1. Detection of Immune Responses in Mucosal T-cells in Kenya Jackton Indangasi 2, Hannah Cheeseman 1, Onyango J. I. Obila 2, Simon Ogola 2, Robert Langat 2,Philip J. Bergin 1, Dilbinder K. Gill 1, Harriet H. Park 3, Jacquelyn Nyange 2, Dagna Laufer 3, Pat Fast 3, Bashir Farah 2, Jill Gilmour 1, Omu Anzala 2. 2 Kenya AIDS Vaccine Initiative (KAVI), Nairobi, Kenya, 1 International AIDS Vaccine Initiative Human Immunology Laboratory (IAVI HIL), Imperial College London, London, UK, 3 International AIDS Vaccine Initiative (IAVI), New York, USA. Introduction The next generation of candidate HIV vaccines include replicating vectors targeted to mucosal sites, which are hoped to induce an efficacious T cell response to limit HIV-1 viral replication in the mucosa. For HIV vaccine clinical trials, it is especially important to ascertain whether there is a mucosal immune response, as this is where the bulk of the viral reservoir is maintained. Immune responses have been shown to differ between racial and geographical populations(1), making it vital to perform comparison studies in the country where a clinical trial will take place, to obtain the relevant background information. The aims of this study were to obtain gut biopsies from HIV- seronegative individuals in Kenya, to ensure that scientists in Kenya were fully trained in the methods to be used in future clinical trials and to demonstrate comparability between assays run in Kenya and the UK. Methods A panel of 10 HIV-negative PBMC, with a range of CMV responses, were used to demonstrate equivalence of the two LSRII cytometers, using the 10-colour intracellular cytokine stimulation (ICS) panel (CD3 APCCy7, CD4 ECD, CD8 Pacific Blue, CD27 PECy7, CD45RO A700, CD107a PECy5, IFN-  APC, IL-2 PE, TNF-  FITC, DUMP [Aqua Blue Viability dye and CD19 AmCyan]) to assess responses to a 6- hour stimulation with Mock, CMV and SEB. Three HIV-positive PBMC samples were stimulated with Mock, CMV, SEB and clade A GAG and NEF pools. Biopsies were taken from the sigmoid colon of 31 HIV-seronegative volunteers and mucosal mononuclear cells (MMC) isolated(2). Parallel blood samples were also taken. MMC and PBMC were rested overnight and stimulated for 6 hours with Mock, CMV, SEB and clade A HIV GAG and NEF pools. PBMC assessed by IFN-  ELISpot were directly compared to pp65 CMV responses detected by flow cytometry. Results Results from the PBMC comparison indicate that the two LSRII cytometers are capable of producing data that is not significantly different. We also show that is it possible to isolate MMC from patients in Kenya and that these cells can be successfully stimulated, stained and acquired. Of the 31 volunteers biopsied, 11 satisfied our pass/fail criteria (Viable CD3 count of >30000 events and CD4/8 count of >10000 events.) and 8 PBMC and 5 MMC samples demonstrated a CMV response (CD4 and/or CD8) by ICS. All 11 responded by ELISpot (PBMC; mean 723 SFU/million; range 95-1440 SFU/million). PBMC ELISpot CMV responses significantly correlated with ICS CMV responses; r 2 = 0.62. Conclusions This study demonstrates that it is feasible to collect biopsies and perform multi- parameter ICS in Kenya and that the data generated is comparable with both data run in London and on an extensively qualified assay (ELISpot). These methods have been successfully transferred and may be used to assess antigen-specific T-cell responses in future vaccine trials. References 1.Reference ranges for the clinical laboratory derived from a rural population in Kericho, Kenya. Kibaya et al. PLoS One. 2008 Oct 3;3(10) 2.Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials. Kaltsidis et al. J Immunol Methods. 2011 Jul 29;370(1-2):43-54. Figure 3: Comparison of CD8+ T-cell IFN-g production. Graph shows 3 HIV positive donors. Data from KAVI (  )  and HIL ( ) are not significantly different, using a Wilcoxon two-tailed matched-pairs signed rank test. Figure 2: Comparison of 10 paired HIV- samples tested at HIL and KAVI. Using a Wilcoxon matched-pairs signed rank test to determine the P values, no significant difference is seen between the data from samples tested at the HIL or at KAVI. Figure 4: a) Viable MMC can be isolated, with an average of 1x10 6 cells per biopsy collected. b) Responses of CD4+ and CD8+ T-cells to CMV stimulation in both MMC and PBMC compartments. Mock responses were low in all cases. Figure 5: ELIspot responses (PBMCs) correlate with flow cytometry assays. PBMC; mean 723 SFU/million; range 95-1440 SFU/million. PBMC ELISpot CMV responses significantly correlated with ICS CMV responses; r 2 = 0.62. Figure 1: Gating strategy used for data analysis in FlowJo. a) b)