HIV-1 Replication and Pathogenesis in vivo

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for ARRA years 1-2 fu nding The goals of this project are to define how HIV-1 interacts with pOC and to elucidate the role of pDC HIV-1 with pDC and the role pOC HIV-1 pathogenesis. cells in HIV-1 replication and pathogenesis. As the major sensor of viral infections, altered pOC level/activity altered pDC playa role during HIV-1 disease progression. However, the role of pOC cells in HIV infection and may play a critical role during HIV-1 disease progression. However, the role of pDC cells in F-IIV infection and poorly understood, mainly due to the lack of robust in vivo models. The OKO-hu HSC pathogenesis is poorly understood, mainly due to the lack of robust in vivo models. The DKO-hu I-4SC model is Ideal ideal for this purpose. With a stable functional human immune system, functional pOC ceUs are developed in functional functional pDC cells are in OKO-hu mice. HIV- 1 normal proportion in all lymphoid organs in DKO-hu mice. HIV-1 establishes persistent infection, with immune with C04 cells. hyperactivation and depletion of human CD4 T cells. We have also shown that, during HIV-1 infection, PDC also shown that, during HIV-1 infection, POC cells are productively infected, activated, depleted and functionally impaired in DKO-hu HSC mice. HIV-1 with in OKO-hu mice. with C04 the pathogenic R3A Env also efficiently activates POC in vitro, correlated with its high binding affinity to CD4 PDC in vitro, correlated with binding receptor and coreceptors. Based on our preliminary findings and reports from Sly-infected monkeys or HIVon from SIV-infected monkeys or HIVinfected patients, I postulate that HIV-1 intimately interacts with POC cells, and chronic engaging of PDC patients, postulate that HIV-1 intimately interacts with PDC cells, and chronic engaging PDC during persistent HIV infection will deplete or impair POC activity. The reduced or altered POC activity wifl PDC activity. The reduced or altered PDC activity infection, hyperimmune activation and AIDS progression. contributes to chronic HIV infection, hyper-immuneactivation and AIDS progression. 'U_ f/1 .y, `<a -d-& n"0 The following modified specific aims are proposed to test these hypotheses. First, we will investigate the we will investigate the these hypotheses. pOC proliferation and survival of pDC cells during early and late-chronic HIV-1 infection in DKO-hu mice (SA1a). and HIV-1 infection OKO-hu Second, we will define the role of each relevant receptor (C04, CCR5, CXCR4, BOCA2, TLR7 and TLR9) in Second. we will define the role of each relevant receptor (CD4, CCR5, CXCR4, BDCA2, TLR7 and TLR9) in pDC activation with genetic approaches. In addition, we win also define the signaling defects in pDC cells In we will the defects in pOC cells pDC Induced by HIV infection, by genetically analyzing the candidate signaling pathways (SA2a). Third, we will induced genetically analyzing the candidate signaling pathways (SA2a). Third, we will pOC-specific ILT7 treat DKO-hu mice with the pDC-specific ILT7 mAb conjugated with the Saporin toxin , which specifically conjugated toxin, which pOC, to test the role of pOC infection (SA3c). depletes pDC, to test the role of pOC during infection (SA3c). thus focus on the most fundamental questions of pOC cells in HIV pathogenesis. Elucidation of We will thus locus on the most fundamental questions of pDC cells in F-IIV pathogenesis. Elucidation of the in HIV-1 infection and AIDS pathogeneSiS mechanism by which HIV-1 interacts with pOC cells and their role in HIV-1 infection and AIDS pathogenesis mechanism by which HIV-1 interacts with pDC cells will facilitate not only our understanding of pDC biology in HIV pathogenesis, but also development of novel not only our understanding pOC biology HIV pathogeneSiS, development novel therapeutic strategies. strategies. (nom .g. shy any dmo S0mwoo :off Cap OIL Q?__. -_a 020 inM ="O E-C o.- 81. Modified Specific Aim Section for first two year ARRA funding first two year ARRA funding BI. Modified Specific Aim As the major sensor of viral infections, altered pDC evel/activity may play a critical role during HIV-1 major sensor viral infections, altered pOC level/activity playa critical role during HIV-1 As disease progression. However, how HIV infection, activates, reduces and impairs pDC cells is poorly disease progression. However, how HIV infection, activates, reduces and impairs pOC cells is poorly understood. The DKO-hu HSC model is ideal for this purpose. With a stable functional human immune system, understood. OKO-hu HSC model is ideal purpose. With developed normal proportion in all lymphoid organs in DKO-hu mice. HIV-1 functional pDC cells functional pDC cells are developed in normal proportion in all lymphoid organs in DKO-hu mice, HIV-1 establishes persistent infection, with immune hyperactivation and depletion of human CD4 cells. Based establishes persistent infection, with immune hyperactivation and depletion of human C04 TI cells. Based on preliminary our pretiminary findings and reports from Sly-infected monkeys or HIV-infected patients, II postulate that HIV-1 and reports from SIV-infected monkeys or HIV-infected patients, postulate that HIV-1 intimately interacts with POC cells (via direct binding to CD4, CCR5, CXCR4 and BDCA2 on PDC cells and and intimately interacts with PDC cells (via direct binding to CD4, CCR5, CXCR4 and BDCA2 on PDC endosomes), TLR7 in endosomes). and chronic exposure of PDC during persistent HIV infection will deplete or impair PDC chronic exposure POC during persistent HIV infection will deplete or PDC activity. reduced or altered PDC activity contributes to aberrant hyper�immune activation and AIDS activity. The reduced or altered PDC activity contributes to aberrant hyper-imrnune activation and AIDS progression. The following modified specific aims are proposed for this 2-year ARRA funded period. progression. following modified aims are proposed this ARRA funded period. N-C N000. a33 0'9M d<= mad human poe (1) To study how HIV-1 infection alters homeostasis and function of human pDC cells in vivo: (1) To study how HIV-1 infection alters homeostasis and function a. Does HIV-1 infection modulate proliferation of pDC cells in lymphoid organs in vivo? vivo? a. HlV1 infection modulate proliferation of pOC cells investigate the proliferation pOC cells during early and late-chronic HIV-1 mice. We will investigate the proliferation pDC cells during early and late-chronic F-11V-1 infection in DKO-hu mice. HIV-1 interacts with pDC and impairs pDC activity: (2) To investigate how l-IIV-1interacts with poe and impairs poe activity: To investigate a. What is the relative role of pDC receptors in activation of pDC cells? a. relative role pDC receptors activation pDC will define the role of each relevant receptor (C04, CCR5, CXCR4, BOCA2, TLR7 and TLR9) in pDC We will define the role of each relevant receptor (CD4, C0R5, CXCR4, BDCA2, TLR7 and TLR9) in pDC We approaches the GEN2.2 human pOC activation with specific inhibitors in PBMC culture and with genetic approaches in the GEN2.2 human pDC activation with specific inhibitors in PBMC cell. We will define the signaling defects in pDC cells induced by HIV infection, by genetically analyzing the define the signaling defects in pDC ceUs induced by HI V infection, by genetically analyzing the cell. candidate signaling pathways. pathways. LL' GUI any ram (3) To define poe in vivo: (3) To define role of pDC cells in HIV-1 infection and pathogenesis in vivo: HIV-1 infection c. Does depletion of pDC in vivo affect HIV-1 infection and pathogeneSiS? pOC vivo HIV-1 c. pathogenesis? >_n depletes pDC, to test the role of pDC during infection. pOC, test role of pOC during infection. 5q. treat DKO-hu mice with the pOC-specific mAb conjugated with the Saporin toxin, which specifically We will treat DKO-hu mice with the pDC-specific mAb conjugated with the Saporin toxin, which specifically ... duo map ANC ... �-' C'O' ... O--_ BSc 0 0._ 00, (�p N._ L-- �o. Oft --' W=o ~�ai Moo 1-6 ELL 0-5 Esc 01O E.., 82. Modified Specific Aim Section for years 3 and 4 NIAID funding f or years 3 NIAlO funding B2. Modified (1) To study how HIV-1 infection alters homeostasis and function of human pOC cells in vivo: poe (1) To study how HIV-1 infection alters and function of b. poe and survival in lymphoid organs in vivo? b. Does HIV-1 infection affect pDC migration and survivat in lymphoid organs in vivo? We will investigate the migration and surviva' poe cells during early and late-chronic HIV-1 infection We will investigate the migration and surviva l of pDC cells during early and late-chronic HIV-1 infection in in DKO-hu mice. (2) To investigat e how HIV-1 interact s with pDC and impairs pOC activity: (2) To investigate how HIV-1 interacts with pOC and impairs pOC activity: b. What genes and signaling pathways are attered in pIJC cells "anergized" by HIV infection in vivo? altered po e Wanergized~ in vivo? b. poe OKO-hu We will purify pDC cells from mock- or HIV-infected DKO-hu mice to identify genes that are deregulated by HIV infection. We will define the signaling defects in pDe cells induced by HIV infection. infection. will the signaling defects in pDC cells induced by HIV ^-o o.3 (3) To define role of pOC cells in HIV-1 infection and pathogenesis in vivo: (3) To define role pOC cells in HIV-1 infection and pathogenesis vivo: a. Does elevated poe activation affect HIV-1 infection and immuno-pathogenesis? a. elevated pDC activation HIV-1 infection and immuno-pathogenesis? b. Does decreased poe activity via TLR7f9 antagonists affect HIV-1 infection and pathogenesis in vivo? b. decreased pDC activity via TLR7/9 antagonists affect infection and pathogenesis in vivo? We will study if stimulation or inhibition of pDC activation with the agonistic or antagonistic ligands of We will study if stimulation or inhibition of po e activation with the agonistic or antagonistic ligands TLR7fTLR9 before or during HIV infection will affect HIV replication and immuno-pathogenesis. TLR7/TLR9 during will replication and immuno-pathogenesis. `<o C.