cART Restores Transient Responsiveness to IFN Type 1 in HIV-Infected Humanized Mice.

The lack of a human immunodeficiency virus (HIV) cure has heightened interest in immunotherapy. As such, type I interferons (IFNs), in particular, IFN alpha (IFN-α), have gained renewed attention. However, HIV pathogenesis is driven by sustained IFN-mediated immune activation, and the use of IFNs is rather controversial. The following questions therein remain: (i) which IFN-α subtype to use, (ii) at which regimen, and (iii) at what time point in HIV infection it might be beneficial. Here, we used IFN-α14 modified by PASylation for its long half-life in vivo to eventually treat HIV infection. We defined the IFN dosing regimen based on the maximum increase in interferon-stimulated gene (ISG) expression 6 h after its administration and a return to baseline of ubiquitin-specific protease 18 (USP18) prior to the next dose. Notably, USP18 is the major negative regulator of type I IFN signaling. HIV infection resulted in increased ISG expression levels in humanized mice. Intriguingly, high baseline ISG levels correlated with lower HIV load. No effect was observed on HIV replication when PASylated IFN-α14 was administered in the chronic phase. However, combined antiretroviral therapy (cART) restored responsiveness to IFN, and PASylated IFN-α14 administered during analytical cART interruption resulted in a transiently lower HIV burden than in the mock-treated mice. In conclusion, cART-mediated HIV suppression restored transient IFN responsiveness and provided a potential window for immunoenhancing therapies in the context of analytical cART interruption. IMPORTANCE cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages. Thus, the HIV research community is exploring novel ways to control HIV infection for longer periods without cART. Here, we explored novel, long-acting IFN-α14 for its efficacy to control HIV replication in HIV-infected humanized mice. We found that IFN-α14 had no effect on chronic HIV infection. However, when mice were treated first with cART, we observed a transiently restored responsiveness to INF and a transiently lower HIV burden after stopping cART. These data emphasize (i) the value of cART-mediated HIV suppression and immune reconstitution in creating a window of opportunity for exploring novel immunotherapies, (ii) the potential of IFNs for constraining HIV, and (iii) the value of humanized mice for exploring novel immunotherapies.

Antiviral Activity of HIV gp120-Targeting Bispecific T Cell Engager Antibody Constructs.

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.IMPORTANCE 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 in vitro and ex vivo 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 ex vivo 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.

Monitoring HIV DNA and cellular activation markers in HIV-infected humanized mice under cART.

BACKGROUND: The major obstacle to cure of HIV type-1 infection is the presence of the HIV reservoir, hidden from the immune system and insensitive to combined antiretroviral therapy (cART). Eradication approaches have been hindered by the difficulty for accurately monitoring its size in vivo, especially in the lymphoid organs. Humanized mouse models are a valuable tool for systematically assess the efficacy of therapeutic interventions in reducing the HIV reservoir. Nonetheless, persistence of the HIV reservoir over time, in the presence of cART, has yet to be analyzed in this in vivo model. FINDINGS: We found that the proviral DNA as well as the total DNA were very stable in the spleen and mesenteric lymph node irrespective of the length of cART. Notably, the amount of proviral DNA was very similar in the spleen and lymph node. Furthermore, we observed a correlation between the percentage of splenic human CD4+ T-cells with total HIV DNA, between the number of human CD38 + CD8+ T-cells in the spleen with the amount of integrated HIV DNA, and eventually between the hCD4/hCD8 ratio in the spleen with integrated as well as total HIV DNA implying that the CD8+ T cells influence the size of the HIV reservoir. CONCLUSIONS: Here, we demonstrated the stability of this reservoir in humanized mice irrespective of the length of cART, confirming the relevancy of this model for HIV latency eradication investigations. Notably, we also found correlates between the frequency of CD4+ T-cells, their activation status and viral parameters, which were analogous to the ones in HIV-infected patients. Thus, hu-mice represent a very valuable HIV latency model.

Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells.

BACKGROUND: Humanized 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. RESULTS: Human 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. CONCLUSIONS: Overall, 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.

Interferon α-Enhanced Clearance of Group A Streptococcus Despite Neutropenia.

BACKGROUND: Neutrophils and monocytes are crucial for controlling bacterial infections. More-frequent bacterial infections are accordingly encountered in neutropenic patients undergoing chemotherapy. This is not the case for pegylated interferon α (IFN-α)-induced neutropenia. We hypothesized that IFN-α induces a compensatory innate antibacterial state that prevents bacterial infections despite the neutropenia. METHODS: To investigate whether patients with hepatitis C virus infection treated with IFN-α killed group A Streptococcus (GAS) better than before initiating therapy, whole blood was used to perform ex vivo GAS killing assays before, during, and after IFN-α therapy. RESULTS: We found that IFN-α therapy enhanced GAS killing in whole blood ex vivo despite the decreased neutrophil and monocyte numbers during IFN-α therapy. IFN-α also boosted neutrophil- and monocyte-mediated GAS killing in vitro. Underlying mechanisms included increased production of the antibacterial properdin, a regulator of the complement activation, as well as reactive oxygen species. CONCLUSIONS: These findings help to explain the rather discrepant facts of neutropenia but preserved antibacterial immune defenses in patients treated with IFN-α.

Triggering TLR2, -3, -4, -5, and -8 reinforces the restrictive nature of M1- and M2-polarized macrophages to HIV.

UNLABELLED: 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.

TLR8 activates HIV from latently infected cells of myeloid-monocytic origin directly via the MAPK pathway and from latently infected CD4+ T cells indirectly via TNF-α.

We previously showed that the TLR7/8 agonist, R-848, activated HIV from cells of myeloid-monocytic origin. In this work, we show that this effect was solely due to triggering TLR8 and that NF-κB was involved in the TLR8-mediated activation of HIV from latently infected cells of myeloid-monocytic origin. Inhibition of Erk1/2 or p38α resulted in attenuation of TLR8-mediated activation of NF-κB. Western blots confirmed that TLR8 triggering activated Erk1/2 and p38α but, surprisingly, not JNK. Although the Erk1/2 inhibitors resulted in a less attenuated TLR8-mediated NF-κB response than did p38α inhibitors, they had a more pronounced effect on blocking TLR8-mediated HIV replication, indicating that other transcription factors controlled by Erk1/2 are involved in TLR8-mediated HIV activation from latently infected cells. TNF-α, which was secreted subsequent to TLR8 triggering, contributed to the activation of HIV from the latently infected cells in an autocrine manner, revealing a bimodal mechanism by which the effect of TLR8 triggering can be sustained. We also found that TNF-α secreted by myeloid dendritic cells acted in a paracrine manner in the activation of HIV from neighboring latently infected CD4(+) T cells, which do not express TLR8. Notably, monocytes from highly active antiretroviral therapy-treated HIV(+) patients with suppressed HIV RNA showed a robust TNF-α secretion in response to TLR8 agonists, pointing to a functional TLR8 signaling axis in HIV infection. Thus, triggering TLR8 represents a very promising strategy for attacking the silent HIV from its reservoir in HIV(+) patients treated successfully with highly active antiretroviral therapy.

HIV interferes with SOCS-1 and -3 expression levels driving immune activation.

HIV infection is characterized by sustained immune activation, which is reflected by activated T cells and, in particular, by increased levels of phosphorylated STAT proteins. Here, we hypothesized that T-cell activation in HIV infection is partially due to the inability of SOCS-1 and SOCS-3 to control the JAK/STAT pathway. We found higher levels of SOCS-1/3 mRNA levels in CD4(+) T cells of HIV-infected patients than in healthy controls. However, SOCS protein levels were lower, explaining the lack of attenuation of the JAK/STAT pathway. Infection of CD4(+) T cells alone did not activate STATs, while ex vivo infection of PBMC did, indicating that non-T cells critical for shaping the immune response, e.g. DC were responsible for the STAT-1 activation. Supernatants from ex vivo-infected PBMC transferred to CD4(+) T cells induced JAK/STAT activation, pointing to a central role of soluble factors. Notably, over-expression of SOCS-1/3 in CD4(+) T cells prevented JAK/STAT activation. Thus, HIV infection interferes with SOCS-1/3 expression driving immune activation. Sustained immune activation disrupts the lymphoid system and favors HIV replication since HIV preferentially infects activated cells. We speculate that regulating SOCS may be a potential way to counteract immune activation in HIV disease.

Inadequate clearance of translocated bacterial products in HIV-infected humanized mice.

Bacterial translocation from the gut and subsequent immune activation are hallmarks of HIV infection and are thought to determine disease progression. Intestinal barrier integrity is impaired early in acute retroviral infection, but levels of plasma lipopolysaccharide (LPS), a marker of bacterial translocation, increase only later. We examined humanized mice infected with HIV to determine if disruption of the intestinal barrier alone is responsible for elevated levels of LPS and if bacterial translocation increases immune activation. Treating uninfected mice with dextran sodium sulfate (DSS) induced bacterial translocation, but did not result in elevated plasma LPS levels. DSS-induced translocation provoked LPS elevation only when phagocytic cells were depleted with clodronate liposomes (clodrolip). Macrophages of DSS-treated, HIV-negative mice phagocytosed more LPS ex vivo than those of control mice. In HIV-infected mice, however, LPS phagocytosis was insufficient to clear the translocated LPS. These conditions allowed higher levels of plasma LPS and CD8+ cell activation, which were associated with lower CD4+/CD8+ cell ratios and higher viral loads. LPS levels reflect both intestinal barrier and LPS clearance. Macrophages are essential in controlling systemic bacterial translocation, and this function might be hindered in chronic HIV infection.

Triggering TLR7 in mice induces immune activation and lymphoid system disruption, resembling HIV-mediated pathology.

Chronic immune activation is a major cause for progressive immunodeficiency in human immunodeficiency virus type-1 (HIV) infection. The underlying trigger, however, remains largely unknown. HIV single-stranded RNA is a potent immune activator by triggering Toll-like receptor (TLR) 7/8. Thus, we hypothesized that sustained TLR7 triggering induces chronic immune activation and thereby contributes to progressive immunodeficiency. We used the synthetic compound R848 or a mixture of uridine-rich HIV single-stranded (ss) RNA oligonucleotides--both are potent TLR7/8 agonists--to explore the effects of sustained TLR7 triggering on the murine lymphoid system. Sustained TLR7 triggering induced an immunopathology reminiscent of progressive lymphoid destruction in HIV disease; we observed lymphopenia, elevated proinflammatory cytokines, splenomegaly, contracted lymphoid subsets, and lymphoid microarchitecture alteration with reduced marginal zone B-lymphocytes. Upon exposure to inactivated vesiculo-stomatitis virus, antibody production was abolished, although splenic lymphocytes were activated and total IgG was elevated. Our data imply that HIV itself may directly contribute to immune activation and dysfunction by stimulating TLR7. Thus, manipulation of TLR7 signaling may be a potential strategy to reduce chronic hyper-immune activation and, thereby, disease progression in HIV infection.

Polymorphisms of SOCS-1 Are Associated With a Rapid HIV Progression Rate.

OBJECTIVES: Immune activation, among others driven by interferon (IFN)-α and IFN-γ activation, is a main feature of progressive HIV infection. Suppressor of cytokine signaling (SOCS) 1 and 3 are negative feedback regulators of the IFN-α and IFN-γ axis. Here, we analyzed the role of 9 single-nucleotide polymorphisms (SNPs) within SOCS-1 and SOCS-3 genes for their association with an HIV progression rate in a cohort of 318 rapid vs 376 slow progressors from the Swiss HIV Cohort Study. DESIGN AND METHODS: We analyzed 9 SNPs, which we have identified in Swiss blood donors, in a cohort of HIV-infected patients (n = 1144), which have been categorized according to the decline in CD4 T-cell counts. In all the conducted analyses, we focused on the comparison between rapid and slow progressors with regard to SNPs in SOCS-1 and SOCS-3 and with regard to haplotypes using multivariate logistic regression models. RESULTS: Three SOCS-1 SNPs (rs193779, rs33989964, and rs4780355) are associated with a risk reduction for rapid progression. Two of these SNPs, rs33989964 and rs4780355, are in strong linkage disequilibrium, forming a frequent haplotype. Homozygous carriers of this haplotype are also associated with a risk reduction for rapid progression. By contrast, the minor TT genotype of rs33977706 is associated with twice the risk for rapid progression. No associations have been observed for the 4 SOCS-3 SNPs or the major SOCS-3 haplotypes. CONCLUSIONS: Our data suggest that SNPs in SOCS-1 are associated with HIV disease progression and speak in favor that immune activation is causal for the progressive immunodeficiency.

EBV renders B cells susceptible to HIV-1 in humanized mice.

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.

RAG2-/- gamma(c)-/- mice transplanted with CD34+ cells from human cord blood show low levels of intestinal engraftment and are resistant to rectal transmission of human immunodeficiency virus.

Rectal transmission is one of the main routes of infection by human immunodeficiency virus type 1 (HIV-1). To efficiently study transmission mechanisms and prevention strategies, a small animal model permissive for rectal transmission of HIV is mandatory. We tested the susceptibility of RAG2(-/-)gamma(c)(-/-) mice transplanted with human cord blood hematopoietic stem cells to rectal infection with HIV. We rectally exposed these humanized mice to cell-free and cell-associated HIV. All mice remained HIV negative as assessed by plasma viral load. The same mice infected intraperitoneally showed high levels of HIV replication. In the gut-associated lymphatic tissue, we found disproportionately smaller numbers of human cells than in other lymphoid organs. This finding may explain the observed resistance to rectal transmission of HIV. To increase the numbers of local HIV target cells and the likelihood of HIV transmission, we treated mice with different proinflammatory stimuli: local application of interleukin-1beta, addition of seminal plasma to the inoculum, or induction of colitis with dextran sodium sulfate. These procedures attracted some human leukocytes, but the transmission rate was still very low. The humanized mice showed low levels of human engraftment in the intestinal tract and seem to be resistant to rectal transmission of HIV, and thus they are an unsuitable model for this application.

Dose-Dependent Differences in HIV Inhibition by Different Interferon Alpha Subtypes While Having Overall Similar Biologic Effects.

Type I interferons (IFNs) are key players in the antiviral immune response. Interferon alpha (IFN-α) belongs to this class of IFNs and comprises 12 subtypes that differ from each other in their binding affinities for a common receptor and, thus, in their signaling potencies. Recent data suggest that IFN-α6 and -α14 are the most potent IFN-α subtypes in restricting HIV replication when applied exogenously. However, in the context of antiviral therapy, IFNs are administered at high doses, which may compensate for differences in potency seen between IFN-α subtypes. In this study, we reexamined whether IFN-α subtypes induce different biological activities, with a focus on how IFN-α treatment dose affects cellular responses to HIV in primary CD4+ T cells, peripheral blood mononuclear cells (PBMCs), and macrophages. We found that the subtypes' antiviral activities were dose dependent, with >90% inhibition of HIV replication at a high dose of all IFN-αs except the weak IFN-α/ß receptor (IFNAR) binder, IFN-α1. The quality of the responses engendered by IFN-α1, -α2, -α6, and -α14 was highly comparable, with essentially the same set of genes induced by all four subtypes. Hierarchal cluster analysis revealed that the individual donors were stronger determinants for the IFN-stimulated-gene (ISG) responses than the specific IFN-α subtype used for stimulation. Notably, IFN-α2-derived mutants with substantially reduced IFNAR2 binding still inhibited HIV replication efficiently, whereas mutants with increased IFNAR1 binding potentiated antiviral activity. Overall, our results support the idea that IFN-α subtypes do not induce different biological responses, given that each subtype is exogenously applied at bioequivalent doses.IMPORTANCE Elucidating the functional role of the IFN-α subtypes is of particular importance for the development of efficacious therapies using exogenous IFN-α. Specifically, this will help define whether IFN therapy should be based on the use of pathogen-dependent IFN subtypes or, rather, IFN mutants with optimized IFNAR binding properties.

Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs.

Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.

Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction.

Gene knockdown using micro RNA (miRNA)-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp), positioning of the targeting strand on the 5' side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC); incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

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/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.

B cells from HIV-infected patients with primary central nervous system lymphoma display an activated phenotype and have a blunted TNF-α response to TLR9 triggering.

Each cell in HIV-associated primary central nervous system lymphoma (PCNSL) harbors latent EBV. Notably, the triggering of TLR9, a key event in HIV pathogenesis, also promotes EBV latency and transformation. We hypothesized that because only a minority of HIV-infected patients develops PCNSL, their B cells exhibit aberrant signaling responses to TLR9 triggering. We found higher levels of IL-6, CD80, and CD86 expression at baseline in B cells of those patients than in B cells of matched controls, whereas TNF-a expression was lower. Notably, on TLR9 triggering with CpG 2006, CD80 and TNF-α were up-regulated to a lesser extent in B cells of the former than in those of matched controls. The reduced up-regulation of CD80 might be explained by its higher baseline expression resulting in a more blunted response rather than a specific deficit of the signaling response to TLR9 triggering. However, this cannot explain the blunted TNF-α response, which warrants further investigation. Finally, since increased IL-6 expression is linked to EBV-associated Hodgkin's lymphoma, the enhanced baseline expression of IL-6 might be important in the pathogenesis of PCNSL in HIV-infected patients.

Anti-HIV activity mediated by natural killer and CD8+ cells after toll-like receptor 7/8 triggering.

We previously found that triggering TLR7/8 either by single stranded HIV RNA or synthetic compounds induced changes in the lymphoid microenvironment unfavorable to HIV. In this study, we used selective TLR7 and 8 agonists to dissect their contribution to the anti-HIV effects. While triggering TLR7 inhibited efficiently HIV replication in lymphoid suspension cells from tonsillar origin, its effect was inconsistent in peripheral blood mononuclear cells (PBMC). In contrast, triggering TLR8 showed a very prominent and overall very consistent effect in PBMC and tonsillar lymphoid suspension cells. Depletion of dendritic cells (DC), Natural killer cells (NK) and CD8+ T-cells from PBMC resulted in the reversal of TLR8 induced anti-HIV effects. Especially noteworthy, depletion of either NK or CD8+ T-cells alone was only partially effective. We interpret these findings that DC are the initiator of complex changes in the microenvironment that culminates in the anti-HIV active NK and CD8+ effector cells. The near lack of NK and the low number of CD8+ T-cells in tonsillar lymphoid suspension cells may explain the lower TLR8 agonist's anti-HIV effects in that tissue. However, additional cell-type specific differences must exist since the TLR7 agonists had a very strong inhibitory effect in tonsillar lymphoid suspension cells. Separation of effector from the CD4+ target cells did not abolish the anti-HIV effects pointing to the critical role of soluble factors. Triggering TLR7 or 8 were accompanied by major changes in the cytokine milieu; however, it appeared that not a single soluble factor could be assigned for the potent effects. These results delineate the complex effects of triggering TLR7/8 for an efficient antiviral defense. While the ultimate mechanism(s) remains unknown, the potent effects described may have therapeutic value for treating chronic viral diseases. Notably, HIV replication is blocked by TLR triggering before HIV integrates into the host chromosome which would prevent the establishment or maintenance of the latent reservoir.