CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

polarization. Gag-specific CD4, but particularly cTfh, were characterized by high expression of multiple co-inhibitory receptors. Longitudinal samples collected before and after ART initiation revealed that this dysregulated phenotype was established during the untreated phase of infection and persisted despite subsequent viral control. More Gag-specific cTfh produced Tfh- (CXCL13, IL-21) and, surprisingly, Th1-cytokines (IL-2, IFNγ, TNFα) after Ag stimulation compared to CMV, suggesting a functionally dysregulated profile. Importantly, this increased production of cytokines by cTfh was mirrored by the functional profile of GC Tfh, as observed in surgical lymph node (LN) biopsies. Conclusion: Our results thus reveal that in contrast to cTfh responses to other viruses in the same subjects, HIV-specific cTfh show a persistently expanded and activated phenotype in HIV+ subjects on ART. This might reflect a functional hyperactivity of GC Tfh in the LN that has been associated with aberrant B cell responses. These permanent alterations may contribute to lack of restoration of effective HIV-specific immunity despite prolonged ART, and complicate HIV cure efforts. 235 NEUTROPHIL DYNAMICS IN SIV-INFECTED PIGTAILED MACAQUES BEFORE AND AFTER ART Egidio Brocca-Cofano 1 , Tianyu He 1 , Ranjit Sivanandham 1 , Benjamin Policicchio 1 , Paola Sette 1 , Tammy Dunsmore 1 , George S. Haret-Richter 1 , Alan Landay 2 , Cristian Apetrei 1 , Ivona Pandrea 1 1 University of Pittsburgh, Pittsburgh, PA, USA, 2 Rush University Medical Center, Chicago, IL, USA Background: Neutrophils are important effectors of innate immunity. They are rapidly mobilized at the site of infection and fight pathogens through phagocytosis, killing of the infected cells or activation of adaptive immune cells. The interaction between platelet and neutrophils is critical for the neutrophil chemotaxis and function at the inflammation sites. During HIV infection, neutrophil counts and function are impaired, which may lead to the increased rates of opportunistic infection. We investigated the dynamics, proliferation, apoptosis and function of neutrophils in a highly pathogenic SIV infection and the impact of antiretroviral treatment (ART) on these parameters. Methods: Seventeen pigtail macaques were included. All animal were infected intravenously with 300TCID50 of SIV. After 42 days, all animals received a triple coformulated antiretroviral therapy (ART) containing tenofovir, emtricitabine and dolutegravir (PMPA+FTC+DTG). Neutrophil absolute counts, apoptosis and proliferation were assessed by flow cytometry. T assess phagocytosis and respiratory burst activity, functional assays were performed, measuring PARs and TF expression on neutrophils after LPS and thrombin stimulation. Cytokines secreted by neutrophils post-PMA stimulation were measured by Luminex. Results: SIV infection induced a significant loss of circulating neutrophils. After ART, neutrophil numbers were partially recovered, but did not reach the baseline levels. High postinfection levels of apoptosis of neutrophils, which persisted post–ART, may be the factor behind neutrophil loss. Significant increases of neutrophil proliferation (Ki-67) occurred early in SIV infection. During late infection, proliferation decreased below the baseline, suggesting a loss of neutrophil replicative capacity. SIV infection significantly decreased phagocytosis and respiratory bust of neutrophils and ART does not provide any short- or long-term benefit. PAR-1 and TF expression on neutrophils increased after LPS stimulation. A clear decrease of neutrophil capacity to secrete cytokines was observed. ART only partially improved neutrophil function. Conclusion: Significant decrease of neutrophil counts and function occur in SIV infection, and are not restored by ART. Phagocytosis and respiratory burst alterations, together with LPS-dependent increases of PARs and TF may contribute to the tissue damage and coagulation abnormalities observed during SIV infection. 236 CHARACTERIZATION OF FDC-BOUND VIRAL PRODUCTS DURING HIV/SIV INFECTION Matthew Bronnimann 1 , Joy M. Folkvord 1 , Pamela Skinner 2 , Eva Rakasz 3 , Elizabeth Connick 1 1 University of Arizona, Tucson, AZ, USA, 2 University of Minnesota, Minneapolis, MN, USA, 3 University of Wisconsin, Madison, WI, USA Background: During chronic HIV/SIV infection, the B-cell follicle is a major site of virus replication, and large numbers of infectious viral particles accumulate on follicular dendritic cells (FDCs) in the form of immune complexes (ICs). Despite the importance of the B-cell follicle in HIV/SIV persistence, the establishment and composition of FDC-bound HIV/SIV ICs remains poorly

characterized. In this study we analyzed FDC-bound viral products during HIV/ SIV infection. Methods: Formalin fixed paraffin embedded (FFPE) lymph nodes (LNs) from acutely infected (7 dpi) and chronically infected (Non SAIDS, 69-118dpi) rhesus macaques or chronically HIV-infected humans were evaluated. Rhesus macaque tissue sections were stained with antibodies to CD20 (B-cells) and smooth muscle myosin heavy chain (FDC networks) and combined with in situ hybridization for SIV RNA (RNAScope ACD). For Human/HIV studies, 1-2 sections of FFPE or snap frozen LNs and tonsils from chronically infected untreated and healthy participants were evaluated. FFPE sections were analyzed for HIV RNA (RNAscope) and snap frozen sections were stained with antibodies to IgD (rings FDCs) and Nef. Imagescope (Leica) was used for quantification. FDC-bound SIV RNA was quantified by positive pixel counts on FDC networks with RNA+ cells excluded. Data were analyzed using Mann-Whitney tests and Spearmen coefficients (r). Results: The percent LN comprised of FDC networks tended to be larger in chronic (n=8) compared to acute (n=6) SIV infection (p=0.08; medians, acute= 5.24% chronic=18.73%). FDC-bound SIV RNA was not observed during acute infection, but was detected in all chronic infections. The size of the FDC-bound RNA reservoir strongly correlated with plasma SIV load in chronic infection (r=0.857, p=0.01). In chronic HIV infection, FDC-bound HIV RNA was detected in all LNs tested. Additionally, HIV-Nef+ staining was evident on FDC networks in most LNs (6/8) and tonsils (1/1), but was absent in healthy controls. Conclusion: No FDC-bound SIV RNA is established during the first 7 days of acute SIV infection despite high viral loads, consistent with the notion that antibodies are necessary for the establishment of this reservoir. The strong relationship between FDC-bound RNA and plasma viral load suggests that they are in equilibriumwith each other. Because Nef is a known immune-modulator, the accumulation of Nef on FDC networks may contribute to dysfunction of Tfh and other follicular cell subsets. 237 HIV-2 IMPACT ON LYMPHOID ORGANS: EVIDENCE OF VIRAL REPLICATION AND TISSUE DISRUPTION Ana Antão, Ana Pires, Cristina Ferreira, Helena Nunes-Cabaço, Sandra Mauricio, Cheila Rocha, Paula Matoso, Rui M. Victorino, Susana M. Fernandes, Ana E. Sousa Universidade de Lisboa, Lisbon, Portugal Background: The ability of HIV to replicate in secondary lymphoid organs (SLO) and disrupt lymph node (LN) architecture are considered main determinants of disease progression. HIV-2+ individuals feature slow rate of CD4 T cell decline and AIDS progression, maintain low to undetectable viremia in the presence of disseminated viral reservoirs, and high titers of broadly neutralizing antibodies throughout the disease course. Thus, this naturally- occurring attenuated infection provides a unique setting to investigate HIV-host interplay in SLO that has been poorly explored. Methods: We conducted a parallel study of 1) archived LN from HIV-2+, HIV-1+ and seronegative individuals using immunochemistry, and 2) in vitro infection of tonsil organ cultures (TOCs) with HIV-2 and HIV-1 primary isolates with different co-receptor usage. Results: HIV-2+ individuals featured very different CD4 T cell counts at the time of lymph node collection (744, 444, 255 and 73 cells/µl), but in all of themwe could find viral replication, as assessed by p27 staining. Using the in vitro model we further confirmed comparable levels of HIV-2 and HIV-1 replication in terms of proviral DNA and gag mRNA levels, although the amount of cells producing virus (KC57+) were significantly lower in HIV-2 X4 than in HIV-1 X4 isolates. Moreover, the pattern of viral impact on the cellular populations was remarkably reproducible between TOCs, with a preferential depletion of follicular CD4 T cells (TFH), particularly upon infection with HIV-2 X4. Interestingly follicular regulatory CD4 T cells (TFR) were less depleted and CXCR5+CD8+ T cells were maintained. Importantly, LN from HIV-2+ patients featured a progressive CD4 T cell depletion in agreement with blood CD4 T cell decline, which was more marked than in the 12 HIV-1+ LN evaluated. Of note, FOXP3+ and CD8 T cells were preserved in non-germinal center areas. Regarding LN architecture, we found significant collagen deposition with reticulin network disruption, even in early stage HIV-2+ individuals, as compared to both seronegatives (n=8) and HIV-1+. Additionally, HIV-2+ LN featured less hyperplasic germinal centres than HIV-1+ LN, but their morphology were more disrupted.

Poster Abstracts

83

CROI 2018