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/γ c null (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.
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. IMPORTANCEMacrophages 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.
BackgroundHumanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points.ResultsHuman cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time.ConclusionsOverall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0209-9) contains supplementary material, which is available to authorized users.
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 HIVresistant 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
Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) as well as in macrophages cocultured with autologous CD8 T cells, the most potent being the human CD4(1 + 2) BiTE [termed CD(1 + 2) h BiTE] antibody construct and the CD4(1 + 2)L17b BiTE antibody construct. The CD4(1 + 2) h BiTE antibody construct promoted HIV infection of human CD4/CD8 T cells. In contrast, the neutralizing B12 and the VRC01 BiTE antibody constructs, as well as the CD4(1 + 2)L17b BiTE antibody construct, did not. Thus, BiTE antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential. HIV is a chronic infection well controlled with the current cART. However, we lack a cure for HIV, and the HIV pandemic goes on. Here, we showed and that a BiTE antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE antibody constructs display efficient killing of gp120-expressing cells and inhibited replication in HIV-infected PBMCs or macrophages. We believe that BiTE antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds which reverse latency.
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, HIVassociated 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.
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.
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