Utilizing Computational Machine Learning Tools to Understand Immunogenic Breadth in the Context of a CD8 T-Cell Mediated HIV Response

Predictive models are becoming more and more commonplace as tools for candidate antigen discovery to meet the challenges of enabling epitope mapping of cohorts with diverse HLA properties. Here we build on the concept of using two key parameters, diversity metric of the HLA profile of individuals within a population and consideration of sequence diversity in the context of an individual's CD8 T-cell immune repertoire to assess the HIV proteome for defined regions of immunogenicity. Using this approach, analysis of HLA adaptation and functional immunogenicity data enabled the identification of regions within the proteome that offer significant conservation, HLA recognition within a population, low prevalence of HLA adaptation and demonstrated immunogenicity. We believe this unique and novel approach to vaccine design as a supplement to vitro functional assays, offers a bespoke pipeline for expedited and rational CD8 T-cell vaccine design for HIV and potentially other pathogens with the potential for both global and local coverage.Fil: McGowan, Ed. Imperial College London; Reino UnidoFil: Rosenthal, Rachel. Francis Crick Institute; Reino UnidoFil: Fiore Gartland, Andrew. Fred Hutchinson Cancer Research Cente; Estados UnidosFil: Macharia, Gladys. Imperial College London; Reino UnidoFil: Balinda, Sheila. Uganda Virus Research Institute; UgandaFil: Kapaata, Anne. Uganda Virus Research Institute; UgandaFil: Umviligihozo, Gisele. Center for Family Health Research; RuandaFil: Muok, Erick. Center for Family Health Research; RuandaFil: Dalel, Jama. Imperial College London; Reino UnidoFil: Streatfield, Claire L.. Imperial College London; Reino UnidoFil: Coutinho, Helen. Imperial College London; Reino UnidoFil: Dilernia, Dario. University of Emory; Estados UnidosFil: Monaco, Daniela C.. University of Emory; Estados UnidosFil: Morrison, David. South Walsham; Reino UnidoFil: Yue, Ling. University of Emory; Estados UnidosFil: Hunter, Eric. University of Emory; Estados UnidosFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gilmour, Jill. Imperial College London; Reino UnidoFil: Hare, Jonathan. International Aids Vaccine Initiative; Estados Unido

Involvement of Hypoxia-Inducible Factor-1 in the Inflammatory Responses of Human LAD2 Mast Cells and Basophils

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli

The effects of PGN and LPS on IgE-induced responses of primary human basophils.

<p>Primary human basophils were exposed to for 4 h to 1 µg/ml PGN or LPS (some of the samples were left untreated). Some of the cell samples (including TLR ligand treated and untreated) were then exposed to 0.1 µg/ml anti-IgE for 2 h. HIF-1α accumulation, VEGF, histamine and IL-4 release were analysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#s2" target="_blank">Materials and methods</a>. Quantitative data are mean values+S.D. of at least three individual experiments. * indicates p<0.01 vs. control. All Western blot data are from one experiment representative of three that gave similar results.</p

Comparative analysis of HIF-1α involvement in inflammatory responses and their cross-interactions in LAD2 human mast cells, primary human basophils and THP-1 human myeloid cells.

<p>This scheme is based on our current findings reported above and also on our previous observations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#pone.0034259-Sumbayev2" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#pone.0034259-Lall1" target="_blank">[18]</a>.</p

LAD2 human mast cells and primary human basophils express detectable amounts of TLRs 2 and 4.

<p>LAD2 cells, primary human basophils (PHB) and THP-1 cells (positive control) were subjected to in-cell TLR2 (<b>A</b>) and TLR4 (<b>B</b>) assays (upper panel). TLRs 2 (<b>A</b>) and 4 (<b>B</b>) were also detected in the cell lysates (lower panel). Quantitative data are mean values+S.D. of at least three individual experiments. * indicates p<0.01 vs. control. All Western blot data shown are from one representative experiment out of three that gave similar results.</p

HIF-1α protein plays a pivotal role in the inflammatory responses of LAD2 mast cells.

<p>(<b>A</b>) Normal and HIF-1α knockdown LAD2 cells were sensitised with 100 ng/ml IgE for 24 h followed by 4 h exposure to 1 µg/ml PGN, LPS or 0.1 µg/ml anti-IgE. HIF-1α accumulation/mRNA levels, VEGF mRNA expression/VEGF release, histamine and TNF-α release, intracellular ATP levels as well as caspase 3 activity were analysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#s2" target="_blank">Materials and methods</a>. Quantitative data are mean values+S.D. of at least three individual experiments. * indicates p<0.01 vs. control. All Western blot data are from one experiment representative of three that gave similar results.</p

HIF-1α protein supports LAD2 mast cell viability during IgE-induced responses.

<p>Normal and HIF-1α knockdown LAD2 cells were sensitised with 100 ng/ml IgE for 24 h followed by 2 h exposure to 0.1 µg/ml anti-IgE (or buffer control). MTS assay was then performed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#s2" target="_blank">Materials and methods</a>). Quantitative data are mean values+S.D. of at least three individual experiments. * indicates p<0.01 vs. control. a – indicates significant (p<0.01) differences between the combined stimulation of basophils with PGN and anti-IgE with PGN or anti-IgE alone.</p

Pam3Cys does not potentiate Anti-IgE response of LAD2 human mast cells.

<p>LAD2 cells were sensitised with 100 ng/ml IgE for 24 h followed by 4 h exposure to 1 µg/ml Pam3Cys. Thereafter, some samples (including TLR ligand treated and untreated) were exposed to 0.1 µg/ml anti-IgE for 2 h. Histamine, VEGF and TNF-α release was analysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#s2" target="_blank">Materials and methods</a>. Quantitative data are mean values+S.D. of at least three individual experiments. * indicates p<0.01 vs. control.</p

Anti-IgE dose-dependent responses of LAD2 human mast cells.

<p>LAD2 human mast cells were sensitised with 100 ng/ml IgE for 24 h followed by 2 h of exposure to increasing concentrations of anti-IgE. HIF-1α accumulation, VEGF mRNA expression/VEGF release, histamine and TNF-α release as well as intracellular ATP levels were analysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034259#s2" target="_blank">Materials and methods</a>. Quantitative data are mean values ± S.D. of at least three individual experiments. * indicates p<0.01 vs. control. All Western blot data are from one experiment representative of three that gave similar results.</p

PGN and LPS induce HIF-1α accumulation in THP-1 but not in LAD2 cells.

<p>THP-1, LAD2 and HEK293 (negative control – lacking TLR2/4 expression) cells were exposed for 4 h to 1 µg/ml PGN or LPS. HIF-1α accumulation was then analysed. Quantitative data are mean values+S.D. of at least three individual experiments. * indicates p<0.01 vs. control. All Western blot data shown are from one representative experiment out of three that gave similar results.</p