CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: An aptamer screen was performed to identify proteins enriched on primary CD4 T-cells infected with HIV. Target enrichment was confirmed by flow cytometry as well as immuno-pulldown in CD4 T-cells from ART-suppressed donors using qPCR and ELISA. Functional assays were performed using recombinant BTN proteins or antibodies to demonstrate the impact of target modulation on HIV latency reversal and T-cell activation. Viral reactivation in a human latency model was measured by GFP or luciferase reporter virus and T-cell activation was evaluated concomitantly via IFNg release in the culture supernatant. Results: BTN immune checkpoint receptors were identified as cell surface proteins overexpressed on in vitro infected HIV+ CD4 T-cells relative to uninfected cells as determined by aptamer screen and flow cytometry. Antibody-pulldowns in CD4 T-cells from ART-suppressed participants demonstrated BTN3A-expressing cells enrich for HIV integrase RNA (4 of 8 participants), LTR DNA (4 of 4), and p24 protein (5 of 8). Recombinant BTN-Fc fusion proteins inhibited activation of human CD4 T-cells following anti-CD3 antibody stimulation, verifying pathway function. In contrast, BTN3A-specific antibodies enhanced T-cell activation and reactivated HIV in response to anti-CD3 antibody; an activity blocked by recombinant BTN3A-Fc proteins. Novel antibodies were generated against three BTN3A protein isoforms using yeast display and characterized for modulation of HIV latency and T-cell activation. Work is ongoing to evaluate Gag-specific CD8 T cell response +/- BTN antibodies. Conclusion: This data collectively implicates BTN3A family members as putative immune targets for HIV transcriptional regulation or T-cell activation. This novel finding warrants further investigation to determine if therapeutically modulating BTNs can impact the latent viral reservoir. 280 BISPECIFIC Au NANOPARTICLES FOR THE ENHANCEMENT OF THE NK IMMUNE RESPONSE AGAINST HIV Antonio Astorga Gamaza 1 , Mireya L. Borrajo 1 , Carla Serra Peinado 1 , Laura Luque-Ballesteros 1 , Oscar Blanch-Lombarte 2 , Julia G Prado 2 , Juan Lorente 3 , Félix Pumarola 3 , Marc Pellicer 3 , Vicenç Falcó 3 , Meritxell Genescà 1 , Víctor Puntes 1 , María J. Buzón 1 1 Vall d'Hebron Research Institute, Barcelona, Spain, 2 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 3 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain Background: An efficient immunological synapse is required for Natural Killer (NK) cells to kill viral infected cells. HIV infection promotes the appearance of dysfunctional NK cells with diminished capacity to kill infected cells. Thus, new tools to reinvigorate and redirect NK-mediated immune effector functions will help to eliminate HIV. Methods: We have developed bispecific gold nanoparticles (BiAb-AuNPs) containing two different polarized antibodies at their surface. BiAb-AuNPs were prepared by conjugating AuNPs with IgG anti-HIVgp120 (A32) and IgG anti-CD16 (3G8) antibodies following a novel controlled, linker-free and polarizing conjugation method. Validation was performed by transmission electron microscopy (TEM), UV-Vis Spectroscopy, Dynamic Light Scattering (DLS) and Zeta-potential measurements. The ability of BiAb-AuNPs to promote specific cell contacts was evaluated by flow cytometry and confocal microscopy. Functionality of BiAb-AuNPs was measured by ADCC assays and cytotoxicity assays performed in tonsil histocultures after ex vivo infection with HIV (n=8). In addition, the killing of viral reactivated cells promoted by BiAb-AuNPs was assessed in a primary cell model of HIV latency (n=5). In all assays we included irrelevant bispecific BiAb-AuNPs as a control. Results: BiAb-AuNPs increased the number of NK-HIV+CD4 T cell doublets by over 7-fold compared to control medium (median %doublets 16.0% vs. 2.5%) (p=0.0143; paired t test). Direct contact zipped by BiAb-AuNPs was confirmed by confocal microscopy. In addition, BiAb-AuNPs increased the percentage of NK cells producing IFN-g and CD107a (median 22.5% vs. 4.9% of medium control) (p<0.05; Friedman test) and triggered a potent cytotoxic response against HIV-expressing cells (median 29.1% vs. 14.9% or 12.7% for irrelevant BiAb- AuNPs and A32, respectively) (p=0.0313 for both comparisons; Wilcoxon test). Moreover, BiAb-AuNPs entered tonsil blocks, measured by the loss of detection of CD16 molecules in NK cells (p=0.0078; Wilcoxon test) and significantly impacted HIV infection in this lymphoid tissue, reaching up to 50% of reduction in some cases (p=0.0313; Wilcoxon test). Furthermore, BiAb-AuNPs enhanced the killing of latent-HIV-infected cells after viral reactivation, inducing a median of 51.5% killing (p=0.0163; One sample t test).

Conclusion: BiAb-AuNPs are a novel molecularly-targeted nanotool that potentiates NK-immune response against HIV. 281 INTERFERON-Α MODULATES THE HOST GLYCOSYLATION MACHINERY DURING TREATED HIV INFECTION Leila B. Giron 1 , Florent Colomb 1 , Emmanouil Papasavvas 1 , Livio Azzoni 1 , Xiangfan Yin 1 , Alitzel Anzurez 1 , Matthew Fair 1 , KaramMounzer 2 , Jay Kostman 2 , Pablo Tebas 3 , Una O'Doherty 3 , Qin Liu 1 , Michael R. Betts 3 , Luis J. Montaner 1 , Mohamed Abdel-Mohsen 1 1 Wistar Institute, Philadelphia, PA, USA, 2 Philadelphia FIGHT, Philadelphia, PA, USA, 3 University of Pennsylvania, Philadelphia, PA, USA Background: A comprehensive understanding of host factors modulated by the key antiviral cytokine interferon-α (IFNα) is imperative for harnessing its beneficial effects while avoiding its detrimental side-effects, during chronic diseases such as HIV infection. Cytokines modulate host glycosylation, and the host glycome (circulating glycans and cell-surface glycans) plays a critical role in mediating several cellular processes and immunological functions. However, the impact of IFNα on host glycosylation machinery has never been characterized. Methods: We assessed the impact of pegylated IFNα2a therapy on circulating IgG glycomes and isolated CD8+T and NK cell-surface glycomes of 18 HIV- mono-infected individuals on suppressive antiretroviral therapy, using capillary electrophoresis and lectin microarrays. Plasma levels of sCD14 and sCD163 were measured by ELISA. CD8+T cell and K562-stimulated NK cell phenotypes were profiled using flow cytometry. Integrated HIV DNA in CD4+T cells was measured by qPCR. Wilcoxon test and Spearman’s correlations were used for statistical analysis. False discovery rates (FDR) were calculated to account for multiple comparisons. Results: Interactome analysis highlighted significant interactions that support a model in which a) IFNα increases the proportion of pro-inflammatory, bisected GlcNAc glycans (known to enhance FcγR binding) within the IgG glycome (FDR<0.02), which in turn b) increases inflammation (as measured by sCD14 and sCD163; p<0.03), which c) leads to lower levels of CD8+T cell functionality (perforin, Eomes, and TNFα expression) but higher degranulation (CD107) (p<0.02, Figure). IFNα-mediated induction of bisected GlcNac associated with a poor reduction of HIV integrated DNA (p=0.02, rho=-0.8). Examining cell- surface glycomes, IFNα increases the levels of T antigen (Gal-GalNAc) on CD8+T cells (FDR=0.01). This induction is associated with lower CD8+T degranulation (p<0.02, rho<-0.8). Last, IFNα increases the levels of fucose on NK cells (p<0.05). This induction is associated with higher expression of Eomes, T-bet, and IFNγ upon K562 stimulation (p=0.048, rho>0.8). Conclusion: IFNα causes host glycomic alterations that are known to mediate inflammatory responses. These alterations are associated with mainly detrimental, but also beneficial, consequences of IFNα on innate and adaptive immune functions. Manipulating glycan-lectin interactions may represent a strategy to enhance the impact of IFNα on immunity while avoiding its detrimental side-effects.

Poster Abstracts

282 PHASE I/II RANDOMIZED STUDY: THERAPEUTIC DENDRITIC CELL VACCINE PLUS PEGYLATED INF-Α Lorna Leal 1 , Elvira Couto Jaime 1 , Yolanda Romero 1 , L Miralles 2 , Tania González 2 , M.J Maleno 2 , Blanca Paño 1 , Pich Judit 1 , Nuria Climent 2 , Sonsoles Sánchez-

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CROI 2020