ABSTRACT
The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.
Subject(s)
AIDS Vaccines/immunology , Cell Separation/methods , Flow Cytometry/methods , Intestinal Mucosa/immunology , T-Lymphocytes/immunology , Adult , Aged , Biopsy , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.