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UnlabelledHLA-B*57:01 and HLA-B*57:03, the most prevalent HLA-B*57 subtypes in Caucasian and African populations, respectively, are the HLA alleles most protective against HIV disease progression. Understanding the mechanisms underlying this immune control is of critical importance, yet they remain unclear. Unexplained differences are observed in the impact of the dominant cytotoxic T lymphocyte (CTL) response restricted by HLA-B*57:01 and HLA-B*57:03 in chronic infection on the Gag epitope KAFSPEVIPMF (KF11; Gag 162 to 172). We previously showed that the HLA-B*57:03-KF11 response is associated with a >1-log-lower viral setpoint in C clade virus infection and that this response selects escape mutants within the epitope. We first examined the relationship of KF11 responses in B clade virus-infected subjects with HLA-B*57:01 to immune control and observed that a detectable KF11 response was associated with a >1-log-higher viral load (P = 0.02). No evidence of HLA-B*57:01-KF11-associated selection pressure was identified in previous comprehensive analyses of >1,800 B clade virus-infected subjects. We then studied a B clade virus-infected cohort in Barbados, where HLA-B*57:03 is highly prevalent. In contrast to findings for B clade virus-infected subjects expressing HLA-B*57:01, we observed strong selection pressure driven by the HLA-B*57:03-KF11 response for the escape mutation S173T. This mutation reduces recognition of virus-infected cells by HLA-B*57:03-KF11 CTLs and is associated with a >1-log increase in viral load in HLA-B*57:03-positive subjects (P = 0.009). We demonstrate functional constraints imposed by HIV clade relating to the residue at Gag 173 that explain the differential clade-specific escape patterns in HLA-B*57:03 subjects. Further studies are needed to evaluate the role of the KF11 response in HLA-B*57:01-associated HIV disease protection.ImportanceHLA-B*57 is the HLA class I molecule that affords the greatest protection against disease progression in HIV infection. Understanding the key mechanism(s) underlying immunosuppression of HIV is of importance in guiding therapeutic and vaccine-related approaches to improve the levels of HIV control occurring in nature. Numerous mechanisms have been proposed to explain the HLA associations with differential HIV disease outcome, but no consensus exists. These studies focus on two subtypes of HLA-B*57 prevalent in Caucasian and African populations, HLA-B*57:01 and HLA-B*57:03, respectively. These alleles appear equally protective against HIV disease progression. The CTL epitopes presented are in many cases identical, and the dominant response in chronic infection in each case is to the Gag epitope KF11. However, there the similarity ends. This study sought to better understand the reasons for these differences and what they teach us about which immune responses contribute to immune control of HIV infection.

Immune responses induced during the early stages of chronic viral infections are thought to influence disease outcome. Using HIV as a model, we examined virus-specific cytotoxic T lymphocytes (CTLs), T helper cells, and viral genetic diversity in relation to duration of infection and subsequent response to antiviral therapy. Individuals with acute HIV-1 infection treated before seroconversion had weaker CTL responses directed at fewer epitopes than persons who were treated after seroconversion. However, treatment-induced control of viremia was associated with the development of strong T helper cell responses in both groups. After 1 yr of antiviral treatment initiated in acute or early infection, all epitope-specific CTL responses persisted despite undetectable viral loads. The breadth and magnitude of CTL responses remained significantly less in treated acute infection than in treated chronic infection, but viral diversity was also significantly less with immediate therapy. We conclude that early treatment of acute HIV infection leads to a more narrowly directed CTL response, stronger T helper cell responses, and a less diverse virus population. Given the need for T helper cells to maintain effective CTL responses and the ability of virus diversification to accommodate immune escape, we hypothesize that early therapy of primary infection may be beneficial despite induction of less robust CTL responses. These data also provide rationale for therapeutic immunization aimed at broadening CTL responses in treated primary HIV infection.

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) [...]

Background: As part of our contribution to the EAVI2020 consortium research project to get a preventive HIV vaccine, we've constructed recombinant Mycobacterium bovis BCG expressing the EAVI2020 T-cell immunogens. Methods: In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVA-CAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic BCG strain (BCG[DELTA]Lys) to generate BCG.[HTI.sup.2auxo.int]. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed and vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for. BALB/c mice were immunized with BCG. [HTI.sup.2auxo.int] prime and MVA.HTI boost was safe and induced HIV-1-specific T-cell responses. T-cell responses were assessed by IFN-[gamma] ELISpot upon stimulation with 17 peptide pools spanning the HTI proteome. Results: The highest total magnitude of IFN-[gamma] spot forming cells (SFC)/[10.sup.6] splenocytes was observed in BCG.[HTI.sup.2auxo.int] primed mice compared to mice receiving MVA.HTI alone or mice primed with BCGwt, although the differences between the vaccination regimens only reached trends. In order to evaluate the differences in the breadth of the immune response between mice primed with BCG.[HTI.sup.2auxo.int] and BCGwt, we examined the number of reactive peptide pools per mouse. Interestingly, both BCG.[HTI.sup.2auxo.int] and BCGwt primed mice recognized an average of 4 peptide pools per mouse. However, the variation was higher in BCG.[HTI.sup.2auxo.int] primed mice with one mouse recognizing 12 peptide pools and three mice recognizing few or no peptide pools. On the other hand when comparing the percentage of responding mice per peptide pool, all BCGwt primed mice respond to the more dominant peptides IKProt and 2BRT, whereas the recognition profile appeared to be more spread out for BCG.[HTI.sup.2auxo.int] primed mice and mice only receiving MVA.HTI, with these groups having a few mice responding to less recognized pools such as 1Dp17, IEp24, 1JProt. Mice were monitored weekly for signs of malaise. No vaccine-related deaths, no loca adverse events, and no associated systemic reactions were observed. Conclusions: This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent paediatric pathogens., OA09.01 N. Saubi (1); A. Kilpelainen (1); Y. Eto (1); C.-W. Chen (1); A. Olvera (2); T. Hanke (3); C. Brander (2) and J. Joseph (4) (1) Vall d'Hebron Institut [...]

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level., Author(s): A J Leslie [1, 12]; K J Pfafferott [1, 12]; P Chetty [2]; R Draenert [3]; M M Addo [3]; M Feeney [3]; Y Tang [3]; E C Holmes [...]