Computational protocol: Immunogenicity of Seven New Recombinant Yellow Fever Viruses 17D Expressing Fragments of SIVmac239 Gag, Nef, and Vif in Indian Rhesus Macaques

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Protocol publication

[…] We performed multi-parameter ICS by incubating freshly isolated PBMC from rhesus macaques with 1.0 µM of separate peptide mixtures spanning the ORFs of Vif, Nef, Tat, and Rev. Vpr and Vpx peptides were combined in one test. We divided the Gag peptides in two tests spanning amino acids 1–291 and 281–510; reactivity to this protein is reported as the sum of these two tests. We used staphylococcal enterotoxin B (SEB) stimulation for our positive control and tissue culture medium devoid of stimulatory peptides for our negative control. We also added anti-CD28 (clone L293; BD Biosciences), anti-CD49d (clone 9g; Pharmigen), and anti-CD107a PE (clone H4A3; BD Biosciences) antibodies and 5.0 µg of Brefeldin A and Golgi Stop (BD Biosciences) to each test. We then placed the samples in a 5.0% CO2 incubator at 37°C. After overnight incubation, we stained the cells at room temperature with antibodies directed against the surface molecules CD4 (PerCP Cy5.5; clone L200; BD Biosciences), CD8 (Pacific Blue; clone RPA-T8; BD Biosciences), CD14 (ECD; clone RMO52; Beckman Coulter), and CD19 (ECD; clone JA.119; Beckman Coulter). After 20 minutes, we washed the cells with FACS buffer (10% of fetal bovine serum in PBS 1X) and fixed them with a 2.0% paraformaldehyde solution. After 30 minutes, we washed off the paraformaldehyde and permeabilized the cells with 0.1% Saponin buffer (FACS buffer containing saponin) and stained them with antibodies directed against the intracellular molecules IFN-γ (PE-Cy7; clone 4S.B3; BD Biosciences), TNF-α (Alexa700; clone MAb11; BD Biosciences), IL-2 (APC; clone MQ1-17H12; BD Biosciences), and MIP-1β (FITC; clone 24006; R&D Systems). After a 45-minute incubation, we washed the cells twice with Saponin buffer and fixed them with 2.0% paraformaldehyde. Samples were acquired using FACS DIVA version 6 on a Special Order Research Product (SORP) BD LSR II equipped with a 50 mW 405 nm violet, a 100 mW 488 nm blue, and a 50 mW 640 nm red laser and were analyzed by FlowJo 9.2 (TreeStar, Inc.). Analysis and presentation of distributions were performed using Pestle and SPICE version 5.22023, downloaded from http://exon.niaid.nih.gov/spice .For data analysis, we first gated on forward scatter height (FSC-H) versus forward scatter area (FSC-A) to remove doublets (). Subsequently, we gated on the lymphocyte population and then created a dump channel by excluding CD14+CD19+ events. At this stage, we separated lymphocyte subsets based on their expression of either CD4 or CD8 (excluding those expressing both markers) and conducted our functional analyses within these two compartments. After making gates for each function (IFN-γ, CD107a, TNF-α, MIP-1β, and IL-2), we used the Boolean gate platform to generate a full array of possible combinations, equating to 32 response patterns when testing five functions (25 = 32). We used three criteria to determine positivity of responses; i) background-subtracted responses had to be at least 2-fold higher than the background itself; ii) Boolean gates for each response pattern had to contain ≥10 events; and iii) response patterns had to be greater than matched values obtained in ICS assays using PBMC from three SIV naïve Indian rhesus macaques under the same stimulation conditions. […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Simian immunodeficiency virus, Macaca mulatta, Homo sapiens, unidentified adenovirus
Diseases Acquired Immunodeficiency Syndrome, Yellow Fever, HIV Infections