth the T cell and antibody response induced by the heterologous DNA/rAd5 prime-boost combination.
rAd5 boosting was well-tolerated with no serious adverse events. Compared to DNA or rAd5 vaccine alone, sequential DNA/rAd5 administration induced 7-fold higher magnitude Env-biased HIV-1-specific CD8(+) T-cell responses and 100-fold greater antibody titers measured by ELISA. There was no significant neutralizing antibody activity against primary isolates. Vaccine-elicited CD4(+) and CD8(+) T-cells expressed multiple functions and were predominantly long-term (CD127(+)) central or effector memory T cells and that persisted in blood for >6 months. Epitopes mapped in Gag and Env demonstrated partial cross-clade recognition.
Heterologous prime-boost using vector-based gene delivery of vaccine antigens is a potent immunization strategy for inducing both antibody and T-cell responses.
ClinicalTrials.gov NCT00102089, NCT00108654.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes ...an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine.
To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10) particles, two logs lower than that used previously.
Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10) rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background. Gene‐based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the ...first phase 1 dose‐escalation study of a multiclade HIV‐1 DNA vaccine. Methods. VRC‐HIVDNA009‐00‐VP is a 4‐plasmid mixture encoding subtype B Gag‐Pol‐Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle‐free intramuscular injection. Humoral responses (measured by enzyme‐linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme‐linked immunospot assay and intracellular cytokine staining after stimulation with antigen‐specific peptide pools) were measured. Results. The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4+ and CD8+ T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine‐induced HIV‐specific T cells evolved over time, with a diminished frequency of interferon‐γ–producing T cells and an increased frequency of interleukin‐2–producing T cells at 1 year. Conclusions. DNA vaccination induced antibody to and T cell responses against 3 major HIV‐1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.
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