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Background: BCN02 was a pilot kick-and-kill clinical trial that combined therapeutic vaccination (MVA.HIVconsv) with the latency reversing agent romidepsin (RMD) followed by a monitored antiretroviral pause (MAP) in 15 early-treated HIV+ individuals (NCT02616874). Out of 12 evaluable participants for an omics sub-analysis, 8 participants showed early (pVL > 2000 copies/mL < 4 weeks) and 4 a more delayed viral rebound (pVL > 2000 copies/mL > 4 weeks) in MAP. Systems biology analyses identified epigenetic and molecular mechanisms associated with response to vaccination and RMD and viral rebound kinetics. Methods: Genome-wide gene expression (Ilumina HiSeq2500) and DNA Methylation (Infinium HumanMethylation450 BeadChip) were assessed in frozen PBMC-pellets at baseline, 1 week after vaccination and 1 week after the 3rd RMD infusion (post-RMD). After pre-processing and normalization steps we applied principal component analyses (PCA) and differential expression/methylation analyses (limma-R/Biocondcutor). GSEA was used for functional analysis, and sPLS-DA (MixOmics-R/Bioconductor), to identify pathways explicative of differential viral rebound kinetics. Results: The largest impact on host transcriptional and DNA methylation (DNAm) profiles was observed after the combined effect of vaccination and RMD, with 733 differentially expressed genes and 5695 differentially methylated positions being detected between baseline and post-RMD (adjusted p < 0.1). Modulated pathways at gene expression and/or DNAm level, were mainly associated with processes including cell cycle, DNA repair or metabolism and HIV, innate and adaptive immunity (GSEA q-value < 0.15). Of note, PCA revealed that only DNAm levels after the combined intervention segregated participants by their early or late viral rebound kinetics in MAP. We summarized the therapy-modulated pathways with eigenvectors of DNAm at post-RMD, and identified HIV, innate immunity and T-cell pathways among the most relevant to discriminate the two viral rebound profiles in MAP. Interestingly, 3 CpG positions in the HIV category, 37 in the innate immunity group and 10 in the T cell category were differentially methylated between individuals with different viral rebound profile (adjusted p < 0.1). Conclusions: Host transcription and epigenetic programs provide a deeper understanding of the molecular mechanisms induced during HIV cure therapies. While DNAm after a kick-and-kill strategy could be used to predict viral rebound kinetics after ART interruption, further study is warranted in future controlled studies., OA14.05LB B. Oriol-Tordera (1,2); A. Esteve-Codina (3); M. Berdasco (4,5); E. Goncalves (6); M. Esteller (4,5,7); T. Hanke (8,9); J. Molto (10); B. Clotet (1,10,11); M.L. Calle (11); B. Combadiere [...]

Background: MHC-E restricted T-cell responses have been observed in SIV-vaccine strategies using CMV-based vectors but their contribution to virus control is unclear. HLA-E was originally identified as a ligand of some NKG2 family receptors expressed by NK cells. Two highly frequent, functional, HLA-E alleles (*01:01 and *01:03) have been defined and T-cell responses to bacterial and viral pathogens restricted by these molecules have been described. Methods: In order to evaluate the interaction of HLA-E-presented viral epitopes with NK- and T-cell receptors, we modeled the structural interaction of HLA-E presenting canonical (VL9) and non-canonical (HIV-derived RL9 and PM9) epitopes with NKG2A/2C or TCR. We determined the ability of 14 peptides (including VL9, 1 CMV-, 1 EBV-and 11 HIV-derived) to stabilize HLA-E *01:01 and *01:03 and how this modified NK degranulation and cytotoxic activity. In vitro inhibition of viral replication by NK cells was assessed in autologous HIV infected CD4+ T-cells from HIV-seronegative individuals (N = 12). HLA-E expression on these target cells was assessed by RT-PCR and flow cytometry. The relationship of HLA-E expression with in-vivo HIV control was tested by measuring HLA-E expression in HIV-controllers (N = 31) and non-controllers (N = 16) by RT-PCR. Results: Our structural models evidenced that TCR have less affinity for HLA-E than NKG2 receptors. Interestingly, HIV peptides RL9 or PM9 presented by HLA-E*01:01 or HLA-E*01:03 showed a predicted increase in affinity to NKG2A and NKG2C, respectively. In vitro experiments indicated that HLA-E*01:01 was generally less stable on the cell surface, but none of HIV-derived peptides stabilized HLA-E. HIV KG9-HLA-E*01*03 and RL9/TP9/VI9/YG9-HLA-E*01*01 decreased NK cytokine secretion. RL9/MD9-HLA-E*01:01 resulted in increased lysis of peptide pulsed target cells. NK-mediated inhibition of viral replication correlated positively with HLA-E expression. Conversely, in HIV-infected individuals, HLA-E expression on total PBMC was significantly higher in non-controller individuals. Conclusions: The elevated expression of HLA-E in uncontrolled HIV infection and its potential differential interaction with NKG2 molecules depending on peptide binding indicates a pivotal role in NK during HIV infection. As such, HLA-E presenting HIV-derived epitopes may sensitize target cells for NK lysis in early infection whereas prolonged, elevated expression of HLA-E may lead to NK reduced viral control., OA05.01 L. Romero-Martin (1); C. Duran-Castells (1); M. Olivella (2); M. Rosas-Umbert (1); M. Ruiz-Riol (1); A. Olvera (1) and C. Brander (1) (1) Institut de Recerca de la Sida [...]

Background: T cell populations showing a particular cytokine polarization may have a key role in inducing and maintaining effective cellular and humoral immunity to HIV. In addition to the commonly assessed Th1 and CTL responses, other T cell profiles involved in the immune response to HIV infection are not well characterized and their contribution to virus control remains unknown. Methods: Cryopreserved PBMCs from HIV-1 controllers (n = 15, median plasma HIV-1 RNA, pVL: 210 copies/mL, median CD4+ T-cell count: 786 cells/[mm.sup.3]) and non-controllers (n = 15, median pVL: 52,435 copies/mL, median CD4+ T-cell count: 406 cells/[mm.sup.3]) were stimulated with 17 peptide pools covering the entire HIV proteome. Effector function profiles of virus-specific T-cells were evaluated by 'boosted flow cytometry'; a novel methodology based on labeling cytokines that characterize specific T cell profiles with the same fluorochrome (Th1/Tc1: INFg, IL-2, TNFa; Th2/Tc2: IL-4, IL-5, IL-13; Th17/Tc17: IL-17A, IL-22; Treg: IL-10, TGFb and Tfh/Tfc: IL-4, IL-21). We also evaluated the humoral response to HIV (IgM, IgA and IgG) to identify T-cell profile associated with humoral response. Finally, cellular and humoral responses correlated with respective clinical parameters (pVL, CD4 count). Results: The total magnitudes of HIV-specific CD4+ Tfh and CD8+ responses with a Tc2 or Tfc profiles were significantly higher in HIV controllers. Total Tfh/Tfc (CD4+ and CD8+) responses correlated negatively with pVL. Different polarization profiles were observed for responses targeting different HIV proteins, and overall a broader range of alternative effector functions were seen in cells from HIV controllers (Rev/Vpr/Env-specific Tc2, Pol/Env-specific Tfc and Pol-specific Tfh responses). HIV controllers also showed higher IgG responses to HIV-gp120 while their virus-specific IgM antibodies titers were lower than in HIV non-controllers. IgM titers correlated positively with pVL and negatively with HIV Tfc-specific responses. The frequency of Tfc cells correlated with the gp120-IgG/IgM ratio, supporting a possibly pivota role of those in HIV control and antibody isotype switching. Conclusions: We have identified a subpopulation of CD8+ T-cells with Tfc-like cytokine profile targeting HIV Pol and Env that correlated with pVL and antibody class switching. Further characterization of those Tfc-like cells is needed to confirm their role in natural HIV control., OA20.01 L. Romero-Martin (1); M.L Rodriguez de la Concepcion (2); J. Carrillo (2); J. Blanco (2); B. Mothe (3); M. Ruiz-Riol (2); C. Brander (2) and A. Olvera (2) (1) [...]