Poly I:C and STING agonist-primed DC increase lymphoid tissue polyfunctional HIV-1-specific CD8+ T cells and limit CD4+ T-cell loss in BLT mice.

Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.

Intra- and extra-cellular environments contribute to the fate of HIV-1 infection.

HIV-1 entry into host cells leads to one of the following three alternative fates: (1) HIV-1 elimination by restriction factors, (2) establishment of HIV-1 latency, or (3) active viral replication in target cells. Here, we report the development of an improved system for monitoring HIV-1 fate at single-cell and population levels and show the diverse applications of this system to study specific aspects of HIV-1 fate in different cell types and under different environments. An analysis of the transcriptome of infected, primary CD4+ T cells that support alternative fates of HIV-1 identifies differential gene expression signatures in these cells. Small molecules are able to selectively target cells that support viral replication with no significant effect on viral latency. In addition, HIV-1 fate varies in different tissues following infection of humanized mice in vivo. Altogether, these studies indicate that intra- and extra-cellular environments contribute to the fate of HIV-1 infection.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

HIV-1 and SIV Infection Are Associated with Early Loss of Lung Interstitial CD4+ T Cells and Dissemination of Pulmonary Tuberculosis.

Lung interstitial CD4+ T cells are critical for protection against pulmonary infections, but the fate of this population during HIV-1 infection is not well described. We studied CD4+ T cells in the setting of HIV-1 infection in human lung tissue, humanized mice, and a Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV) nonhuman primate co-infection model. Infection with a CCR5-tropic strain of HIV-1 or SIV results in severe and rapid loss of lung interstitial CD4+ T cells but not blood or lung alveolar CD4+ T cells. This is accompanied by high HIV-1 production in these cells in vitro and in vivo. Importantly, during early SIV infection, loss of lung interstitial CD4+ T cells is associated with increased dissemination of pulmonary Mtb infection. We show that lung interstitial CD4+ T cells serve as an efficient target for HIV-1 and SIV infection that leads to their early depletion and an increased risk of disseminated tuberculosis.

An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.

HIV-1 Balances the Fitness Costs and Benefits of Disrupting the Host Cell Actin Cytoskeleton Early after Mucosal Transmission.

HIV-1 primarily infects T lymphocytes and uses these motile cells as migratory vehicles for effective dissemination in the host. Paradoxically, the virus at the same time disrupts multiple cellular processes underlying lymphocyte motility, seemingly counterproductive to rapid systemic infection. Here we show by intravital microscopy in humanized mice that perturbation of the actin cytoskeleton via the lentiviral protein Nef, and not changes to chemokine receptor expression or function, is the dominant cause of dysregulated infected T cell motility in lymphoid tissue by preventing stable cellular polarization required for fast migration. Accordingly, disrupting the Nef hydrophobic patch that facilitates actin cytoskeletal perturbation initially accelerates systemic viral dissemination after female genital transmission. However, the same feature of Nef was subsequently critical for viral persistence in immune-competent hosts. Therefore, a highly conserved activity of lentiviral Nef proteins has dual effects and imposes both fitness costs and benefits on the virus at different stages of infection.

Humanized mouse models of latent HIV infection.

Antiretroviral therapy can efficiently control HIV viral replication, resulting in low viral loads and sustained CD4+ T cell counts in HIV-infected persons. However, fast viral rebound occurs in most infected persons when therapy is interrupted. The principal component of persistent infection is a latent but replication-competent HIV reservoir. The long half-life of this reservoir is a major barrier to cure, and its elimination is the target of important research efforts. Animal models that can recapitulate this aspect of human infection are needed to examine the HIV reservoir in tissues in vivo, and to test eradication strategies. In this review, we will summarize recent studies using humanized mouse models to examine different aspects of the viral reservoir.

Chemoattractant-mediated leukocyte trafficking enables HIV dissemination from the genital mucosa.

HIV vaginal transmission accounts for the majority of newly acquired heterosexual infections. However, the mechanism by which HIV spreads from the initial site of viral entry at the mucosal surface of the female genital tract to establish a systemic infection of lymphoid and peripheral tissues is not known. Once the virus exits the mucosa it rapidly spreads to all tissues, leading to CD4+ T cell depletion and the establishment of a viral reservoir that cannot be eliminated with current treatments. Understanding the molecular and cellular requirements for viral dissemination from the genital tract is therefore of great importance, as it could reveal new strategies to lengthen the window of opportunity to target the virus at its entry site in the mucosa where it is the most vulnerable and thus prevent systemic infection. Using HIV vaginal infection of humanized mice as a model of heterosexual transmission, we demonstrate that blocking the ability of leukocytes to respond to chemoattractants prevented HIV from leaving the female genital tract. Furthermore, blocking lymphocyte egress from lymph nodes prevented viremia and infection of the gut. Leukocyte trafficking therefore plays a major role in viral dissemination, and targeting the chemoattractant molecules involved can prevent the establishment of a systemic infection.

Protection of Humanized Mice From Repeated Intravaginal HIV Challenge by Passive Immunization: A Model for Studying the Efficacy of Neutralizing Antibodies In Vivo.

Humanized mice reconstituted with a human immune system can be mucosally infected with human immunodeficiency virus (HIV), opening up the possibility of studying HIV transmission in a small-animal model. Here we report that passive immunization with the broadly neutralizing antibody b12 protected humanized mice against repetitive intravaginal infection in a dose-dependent manner. In addition, treatment with the antibody PGT126, which is more potent in vitro, was more efficacious in vivo and provided sterilizing protection. Our results demonstrate that humanized mice can be used as a small-animal model to study the efficacy and mechanism of broadly neutralizing antibody protection against HIV acquisition.

IL-21 induces antiviral microRNA-29 in CD4 T cells to limit HIV-1 infection.

Initial events after exposure determine HIV-1 disease progression, underscoring a critical need to understand host mechanisms that interfere with initial viral replication. Although associated with chronic HIV-1 control, it is not known whether interleukin-21 (IL-21) contributes to early HIV-1 immunity. Here we take advantage of tractable primary human lymphoid organ aggregate cultures to show that IL-21 directly suppresses HIV-1 replication, and identify microRNA-29 (miR-29) as an antiviral factor induced by IL-21 in CD4 T cells. IL-21 promotes transcription of all miR-29 species through STAT3, whose binding to putative regulatory regions within the MIR29 gene is enriched by IL-21 signalling. Notably, exogenous IL-21 limits early HIV-1 infection in humanized mice, and lower viremia in vivo is associated with higher miR-29 expression. Together, these findings reveal a novel antiviral IL-21-miR-29 axis that promotes CD4 T-cell-intrinsic resistance to HIV-1 infection, and suggest a role for IL-21 in initial HIV-1 control in vivo.

Chemokine-mediated immune responses in the female genital tract mucosa.

The genital tract mucosa is the site where sexually transmitted infections gain entry to the host. The immune response at this site is thus critical to provide innate protection against pathogens that are seen for the very first time as well as provide long-term pathogen-specific immunity, which would be required for an effective vaccine against sexually transmitted infection. A finely regulated immune response is therefore required to provide an effective barrier against pathogens without compromising the capacity of the genital tract to allow for successful conception and fetal development. We review recent developments in our understanding of the immune response in the female genital tract to infectious pathogens, using herpes simplex virus-2, human immunodeficiency virus-1 and Chlamydia trachomatis as examples, with a particular focus on the role of chemokines in orchestrating immune cell migration necessary to achieve effective innate and adaptive immune responses in the female genital tract.

Electron tomography of HIV-1 infection in gut-associated lymphoid tissue.

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.

BLT humanized mice as model to study HIV vaginal transmission.

The majority of human immunodeficiency virus type 1 (HIV-1) infections occur by sexual exposure, and vaginal transmission accounts for more than half of all newly acquired infections. Studies of vaginal transmission of simian immunodeficiency virus to nonhuman primates (NHPs) have suggested an important role for immune cell trafficking in the establishment of infection as well is in the process of viral dissemination. However, NHP models do not permit the study of HIV transmission and dissemination. The improvement of humanized mouse models with robust human immune cell reconstitution of the female genital tract renders these mice susceptible to intravaginal HIV infection. Thus humanized mouse models of HIV vaginal infection will allow the study of the mechanisms involved in HIV transmission and dissemination in vivo.

Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

Treatment with Evasin-3 reduces atherosclerotic vulnerability for ischemic stroke, but not brain injury in mice.

Neutrophilic inflammation might have a pathophysiological role in both carotid plaque rupture and ischemic stroke injury. Here, we investigated the potential benefits of the CXC chemokine-binding protein Evasin-3, which potently inhibits chemokine bioactivity and related neutrophilic inflammation in two mouse models of carotid atherosclerosis and ischemic stroke, respectively. In the first model, the chronic treatment with Evasin-3 as compared with Vehicle (phosphate-buffered saline (PBS)) was investigated in apolipoprotein E-deficient mice implanted of a 'cast' carotid device. In the second model, acute Evasin-3 treatment (5 minutes after cerebral ischemia onset) was assessed in mice subjected to transient left middle cerebral artery occlusion. Although CXCL1 and CXCL2 were upregulated in both atherosclerotic plaques and infarcted brain, only CXCL1 was detectable in serum. In carotid atherosclerosis, treatment with Evasin-3 was associated with reduction in intraplaque neutrophil and matrix metalloproteinase-9 content and weak increase in collagen as compared with Vehicle. In ischemic stroke, treatment with Evasin-3 was associated with reduction in ischemic brain neutrophil infiltration and protective oxidants. No other effects in clinical and histological outcomes were observed. We concluded that Evasin-3 treatment was associated with reduction in neutrophilic inflammation in both mouse models. However, Evasin-3 administration after cerebral ischemia onset failed to improve poststroke outcomes.

HIV-infected T cells are migratory vehicles for viral dissemination.

After host entry through mucosal surfaces, human immunodeficiency virus-1 (HIV-1) disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during the early stages of transmission and systemically during subsequent dissemination are not known. In vitro studies suggest that the formation of virological synapses during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer. It is unclear, however, whether T-cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial and lymphoid tissues. Here, using multiphoton intravital microscopy, we examine the dynamic behaviour of HIV-infected T cells in the lymph nodes of humanized mice. We find that most productively infected T cells migrate robustly, resulting in their even distribution throughout the lymph node cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope-dependent cell fusion. Both uncoordinated motility of syncytia and adhesion to CD4(+) lymph node cells led to the formation of long membrane tethers, increasing cell lengths to up to ten times that of migrating uninfected T cells. Blocking the egress of migratory T cells from the lymph nodes into efferent lymph vessels, and thus interrupting T-cell recirculation, limited HIV dissemination and strongly reduced plasma viraemia. Thus, we have found that HIV-infected T cells are motile, form syncytia and establish tethering interactions that may facilitate cell-to-cell transmission through virological synapses. Migration of T cells in lymph nodes therefore spreads infection locally, whereas their recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras.

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⁺ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.

Single administration of the CXC chemokine-binding protein Evasin-3 during ischemia prevents myocardial reperfusion injury in mice.

OBJECTIVE: Evasins (chemokine-binding proteins) have been shown to selectively neutralize chemokine bioactivity. We investigated the potential benefits of Evasin-3 on mouse myocardial ischemia/reperfusion injury. METHODS AND RESULTS: In vivo and ex vivo (Langendorff model) left coronary artery ligature was performed in C57Bl/6 mice. Coronary occlusion was maintained for 30 minutes, followed by different times (up to 24 hours) of reperfusion. Five minutes after coronary occlusion, mice received 1 intraperitoneal injection of Evasin-3 or vehicle. Infarct size was assessed histologically and by serum cardiac troponin I ELISA. In vitro neutrophil chemotaxis, immunohistology, oxidative stress quantification, real-time RT-PCR analysis of leukocyte chemoattractants, and Western blots for cardioprotective intracellular pathway activation were performed. Evasin-3 reduced infarct size and cardiac troponin I levels compared with vehicle. This effect was associated with the reduction of neutrophil infiltration and reactive oxygen species production within the infarcted myocardium. Evasin-3 did not reduce infarct size in the absence of circulating neutrophils (Langendorff model). Evasin-3 did not influence the activation of intracellular cardioprotective pathways or the expression of leukocyte chemoattractants during early phases of reperfusion. CONCLUSIONS: Single administration of Evasin-3 during myocardial ischemia significantly reduced infarct size by preventing CXC chemokine-induced neutrophil recruitment and reactive oxygen species production in myocardial ischemia/reperfusion.

Structural basis of chemokine sequestration by a tick chemokine binding protein: the crystal structure of the complex between Evasin-1 and CCL3.

BACKGROUND: Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins. METHODOLOGY/PRINCIPAL FINDINGS: We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1:1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent pi-pi interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine. CONCLUSIONS/SIGNIFICANCE: However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the "address" whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.