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P15.03 Development of a new cem reporter t-cells (gxr-cells) viral inhibition assay (via) for elucidating the role of class-i-hla alleles on the inhibitory capacity of hiv-1-specific cd8+t-cells
  1. F Ogunshola1,
  2. N Mewala1,
  3. JK Wright1,
  4. N Ismail1,
  5. MA Brockman2,3,
  6. BD Walker1,2,
  7. T Ndung’u1,2,
  8. ZM Ndhlovu1,2
  1. 1HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa
  2. 2Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
  3. 3Simon Fraser University, Burnaby, Canada


Introduction Standard immunogenicity assays, such as ELISpot and intracellular cytokine staining, fail to correlate HIV-1-specific CD8+T-cells responses with HIV-1 replication in-vivo. Therefore, it is essential to develop assays that can determine antiviral potential of vaccine elicited CD8+T-cells. The current ELISA-VIA measures HIV-1-p24 production overtime in autologous CD4+T-cells. However, it is not designed to identify the class-I-HLA-allele involved in mediating the response. We developed a new FACS based VIA that can investigate CD8+T-cells antiviral potential in the context of restricting class-I-HLA alleles. The assay measures the ability of CD8+T-cells to kill HIV-1 infected GXR-cells over-expressing class-I-HLA allele of interest. The assay utilises a GXR-cell engineered to express GFP upon HIV-1 infection.

Methods CD8+T-cells were co-cultured with HIV-1 infected GXR-cells for 3 days. Reduction in the infected GXR-cells expressing GFP measured by FACS was used to evaluate the CD8+T-cells killing activity. The assay was validated using a panel of 9 HIV-infected samples and were concurrently assayed with the ELISA-VIA. The tested results on each assay were categorised into four groups namely: true-inhibition (TI ≥50%), doubtful-inhibition (DI ≥20% to ≤49.99%), false-inhibition (FI ≥10% to ≤19.99%) and non-inhibition (NI≤ 9.99%). These results were used in a 2 by 2 table to compute sensitivity (TI/TI+DI) and specificity (FI/FI+NI).

Results True inhibition was observed in 44% of samples analysed using GXR-VIA compared to 33% with ELISA-VIA. 11% with GXR-VIA had doubtful result compared to 33% with ELISA-VIA. 22% with GXR-VIA were categorised as false inhibition compared to 33% with ELISA-VIA. Interestingly, no sample showed non-inhibition with GXR-VIA whereas 22% showed no inhibition by ELISA-VIA. Collectively, GXR-VIA is very specific (100%) but less sensitive (57%) at detecting virus inhibition activity.

Conclusion The specificity of GXR-VIA and its marginal sensitivity indicates that the assay is capable of identifying CD8+T-cells-mediated inhibition of HIV-1 replication. Overall, the GXR-VIA provides a platform to assess the influence of different restricting class-I-HLA alleles on HIV-1-specific CD8+T-cells antiviral function.

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