Ines Frank

Blockade of Attachment and Fusion Receptors Inhibits HIV-1 Infection of Human Cervical Tissue

Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell–specific intercellular adhesion molecule–grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3+HLA-DR− and CD3−HLA-DR+ cells, with a significant proportion of the latter also expressing dendritic cell–specific intercellular adhesion molecule–grabbing integrin. Bead depletion studies demonstrated that such HLA-DR+ cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.

HSV-2 Infection of Dendritic Cells Amplifies a Highly Susceptible HIV-1 Cell Target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α4β7 on lymphocytes. α4β7 can be engaged by HIV-1 on the cell-surface and CD4+ T cells expressing high levels of this integrin (α4β7 high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α4β7 high CD4+ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (nu200a=u200a11), but not UV-treated (nu200a=u200a8), HSV-2. We found that CD11c+ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α4β7 expression on CD4+ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α4β7 highCD4+ T cells. These factors may play a role in increasing the susceptibility to HIV-1.

Immunodeficiency virus exploitation of dendritic cells in the early steps of infection

The unique capacity of dendritic cells (DCs) to capture and process pathogens for presentation to the immune system, combined with their capacity to express costimulatory and adhesion molecules as well as cytokines and chemokines, renders them powerful antigen‐presenting cells. However, immunodeficiency viruses hijack DCs to facilitate virus dissemination while subverting effective immune activation. Depending on the activation level of the DC subset, human immunodeficiency virus can use different receptors (CD4, chemokine, and C‐type lectin receptors) to bind to DCs. These aspects likely impact whether a DC is productively infected by or simply carries virus for transmission to more permissive targets. DCs efficiently transmit virus to CD4+ T cells, driving virus growth as well as providing signals to trigger virus expansion in virus‐bearing CD4+ T cells. There is accumulating evidence that viral determinants (nef, tat) selectively modulate immature DC biology, fostering DC–T cell interactions and virus replication without up‐regulating costimulatory molecules for effective immune function. In addition, virus‐loaded, immature DCs activate CD4+ virus‐specific T cells, and mature DCs stimulate CD4+ and CD8+ T cells. Thus, even if immature DCs entrap virus as it crosses the mucosae and initiate a CD4+ T cell response, this is likely insufficient to control infection. Appreciating how virus modulates DC function and what determines whether virus is processed for immune stimulation or transmitted between cells will unveil the exact role of these cells in the onset of infection and advance preventative microbicide and vaccine/therapeutic approaches.

Entry inhibitors SCH-C, RANTES, and T-20 block HIV type 1 replication in multiple cell types.

The small-molecule CCR5 antagonist SCH-C (SCH 351125) was tested for its ability to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells, immature dendritic cells (DCs), and macrophages. Inhibition of infection of PBMCs by virus associated with mature DC in trans was also studied. For comparison, the peptide-based fusion inhibitor T-20 and the CC-chemokine RANTES were also evaluated. Although some cell type-dependent differences in potency were observed, each of the three entry inhibitors was active against the replication of three different CCR5-using primary isolates in each cell type. CCR5-dependent HIV-1 infectivity, whether DC associated or not, is thus vulnerable to inhibitors that block the virus-cell fusion process by different mechanisms. Together, these results suggest that SCH-C and other entry inhibitors should be evaluated for their clinical potential as inhibitors of HIV-1 replication in several settings, including the prevention of maternal-infant transmission and the prevention of sexual transmission by topical application as a microbicide.

A Fusion Inhibitor Prevents Spread of Immunodeficiency Viruses, but Not Activation of Virus-Specific T Cells, by Dendritic Cells

ABSTRACT Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4+ T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4+, not CD8+, T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4+ T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4+ and CD8+ T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4+ T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.

Retinoic Acid Imprints a Mucosal-like Phenotype on Dendritic Cells with an Increased Ability To Fuel HIV-1 Infection

The tissue microenvironment shapes the characteristics and functions of dendritic cells (DCs), which are important players in HIV infection and dissemination. Notably, DCs in the gut have the daunting task of orchestrating the balance between immune response and tolerance. They produce retinoic acid (RA), which imprints a gut-homing phenotype and influences surrounding DCs. To investigate how the gut microenvironment impacts the ability of DCs to drive HIV infection, we conditioned human immature monocyte-derived DCs (moDCs) with RA (RA-DCs), before pulsing them with HIV and mixing them with autologous T cells. RA-DCs showed a semimature, mucosal-like phenotype and released higher amounts of TGF-β1 and CCL2. Using flow cytometry, Western blot, and microscopy, we determined that moDCs express the cell adhesion molecule mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and that RA increases its expression. MAdCAM-1 was also detected on a small population of DCs in rhesus macaque (Macaca mulata) mesenteric lymph node. RA-DCs formed more DC–T cell conjugates and promoted significantly higher HIV replication in DC–T cell mixtures compared with moDCs. This correlated with the increase in MAdCAM-1 expression. Blocking MAdCAM-1 partially inhibited the enhanced HIV replication. In summary, RA influences DC phenotype, increasing their ability to exacerbate HIV infection. We describe a previously unknown mechanism that may contribute to rapid HIV spread in the gut, a major site of HIV replication after mucosal exposure.

A Small Molecule, Which Competes with MAdCAM-1, Activates Integrin α4β7 and Fails to Prevent Mucosal Transmission of SHIV-SF162P3

Mucosal HIV-1 transmission is inefficient. However, certain viral and host characteristics may play a role in facilitating HIV acquisition and systemic expansion. Cells expressing high levels of integrin α4β7 have been implicated in favoring the transmission process and the infusion of an anti-α4β7 mAb (RM-Act-1) prior to, and during a repeated low-dose vaginal challenge (RLDC) regimen with SIVmac251 reduced SIV acquisition and protected the gut-associated lymphoid tissues (GALT) in the macaques that acquired SIV. α4β7 expression is required for lymphocyte trafficking to the gut lamina propria and gut inductive sites. Several therapeutic strategies that target α4β7 have been shown to be effective in treating inflammatory conditions of the intestine, such as inflammatory bowel disease (IBD). To determine if blocking α4β7 with ELN, an orally available anti-α4 small molecule, would inhibit SHIV-SF162P3 acquisition, we tested its ability to block MAdCAM-1 (α4β7 natural ligand) and HIV-gp120 binding in vitro. We studied the pharmacokinetic profile of ELN after oral and vaginal delivery in macaques. Twenty-six macaques were divided into 3 groups: 9 animals were treated with ELN orally, 9 orally and vaginally and 8 were used as controls. All animals were challenged intra-vaginally with SHIV-SF162P3 using the RLDC regimen. We found that ELN did not protect macaques from SHIV acquisition although it reduced the SHIV-induced inflammatory status during the acute phase of infection. Notably, integrins can exist in different activation states and, comparing the effect of ELN and the anti-α4β7 mAb RM-Act-1 that reduced susceptibility to SIV infection, we determined that ELN induces the active conformation of α4β7, while RM-Act-1 inhibits its activation through an allosteric mechanism. These results suggest that inhibition of α4β7 activation may be necessary to reduce susceptibility to SIV/SHIV infection and highlight the complexity of anti-integrins therapeutic approach in HIV as well as in IBD and other autoimmune diseases.

Consequences of dendritic cell (DC)-immunodeficiency virus interactions: Chemically inactivated virus as a model for studying antigen presentation and virus transmission by primate DCs

We have established a model approach to study DC-virus communication, in which classically immature and mature DCs can be compared. The striking observation that macropinocytically poor mature DCs capture and then internalize whole virus particles has substantial implications for both antigen processing and presentation as well as cell to cell transmission of virus from DCs to nearby T cells (as well as other cell types). Studies are ongoing using this system to determine what molecules and mechanisms are involved in virus binding and internalization by immature versus mature DCs. Discerning the consequences of the differential fates of virus in immature versus mature monocyte-derived DCs should provide important information on how virus captured by DCs is processed for immune activation versus virus dissemination. While there are many features shared by monocyte-derived DCs and DCs directly isolated from blood or tissues, there are some important distinctions between these DC subsets and their activation stage. These traits have considerable influence on immune activation by DCs as well as how the individual DC subset handles an immunodeficiency virus. Thus, this approach is ultimately being applied to specific DC subsets, especially those found at the body surfaces where the first DC-virus interactions most frequently occur in vivo. Utilizing this system, we hope to better comprehend the initial events of DC-virus communication to (i) facilitate the development of strategies to block these events and prevent the onset of infection and (ii) identify how to augment the generation of broad anti-viral immunity.

A model of genital herpes simplex virus Type 1 infection in Rhesus Macaques

Although HSV‐2 is the major cause of genital lesions, HSV‐1 accounts for half of new cases in developed countries.

Integrin α4β7 Blockade Preferentially Impacts CCR6+ Lymphocyte Subsets in Blood and Mucosal Tissues of Naive Rhesus Macaques

Infusion of a simianized anti-α4β7 mAb (Rh-α4β7) just before and following SIV infection protected rhesus macaques from developing AIDS and partially from vaginal SIV acquisition. Recently, short-term treatment with Rh-α4β7 in combination with cART was found to lead to prolonged viral suppression after withdrawal of all therapeutic interventions. The humanized form of Rh-α4β7, vedolizumab, is a highly effective treatment for inflammatory bowel disease. To clarify the mechanism of action of Rh-α4β7, naive macaques were infused with Rh-α4β7 and sampled in blood and tissues before and after treatment to monitor several immune cell subsets. In blood, Rh-α4β7 increased the CD4+ and CD8+ T cell counts, but not B cell counts, and preferentially increased CCR6+ subsets while decreasing CD103+ and CD69+ lymphocytes. In mucosal tissues, surprisingly, Rh-α4β7 did not impact integrin α4+ cells, but decreased the frequencies of CCR6+ and CD69+ CD4+ T cells and, in the gut, Rh-α4β7 transiently decreased the frequency of memory and IgA+ B cells. In summary, even in the absence of inflammation, Rh-α4β7 impacted selected immune cell subsets in different tissues. These data provide new insights into the mechanisms by which Rh-α4β7 may mediate its effect in SIV-infected macaques with implications for understanding the effect of treatment with vedolizumab in patients with inflammatory bowel disease.