Computational protocol: A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

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Protocol publication

[…] The magnitude, polyfunctionality and phenotypes of the HIV-1-specific T-cell adaptive and memory responses were analyzed by flow cytometry and intracellular cytokine staining (ICS) as previously described [, ]. Briefly, splenocytes were rested overnight and the following day 4×106 splenocytes were stimulated with 5 μg/ml of HIV-1 Env peptide pool spanning the full length gp120 from BX08 isolate in addition to 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences), and monensin (1X; eBioscience) in RPMI 1640 media supplemented with 10% FCS for 6 h in a 96 well plate. Peptides were provided by the Eurovacc Foundation and were previously described []. Also, splenocytes were stimulated with A20 cells transfected with DNA-gp120 (4 x 105 gp120 transfected A20 cells in 4 x 106 splenocytes; the ratio of gp120-transfected A20 cells to splenocytes was equal to 1:10). Following stimulation, cells were washed, Fc receptors were blocked using anti CD16/CD32 (BD Biosciences), stained for the surface markers, fixed, permeabilized (Cytofix/Cytoperm kit; BD Biosciences), and stained intracellularly for cytokines with the appropriate fluorochromes. Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen). Cells were stained with different mouse antibodies, such as CD3-PE-CF594, CD4-APC-Cy7, CD8-V500, IFN-γ-PE-Cy7, IL-2-APC and TNF-α-PE (all from BD Biosciences). In addition, for differentiating memory T-cells the following antibodies were used: CD62L-Alexa 700 (BD Biosciences) and CD127-PerCP-Cy5.5 (eBioscience). A million cells were then passed through GALLIOS flow cytometer (Beckman Coulter) and the data was analyzed with FlowJo (Tree Star. Inc) and Spice (version 5.0). Appropriate controls were used and the values from unstimulated samples were subtracted. […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Vaccinia virus, Human immunodeficiency virus 1, Cricetulus griseus, Homo sapiens, Mus musculus
Diseases HIV Infections