Mary-Aude Rochat

Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting anti-retroviral drugs for treating HIV-1

Background Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. Methods/Principal Findings NOD/shi-scid/γcnull (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. Conclusions/Significance This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.

Triggering TLR2, -3, -4, -5, and -8 Reinforces the Restrictive Nature of M1- and M2-Polarized Macrophages to HIV

ABSTRACT Macrophages must react to a large number of pathogens and their effects. In chronic HIV infection, the microenvironment changes with an influx of microbial products that trigger Toll-like receptors (TLRs). That dynamic nature can be replicated ex vivo by the proinflammatory (M1-polarized) and alternatively activated (M2-polarized) macrophages. Thus, we determined how polarized macrophages primed by various TLR agonists support HIV replication. Triggering of TLR2, -3, -4, -5, and -8 reinforced the low level of permissiveness in polarized macrophages. HIV was inhibited even more in M1-polarized macrophages than in macrophages activated only by TLR agonists. HIV was inhibited before its integration into the host chromosome. Polarization and triggering by various TLR agonists resulted in distinct cytokine profiles, endocytic activity, and distinct upregulation of restriction factors of HIV. Thus, different mechanisms likely contribute to the HIV-inhibitory effects. In chronic HIV infection, macrophages might become less permissive to HIV due to changes in the microenvironment. The high level of reactivity of polarized macrophages to TLR triggering may be exploited for immunotherapeutic strategies. IMPORTANCE Macrophages are a major target of HIV-1 infection. Different cell types in this very heterogeneous cell population respond differently to stimuli. In vitro, the heterogeneity is mimicked by their polarization into proinflammatory and alternatively activated macrophages. Here we explored the extent to which agonists triggering the TLR family affect HIV replication in polarized macrophages. We found that a number of TLR agonists blocked HIV replication substantially when given before infection. We also report the mechanisms of how TLR agonists exert their inhibitory action. Our findings may advance our understanding of which and how TLR agonists block HIV infection in polarized macrophages and may facilitate the design of novel immunotherapeutic approaches.

Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

ABSTRACT Gene-engineered CD34+ hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+ T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+ cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+ T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

Promising Role of Toll-Like Receptor 8 Agonist in Concert with Prostratin for Activation of Silent HIV

ABSTRACT The persistence of latently HIV-infected cells in patients under combined antiretroviral treatment (cART) remains the major hurdle for HIV eradication. Thus far, individual compounds have not been sufficiently potent to reactivate latent virus and guarantee its elimination in vivo. Thus, we hypothesized that transcriptional enhancers, in concert with compounds triggering the innate immune system, are more efficient in reversing latency by creating a Th1 supportive milieu that acts against latently HIV-infected cells at various levels. To test our hypothesis, we screened six compounds on a coculture of latently infected cells (J-lat) and monocyte-derived dendritic cells (MDDCs). The protein kinase C (PKC) agonist prostratin, with a Toll-like receptor 8 (TLR8) agonist, resulted in greater reversion of HIV latency than any single compound. This combinatorial approach led to a drastic phenotypic and functional maturation of the MDDCs. Tumor necrosis factor (TNF) and cell-cell interactions were crucial for the greater reversion observed. Similarly, we found a greater potency of the combination of prostratin and TLR8 agonist in reversing HIV latency when applying it to primary cells of HIV-infected patients. Thus, we demonstrate here the synergistic interplay between TLR8-matured MDDCs and compounds acting directly on latently HIV-infected cells, targeting different mechanisms of latency, by triggering various signaling pathways. Moreover, TLR8 triggering may reverse exhaustion of HIV-specific cytotoxic T lymphocytes that might be essential for killing or constraining the latently infected cells. IMPORTANCE Curing HIV is the Holy Grail. The so-called “shock and kill” strategy relies on drug-mediated reversion of HIV latency and the subsequent death of those cells under combined antiretroviral treatment. So far, no compound achieves efficient reversal of latency or eliminates this latent reservoir. The compounds may not target all of the latency mechanisms in all latently infected cells. Moreover, HIV-associated exhaustion of the immune system hinders the efficient elimination of the reactivated cells. In this study, we demonstrated synergistic latency reversion by combining agonists for protein kinase C and Toll-like receptor 8 in a coculture of latently infected cells with myeloid dendritic cells. The drug prostratin stimulates directly the transcriptional machinery of latently infected cells, and the TLR8 agonist acts indirectly by maturing dendritic cells. These findings highlight the importance of the immune system and its activation, in combination with direct-acting compounds, to reverse latency.

Differential Dynamics of HIV Infection in Humanized MISTRG versus MITRG Mice

Humanized mice are a powerful tool to study HIV in vivo. The recently generated mouse strains MITRG and MISTRG, which differ in human SIRPα expression, support an improved human myeloid lineage development from human hematopoietic stem and progenitor cells. The rationale of the study was the characterization of the two mouse strains during an HIV infection with CCR5- and CXCR4-tropic viruses. Upon HIV infection, we observed HIV dissemination and sustained viral load over 20 wk in peripheral blood in both reconstituted mouse strains. However, HIV RNA levels were significantly lower in MITRG mice compared with MISTRG mice during the first 8 wk postinfection. HIV-infected MISTRG mice showed lymphocyte activation and changes in lymphocyte subsets in blood and spleen, recapitulating hallmarks of HIV infection in humans. Depletion of murine tissue-resident macrophages in MITRG mice led to significantly elevated viral loads, and lymphocyte levels were similar to those in HIV-infected MISTRG mice. Depletion of CD8+ T cells in MISTRG mice before HIV infection resulted in substantially decreased CD4+ T cell levels, indicating functionality of human CD8+ T cells; depletion of CD4+CD8+ thymocytes may have contributed, in part, to the latter finding. In summary, MITRG and MISTRG mice represent novel HIV mouse models, despite differential HIV dynamics.

448. Lentivector Knock-Down of CCR5 in Hematopoietic Stem Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

Gene engineered CD34+ hematopoietic stem cells (HSCs) can be used to generate an HIV-1 resistant immune system. In this study, we combine CCR5 knock-down from a highly efficient miRNA lentivector and pre-transplantation selection of transduced HSCs. Low level transduction of HSCs and subsequent sorting by flow cytometry yielded >90% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain (YU-2): viral load was significantly decreased over months, and human CD4+ T cells were preserved. In one mouse, viral mutations, presumably to a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSC-based CCR5 knock-down may lead to efficient control of HIV in vivo. As a certain threshold of transduced HSCs is required, improved transduction procedures or in vitro/in vivo selection strategies might be necessary if this is to be translated to clinics. We overcome a major limitation of previous HIV gene therapy humanized mice studies, namely the generation of chimeric mice in which only a proportion of the cells in vivo are anti-HIV engineered. We produce mice where practically all CD4+ T-cells are transduced resulting in long-term suppression of HIV. This underlines the promising future of gene engineering HIV resistant CD34+ cells which produce a constant supply of HIV resistant progeny.