In this cross-sectional study we evaluated T-cell responses using several assays to determine immune correlates of HIV control that distinguish untreated viraemic controllers (VC) from noncontrollers (NC) with similar CD4 counts. Samples were taken from 65 ART-naïve chronically HIV-infected VC and NC from Thailand with matching CD4 counts in the normal range (>450 cells/μl). We determined HIVp24-specific T-cell responses using standard Interferon-gamma (IFNγ) ELISpot assays, and compared the functional quality of HIVp24-specific CD8+ T-cell responses using polychromatic flow cytometry. Finally, in vitro HIV suppression assays were performed to evaluate directly the activity of CD8+ T cells in HIV control. Autologous CD4+ T cells were infected with primary patient-derived HIV isolates and the HIV suppressive activity of CD8+ T cells was determined after co-culture, measuring production of HIVp24 Ag by ELISA. The HIVp24-specific T-cell responses of VC and NC could not completely be differentiated through measurement of IFNγ-producing cells using ELISpot assays, nor by the absolute cell numbers of polyfunctional HIVp24-specific CD8+ T cells. However, in vitro HIV suppression assays showed clear differences between VC and NC. HIV suppressive activity, mediated by either ex vivo unstimulated CD8+ T cells or HIVp24-specific T-cell lines, was significantly greater using cells from VC than NC cells. Additionally, we were able to demonstrate a significant correlation between the level of HIV suppressive activity mediated by ex vivo unstimulated CD8+ T cells and plasma viral load (pVL) (Spearman r = -0.7345, p = 0.0003). This study provides evidence that in vitro HIV suppression assays are the most informative in the functional evaluation of CD8+ T-cell responses and can distinguish between VC and NC.
In this study, a first food-grade mucosal vaccine against leptospirosis was developed without the use of antibiotic resistance gene. This expression system is based on a food-grade host/vector system of Lactobacillus plantarum and a new vaccine candidate antigen, a leucine-rich repeat (LRR) protein of Leptospira borgpetersenii. The LRR of interest from serovar Sejroe is encoded by two overlapping genes and these genes were fused together by site-directed mutagenesis. The mutant gene thus obtained could be successfully expressed in this system as was shown by western blot analysis and liquid chromatography-mass spectrometry (LC-MS/MS) analysis. In addition, this analysis showed that the mutant LRR protein fused to a homologous signal peptide of L. plantarum could be exported to the cell surface as a result of the native LPXAG motif of the heterologous LRR protein, which presumably is responsible for anchoring the protein to the cell wall of L. plantarum. This new strategy could be an essential tool for further studies of leptospirosis mucosal vaccine delivery.
We concluded that an adequate number of high functional quality Gag p24-specific CD8 T cells is strongly associated with a natural HIV controller status.
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