CROI 2016 Abstract eBook
Conclusions: This study identified a previously unrecognized subpopulation of cDCs induced preferentially in ECs with improved ability to mount cell-intrinsic immune responses to HIV-1 and expand antiviral T cell immune responses. Manipulation of this dendritic cell subpopulation by TLR agonists may provide novel opportunities for improving immune- based approaches for HIV treatment and prevention in a broader patient population. 20 The Sooty Mangabey Genome Sequence Reveals New Insights in Natural SIV Infections David J. Palesch 1 ; Steven E. Bosinger 1 ; GregoryTharp 2 ;Yue Liu 3 ; Muthuswamy Raveendran 3 ; Donna Muzny 3 ; Richard Gibbs 3 ; Kim C.Worley 3 ; Jeffrey Rogers 3 ; Guido Silvestri 1 1 Yerkes Natl Primate Rsr Cntr, Emory Univ, Atlanta, GA, USA; 2 Emory Univ, Atlanta, GA, USA; 3 Baylor Coll of Med, Houston, TX, USA Background: In contrast to HIV infection in humans and experimental SIV infection of rhesus macaques, SIV infection of natural host sooty mangabeys ( Cercocebus atys ) is typically non-pathogenic despite high levels of virus replication. To identify host genetic factors that allow sooty mangabeys (SM) to avoid SIV-related pathogenesis, we produced a de novo whole genome assembly for the SM and performed comparative analysis with AIDS-susceptible species. Methods: DNA from a female SM was sequenced at the Baylor Human Genome Sequencing Center using Illumina short read technology and Pacific Biosciences long reads. The assembly was produced using ALLPATHS-LG to generate contigs and scaffolds, then Atlas-Gapfill and PBJelly for filling gaps and extending contigs. Annotation was performed through the NCBI Genome Annotation Pipeline. To identify immune molecules with divergence between SM and AIDS-susceptible primates, we aligned multiple protein sequences using the MultAlin tool. To identify assembly/annotation errors, we used RNA-seq data frommultiple SM tissues. Raw reads were aligned to the SM assembly Caty1.0 and to the rhesus assembly MacaM using the STAR Aligner tool. Results: The final assembly has deep coverage (mean 192x) across 2.85 Gb of the SM genome with contig N50 of 113 kb and scaffold N50 of 12.8 Mb. We identified several candidate genes with major sequence differences between human, rhesus macaque and SM, which were confirmed by RNA-Seq. A key phenotypic feature of SMs is the significantly lower innate responses to lipopolysaccharide and other components of gram-negative bacteria compared to AIDS-susceptible species This may help to limit overall immune activation after SIV infection. Of interest, we identified a C-terminal frame shift mutation in the toll-like receptor 4 (TLR4) locus of SM, a crucial sensor of bacterial products that induces a proinflammatory cytokine response. The same frame shift mutation was also observed in the coding region of TLR4 of African Green Monkeys. Lastly, we also identified major structural variations in exons 3 and 4 of the immune regulatory protein intercellular adhesion molecule 2 (ICAM2). Conclusions: We identified several mutations predicted to have substantial effect on protein function in SM and AGM relative to non-natural SIV hosts. Identification of a TLR4- specific mutation in the SM genome provides the mechanistic basis for attenuated responses to Gram-negative bacteria, an important aspect of the natural host phenotype. 21 Hyperacute Microbial Translocation During Pathogenic SIV Infection Adam J. Ericsen 1 ; Michael Lauck 1 ; Mariel S. Mohns 1 ; Sarah R. DiNapoli 2 ; James P. Mutschler 1 ; Justin M. Greene 3 ; Benjamin J. Burwitz 3 ; Jonah B. Sacha 3 ;Thomas C. Friedrich 1 ; Jason M. Brenchley 4 ; David H. O’Connor 1 1 Univ of Wisconsin-Madison, Madison, WI, USA; 2 NIH, Bethesda, MD, USA; 3 Oregon Hlth & Sci Univ, Beaverton, OR, USA; 4 Frederick Natl Lab for Cancer Rsr, Frederick, MD, USA Background: During chronic human immunodeficiency virus (HIV) infection, microbial products migrate into the blood from the gastrointestinal tract concomitant with systemic immune activation. Whether this pathological microbial translocation occurs during acute immunodeficiency virus infection remains unknown, but given the immunomodulatory activity of microbial products, acute-phase translocation could drive early virus replication. Methods: Using simian immunodeficiency virus (SIV)-infected cynomolgus macaques, we performed 16S ribosome deep-sequencing and quantitative PCR to investigate SIV-associated perturbation to the composition and abundance of microbial products within stool and blood plasma. ELISA-based assays were used to monitor fluctuations in peripheral inflammation (MCP-1 and SAA-1), the bacteria-specific host response (sCD14 and EndoCab), and intestinal permeability (IFABP). We used flow cytometry to track changes to peripheral blood lymphocyte populations. Results: We found that within the first week of infection, prior to the peak of viremia, plasma levels of bacterial DNA increased as much as 1,390-fold over baseline while plasma levels of soluble CD14 (sCD14) correlated with set-point levels of virus replication. Translocation was accompanied by peripheral inflammation and an increase in CD4+CCR5+ T cells, which are the primary targets of virus replication. Conclusions: Altogether, our results identify hyperacute microbial translocation as one of the earliest pathological phenomena to occur during immunodeficiency virus infection, and suggest that it may promote early virus replication. Prophylactic strategies may benefit from better understanding the hyperacute-phase relationship between host commensals and incipient immunodeficiency virus. 22 CD8 T Cells Are Required to Suppress Viremia in SIV-Infected ART Treated Macaques Emily K. Cartwright 1 ;Thomas H.Vanderford 2 ; Kirk A. Easley 3 ; Joern E. Schmitz 4 ; Ann Chahroudi 5 ; Steven E. Bosinger 2 ; Mirko Paiardini 2 ; Jacob D. Estes 6 ; Guido Silvestri 2 1 Emory Univ, Atlanta, GA, USA; 2 Yerkes Natl Primate Rsr Cntr, Emory Univ, Atlanta, GA, USA; 3 Emory Univ Rollins Sch of PH, Atlanta, GA, USA; 4 Beth Israel Deaconess Med Cntr, Harvard Med Sch, Boston, MA, USA; 5 Emory Univ Sch of Med, Atlanta, GA, USA; 6 Frederick Natl Lab, Leidos Biomed Rsr, Frederick, MD, USA Background: HIV infection persists despite suppressive antiretroviral therapy (ART) and treatment interruption results in rapid viral rebound. Previous studies using antibody- mediated CD8+ lymphocyte depletion in simian immunodeficiency virus (SIV)-infected rhesus macaques (RM) showed that these cells suppress viremia in ART-naïve animals. However, the role of CD8+ lymphocytes during ART-mediated virus suppression is unknown. Using in vivo CD8+ lymphocyte depletion, we sought to elucidate whether CD8+ lymphocytes are required to maintain virus suppression in ART-treated SIV-infected RM. Methods: 13 rhesus macaques were infected IV with 3000 TCID50 of SIV mac239 . At 8 weeks post SIV infection animals were put on a 4 drug ART regimen consisting of Tenofovir, Emtricitabine, Raltegravir, and Darunavir for the duration of the study. Once virus was undetectable, we administered the anti-CD8 antibody MT-807R1 intravenously (50 mg/ kg). Plasma viral load and quantification of cell-associated SIV mac gag DNA was determined by RT- PCR. A next-generation, ultra-sensitive RNA in situ hybridization technology, RNAscope was used to visualize SIV-RNA+ cells in lymph nodes. Multiparametric flow cytometry was performed to monitor the immunological effects of the used interventions. Results: We found that in SIV-infected, ART-treated RMs, depletion of CD8+ lymphocytes results in increased virus production in both plasma and lymph nodes in 100% (13 out of 13 RM) of the treated animals. We also show that repopulation of CD8+ T cells (but not CD8+ NK cells) is associated with reestablishment of virus control. In addition, we found that the level of SIV-specific CD8+ T cells pre-CD8 depletion correlate with virus production after CD8 depletion. While the levels of SIV-DNA-positive cells remained unchanged after CD8+ lymphocyte depletion and reconstitution, the frequency of SIV-infected CD4+ T-cells pre-depletion positively correlates with both peak and area-under-the-curve of viremia post-depletion. Conclusions: This study is the first to examine the effects of CD8+ lymphocyte depletion on SIV-infected rhesus macaques undergoing continuous ART. Our findings reveal a previously underappreciated role of CD8+ T cells in cooperating with ART to maintain virus suppression during therapy. We believe this study provides important rationale to further explore the potential effect of therapeutic vaccinations and check-point blockade inhibitors in ART-treated HIV-infected humans. 23 Lack of CTL Attenuates, but Does Not Ablate Compartmentalization of SIV Replication Joy M. Folkvord 1 ; MelissaThomas 1 ; Eva Rakasz 2 ; Shengbin Li 3 ; Pamela Skinner 3 ; Elizabeth Connick 4 1 Univ of Colorado Anschutz Med Campus, Aurora, CO, USA; 2 Wisconsin Natl Primate Rsr Cntr, Madison, WI, USA; 3 Univ of Minnesota, Minneapolis, MN, USA; 4 Univ of Colorado, Denver, CO, USA Background: In chronic, asymptomatic SIV disease, SIV RNA+ cells are concentrated within B cell follicles. As CTL are abundant in extrafollicular regions (EF) of secondary lymphoid tissues, but scarce inside the follicle (F), we hypothesized that in acute infection prior to CTL development or in chronic infection after CD8 depletion, compartmentalization of SIV RNA+ cells would diminish.
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