The Cellular Cytotoxicity Laboratory has developed, optimized, and pre-validated, and validated assays that can quantify the cytotoxic cellular subsets in regard to their ability to eliminate the HIV-1 infected cells. In this context, the Interferon Gamma (IFN-γ) Enzyme Linked ImmunoSpot (ELISpot) has been proven to be an excellent surrogate of the cytotoxic assay to quantify the total T cell response and perform epitope mapping. Moreover, we have implemented the detection of granzyme B (GzB) delivered by the effector cells into the target cells of interest to detect the cytotoxic activity of NK and HIV-1-specific CD8+ T cells. We have also optimized a novel approach to quantify the elimination of HIV-1-infected cells by cytotoxic CD8+ T cells, NK cells, and Antibody Dependent Cellular Cytotoxicity (ADCC)-mediating antibodies using reduction in the luciferase activity as final readout.
We have implemented these assays in a variety of studies to detect Cytotoxic T and NK cell responses, as well as ADCC induced by natural HIV-1 infection or by vaccine candidates.
The assays are currently performed as optimized or validated assays under Good Clinical Laboratory Practice (GCLP) guidelines. The laboratory can perform these assays in support of both pre-clinical studies and clinical trials, as well as for basic research purposes.
I. Interferon Gamma (IFN-γ) Enzyme Linked ImmunoSpot (ELISpot).
The principle of this assay is based on the detection of the IFN-γ released by the T cells upon stimulation with antigens of interest, and quantified as the formation of spot son a solid membrane. Each spot represents an antigen-specific T cell. Data are reported as Spot Forming Cells per 2x105 PBMC.
II. Granzyme B flow-based Cytotoxicity Assay.
In our assay platform, active Granzyme B (GzB) is identified by its ability to cleave a selective peptide-linked fluorogenic substrate containing a GzB recognition motif. Fluorescence is conferred upon hydrolysis of the substrate, thereby producing a signal that can be used to directly identify individual cells targeted by the cytotoxic effector cells. Enumeration of these cells by high-throughput flow cytometry provides a rapid and quantitative measure of cytotoxic activity as mediated by NK cell, T cell, and ADCC responses.
III. Luciferase- based Cytotoxicity Assay.
The principle of this assay is based on the utilization of HIV-1 Infectious Molecular Clones (IMC) that either represent a full length infectious HIV-1 isolate or designed to encode HIV-1 env genes of interest in cis within an isogenic common backbone that also expresses the Renilla luciferase reporter gene and preserves all viral open reading frames. The luciferase reporter is under the control of the HIV-1 Tat gene. Upon HIV-1 infection of any cellular subset of interest, expression of Tat during HIV-1 replication will induce expression of the luciferase and the presence of infected cells can be easily quantified as Relative Luminescence Units. In the presence of the cytotoxic cells of interest, the elimination of infected target cells can quantified after a minimum of two hours by evaluating the residual luminescence units.
I. Center for HIV/AIDS Vaccine Immunology (CHAVI), Focus 1: ADCC
The major goal of this project is to understand the role of ADCC responses in protection from natural infection in order to develop an effective preventative HIV-1 vaccine.
II. Comprehensive Antibody-Vaccine Immune Monitoring Consortium, ADCC core
The major goal of this project is to conduct an evaluation of ADCC elicited in the context of pre-clinical studies and clinical HIV vaccine trials.
III. Primate AIDS Vaccine Evaluation Group
The objective is to perform as a Non-Human Primate Core Humoral Immunology Laboratory for AIDS Vaccine Research and Development characterizing ADCC responses.
Guido Ferrari, Director: gflmp@duke.edu
Justin Pollara, Co-director: Justin.pollara@duke.edu
Mark Berrong, ELISpot Lab Manager: mark.berrong@duke.edu
Sherry Stanfield-Oakley, ADCC Lab Manager: sherry.oakley@duke.edu