Annette Audigé

TLR7/8 Triggering Exerts Opposing Effects in Acute versus Latent HIV Infection

TLRs trigger innate immunity by recognizing conserved motifs of microorganisms. Recently, ssRNAs from HIV and influenza virus were shown to trigger TLR7 and 8. Thus, we hypothesized that HIV ssRNA, by triggering TLR7/8, affects HIV pathogenesis. Indeed, HIV ssRNA rendered human lymphoid tissue of tonsillar origin or PBMC barely permissive to HIV replication. The synthetic compound R-848, which also triggers TLR7/8, showed similar anti-HIV activity. Loss of R-848’s activity in lymphoid tissue depleted of B cells suggested a role for B cells in innate immunity. TLR7/8 triggering appears to exert antiviral effects through soluble factors: conditioned medium reduced HIV replication in indicator cells. Although a number of cytokines and chemokines were increased upon adding R-848 to lymphoid tissue, blocking those cytokines/chemokines (i.e., IFN-α receptor, IFN-γ, MIP-1α, -1β, RANTES, and stromal cell-derived factor-1) did not result in the reversal of R-848’s anti-HIV activity. Thus, the nature of this soluble factor(s) remains unknown. Unlike lymphoid tissue acutely infected with HIV, triggering latently infected promonocytic cells induced the release of HIV virions. The anti-HIV effects of triggering TLR7/8 may inhibit rapid killing, while pro-HIV effects may guarantee a certain replication level. Compounds triggering TLR7/8 may be attractive drug candidates to purge latent HIV while preventing new infections.

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.

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

ABSTRACT Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4+ T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4+ T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4+ T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.

Anti-HIV State but Not Apoptosis Depends on IFN Signature in CD4+ T Cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4+ T cell target population, we examined gene expression in CD4+ T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-α-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-αR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-α. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4+ T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4+ T cells, which induces an anti-HIV state but not apoptosis.

Modeling HIV infection and therapies in humanized mice

The human immunodeficiency virus (HIV) type-1 is a human-specific virus. The lack of a widely available small-animal model has seriously hampered HIV research. In 2004, a new humanised mouse model was reported. It was based on the intrahepatic injection of human CD34+ cord blood cells into newborn, highly immunodeficient mice. These mice develop a lymphoid system of human origin and are highly susceptible to HIV infection and showed disseminated infection, persistent viraemia and characteristic helper CD4+ T-cell loss. Here, we will briefly review the various existing humanised mouse models and highlight their value to the study of HIV infection.

HIV-1 Does Not Provoke Alteration of Cytokine Gene Expression in Lymphoid Tissue after Acute Infection Ex Vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1β, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1β, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1β, -2, -4, -6, -8, -10, -12, -15, IFN-γ, TNF-α, TGF-β, and β-chemokines in tissue blocks or HLAC. The increased expression of IL-1β, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.

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.

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.

Uncoupled Anti-HIV and Immune-Enhancing Effects when Combining IFN-α and IL-7

Cytokine-based therapies have been examined for purging viral reservoirs and immunomodulation in HIV infection. However, single cytokines did not result in either HIV eradication or an efficient HIV-specific immune response. We hypothesize that cytokines with distinct biologic effects need to be combined for immunotherapy of HIV infection. In this study, we investigated the anti-HIV activity and immune-enhancing effects of the combination of IFN-α and IL-7. In human lymphocyte aggregate cultures infected ex vivo with the X4 HIV strain NL4-3, IFN-α/IL-7 potently inhibited HIV replication and preserved CD4+ T cells, probably by up-regulating Bcl-2. IFN-α/IL-7 also strongly inhibited R5 HIV replication. Furthermore, in allogeneic MLRs, IFN-α/IL-7 increased T cell proliferation and IFN-γ production. IFN-α alone also had strong anti-HIV activity, but neither preserved CD4+ T cells nor increased T cell responses in MLRs. IL-7 alone maintained T cells and enhanced T cell activation in MLRs, but only moderately inhibited or increased HIV replication. Thus, coadministration of IFN-α/IL-7 combines the potent anti-HIV activity of IFN-α with the beneficial effects of IL-7 on T cell survival and function. We speculate that IFN-α will block viral replication, activate APCs, and up-regulate MHC molecules, thus allowing IL-7 to display its effects for generating an efficient immune response. In this scenario, the known reactivation of latent HIV by IL-7 may be advantageous.

Evaluation of the Immunomodulatory and Antiviral Effects of the Cytokine Combination IFN-α and IL-7 in the Lymphocytic Choriomeningitis Virus and Friend Retrovirus Mouse Infection Models

Existing therapies for chronic viral infections are still suboptimal or have considerable side effects, so new therapeutic strategies need to be developed. One option is to boost the hosts immune response with cytokines. We have recently shown in an acute ex vivo HIV infection model that co-administration of interferon (IFN)-α and interleukin (IL)-7 allows us to combine the potent anti-HIV activity of IFN-α with the beneficial effects of IL-7 on T-cell survival and function. Here we evaluated the effect of combining IFN-α and IL-7 on viral replication in vivo in the chronic lymphocytic choriomeningitis virus (LCMV) and acute Friend retrovirus (FV) infection models. In the chronic LCMV model, cytokine treatment was started during the early replication phase (i.e., on day 7 post-infection [pi]). Under the experimental conditions used, exogenous IFN-α inhibited FV replication, but had no effect on viral replication in the LCMV model. There was no therapeutic benefit of IL-7 either alone or in combination with IFN-α in either of the two infection models. In the LCMV model, dose-dependent effects of the cytokine combination on T-cell phenotype/function were observed. It is possible that these effects would translate into antiviral activity in re-challenged mice. It is also possible that another type of IFN-α/β or induction of endogenous IFN-α/β alone or in combination with IL-7 would have antiviral activity in the LCMV model. Furthermore, we cannot exclude that some effect on viral titers would have been seen at later time points not investigated here (i.e., beyond day 34 pi). Finally, IFN-α/IL-7 may inhibit the replication of other viruses. Thus it might be worth testing these cytokines in other in vivo models of chronic viral infections.