Inhibition of the binding of HCV serum particles to human hepatocytes by E1E2‐specific D32.10 monoclonal antibody

The aim of this study was to determine the inhibition of binding activity of the monoclonal antibody (mAb) D32.10 which recognizes a highly conserved discontinuous antigenic determinant (E1:297–306, E2:480–494, and E2:613–621) expressed on the surface of serum‐derived HCV particles (HCVsp) of genotypes 1a, 1b, 2a, and 3a. To this end, an in vitro direct cell‐binding assay based on the attachment of radiolabeled HCVsp was developed, and Scatchard plots were used to analyze ligand–receptor binding data. HCV adsorption was also assessed by quantitating cell‐associated viral RNA by a real‐time RT‐PCR method. Saturable concentration‐dependent specific binding of HCVsp to Huh‐7 or HepaRG cells was demonstrated. The Scatchard transformed data showed two‐site interaction for Huh‐7 and proliferative HepaRG cells: the high‐affinity binding sites (K_d1 = 0.1–0.5 µg/ml) and the low‐affinity binding sites (K_d1 = 5–10 µg/ml), and one‐site high‐affinity binding model between E1E2/D32.10‐positive HCVsp and hepatocyte‐like differentiated HepaRG cells. The E1E2‐specific mAb D32.10 inhibited efficiently (>60%) and selectively the binding with an IC₅₀ ≤0.5 µg/ml in all the experimental approaches using serum HCV of genotype either 3 or 1b. This supports the involvement of the E1E2/D32.10 discontinuous antigenic determinant in the interactions between human hepatocytes and HCVsp, and suggests that D32.10‐like antibodies present in sera from patients infected with HCV could play a protective role. J. Med. Virol. 81:1726–1733, 2009. © 2009 Wiley‐Liss, Inc.     

Dominant CD4‐dependent RNA‐dependent RNA polymerase‐specific T‐cell responses in children acutely infected with human enterovirus 71 and healthy adult controls

Human enterovirus 71 (EV71) is one of the major causes of hand, foot and mouth disease (HFMD), which leads to significant mortality in infected children. A prophylactic vaccine is urgently needed. However, little is known about the protective T‐cell immunity in individuals infected with the EV71 virus. In this study, we performed a comprehensive ex vivo interferon‐γ ELISPOT analysis in 31 children infected with EV71 as well as in 40 healthy adult controls of the CD4⁺ and CD8⁺ T‐cell responses to overlapping peptides spanning the VP1 structural protein and RNA‐dependent RNA polymerase (RdRp) non‐structural protein. EV71‐specific CD4 T‐cell responses were detected in most of the acute patients and were mostly CD4‐dependent RdRp‐specific responses. CD8‐dependent VP1 and RdRp‐specific responses were also detected in a small proportion of recently infected children. There was no significant association between the strength of the T‐cell responses and disease severity observed during the acute EV71 infection phase. Interestingly, an RdRp‐specific, but no VP1‐specific, CD4‐dependent T‐cell response was detected in 30% of the adult controls, and no T‐cell responses were detected in healthy children. In addition, 24 individual peptides containing potential T‐cell epitope regions were identified. The data suggest that CD4‐dependent RdRp‐specific T‐cell responses may play an important role in protective immunity, and the epitopes identified in this study should provide valuable information for future therapeutic and prophylactic vaccine design as well as basic research.     

Reversal of chronic to resolved infection by IL‐10 blockade is LCMV strain dependent

Chronic viral infections lead to CD8⁺ T‐cell exhaustion, characterized by impaired cytokine secretion. The immune‐regulatory cytokine IL‐10 promotes chronicity of infection with lymphocytic choriomeningitis virus (LCMV) Clone 13, as absence of IL‐10 or blocking of IL‐10R during early LCMV Clone 13 infection results in viral clearance. Thus, treatment of humans suffering from chronic viral infections with IL‐10 neutralizing or IL‐10R blocking antibodies was proposed to boost virus‐specific T‐cell responses to enhance control or even clear the viral infection. Here we demonstrate that although CD4⁺ and CD8⁺ T cells can produce elevated levels of cytokines in IL‐10^?/? mice early after infection compared with WT mice, IL‐10^?/? mice cannot clear an infection with the quicker replicating LCMV strain Docile, eventually resulting in T‐cell exhaustion. These data suggest that the success of IL‐10 blockade to control chronic viral infections may critically depend on the virulence of the infecting strain.     

NK cells are primed by ANRS MVAHIV‐infected DCs,via a mechanism involving NKG2D and membrane‐bound IL‐15, to control HIV‐1 infection in CD4+ T cells

Natural killer (NK) cells are the major antiviral effector cell population of the innate immune system. It has been demonstrated that NK‐cell activity can be modulated by the interaction with dendritic cells (DCs). The HIV‐1 vaccine candidate Modified Vaccinia Ankara encoding an HIV polypeptide (MVA_HIV), developed by the French National Agency for Research on AIDS (ANRS), has the ability to prime NK cells to control HIV‐1 infection in DCs. However, whether or not MVA_HIV‐primed NK cells are able to better control HIV‐1 infection in CD4⁺ T cells, and the mechanism underlying the specific priming, remain undetermined. In this study, we show that MVA_HIV‐primed NK cells display a greater capacity to control HIV‐1 infection in autologous CD4⁺ T cells. We also highlight the importance of NKG2D engagement on NK cells and DC‐produced IL‐15 to achieve the anti‐HIV‐1 specific priming, as blockade of either NKG2D or IL‐15 during MVA_HIV‐priming lead to a subsequent decreased control of HIV‐1 infection in autologous CD4⁺ T cells. Furthermore, we show that the decreased control of HIV‐1 infection in CD4⁺ T cells might be due, at least in part, to the decreased expression of membrane‐bound IL‐15 (mbIL‐15) on DCs when NKG2D is blocked during MVA_HIV‐priming of NK cells.     

Increased frequency and function of KIR2DL1–3+ NK cells in primary HIV‐1 infection are determined by HLA‐C group haplotypes

The acquisition and maintenance of NK‐cell function is mediated by inhibitory killer‐cell immunoglobulin‐like receptors (KIRs) through their interaction with HLA class I molecules. Recently, HLA‐C expression levels were shown to be correlated with protection against multiple outcomes of HIV‐1 infection; however, the underlying mechanisms are poorly understood. As HLA‐C is the natural ligand for the inhibitory receptors KIR2DL1 and KIR2DL2/3, we sought to determine whether HLA‐C group haplotypes affect NK‐cell responses during primary HIV‐1 infection. The phenotypes and functional capacity of NK cells derived from HIV‐1‐positive and HIV‐1‐negative individuals were assessed (N = 42 and N = 40, respectively). HIV‐1 infection was associated with an increased frequency of KIR2DL1–3⁺ NK cells. Further analysis showed that KIR2DL1⁺ NK cells were selectively increased in individuals homozygous for HLA‐C2, while HLA‐C1‐homozygous individuals displayed increased proportions of KIR2DL2/3⁺ NK cells. KIR2DL1–3⁺ NK cells were furthermore more polyfunctional during primary HIV‐1 infection in individuals also encoding for their cognate HLA‐C group haplotypes, as measured by degranulation and IFN‐γ and TNF‐α production. These results identify a novel relationship between HLA‐C and KIR2DL⁺ NK‐cell subsets and demonstrate that HLA‐C‐mediated licensing modulates NK‐cell responses to primary HIV‐1 infection.     

293 cells over‐expressing human ADI1 and CD81 are permissive for serum‐derived hepatitis C virus infection

Human aci‐reductone dioxygenase 1 (ADI1) is a member of the Cupin superfamily. It binds to and inhibits the activities of membrane‐type 1 matrix metalloproteinase, a protein known to interact with the tight junction protein, claudin‐1. Previously, a variant protein, named submergence‐induced protein‐like factor (Sip‐L), consisting of ADI1 amino acids 64–179, was found to support hepatitis C virus (HCV) infection and replication in 293 cells. In the present study, it was discovered that over‐expression of human ADI1 in 293 cells (293‐ADI1 cells) also supported HCV infection and replication. Using serum‐derived HCV as an infectious source, enhanced cell uptake of HCV to a Northern blot detectable level was found in 293 cells over‐expressing both CD81 and ADI1 (293‐ADI1‐CD81 cells). The enhanced cell entry was confirmed by the use of the vesicular stomatitis virus‐based HCV pseudotype particles. However, transfection of HCV replicon RNA by electroporation into naïve 293 and 293‐ADI1 cells revealed no difference in replication efficiency. Using the infectious J6/JFH chimera as an infectious source, the infectivity was compared between 293‐ADI1‐CD81 and Huh‐7.5 cells. More infection foci were formed in the 293‐ADI1‐CD81 cells in the first round of infection. In conclusion, human ADI1 over‐expression in 293 cells enhances cell entry but not replication of HCV. 293‐ADI1‐CD81 cells are permissive for serum‐derived HCV infection. J. Med. Virol. 81:1560–1568, 2009. © 2009 Wiley‐Liss, Inc.     

Respiratory syncytial virus infection augments NOD2 signaling in an IFN‐β‐dependent manner in human primary cells

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, with remarkable variability in disease severity. An exaggerated proinflammatory response and influx of leukocytes is part of the pathogenesis of severe RSV disease. Here, we show an increase in proinflammatory cytokine production by human immune cells after stimulation with RSV and muramyl dipeptide (MDP), which is recognized by nucleotide‐binding oligomerization domain containing 2 (NOD2). PBMCs from Crohn's disease patients homozygous for the 3020insC mutation in the NOD2 gene did not show a synergistic response to stimulation with RSV and MDP, suggesting that NOD2 is essential for the observed synergy. Further experiments aimed at identifying the viral ligand indicated that viral RNA plays an essential role in the recognition of RSV. Stimulation with RSV or Poly(I:C) induced IFN‐β expression, which resulted in an increased expression of the viral receptors TLR3 and RIG‐I, as well as an increased NOD2 expression. Our data indicate that IFN‐β induction by viral RNA is an essential first step in the increased proinflammatory response to MDP. We hypothesize that the enhanced proinflammatory response to MDP following RSV infection may be an important factor in determining the outcome of the severity of disease.     

Vessel co‐option is common in human lung metastases and mediates resistance to anti‐angiogenic therapy in preclinical lung metastasis models

Anti‐angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti‐angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre‐existing vessels from surrounding tissue (vessel co‐option). As anti‐angiogenic therapies were designed to target only new blood vessel growth, vessel co‐option has been proposed as a mechanism that could drive resistance to anti‐angiogenic therapy. However, vessel co‐option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti‐angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co‐option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co‐opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co‐opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co‐option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co‐option mediates resistance to the anti‐angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co‐option in lung metastases occurs through at least three distinct mechanisms, that vessel co‐option occurs frequently in lung metastases, and that vessel co‐option could mediate resistance to anti‐angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co‐option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross‐reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5‐D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross‐reactivity of clone 5‐D8/1 with normal islet proteins. Western blotting, two‐dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5‐D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B‐type and ATP synthase beta subunit. Immunohistochemistry using the clone 5‐D8/1 revealed a granular cytoplasmic staining pattern in mitochondria‐rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent‐onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5‐D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5‐D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent‐onset type 1 diabetes patients could reflect cross‐reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus‐infected patients after 5 years of highly active anti‐retroviral therapy may be due to increased thymic production of naive Tregs

This study determines levels of regulatory T cells (T_regs), naive T_regs, immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)‐infected patients receiving prolonged highly active anti‐retroviral therapy (HAART) who have known thymic output, and explores if naive T_regs may represent recent thymic emigrant T_regs. HIV‐infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T_regs (CD3⁺CD4⁺CD25⁺CD127^low), naive T_regs (CD3⁺CD4⁺CD25⁺CD45RA⁺) and activation markers (CD38⁺human leucocyte antigen D‐related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T_REC) content in CD4⁺ cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T_regs were significantly higher in HIV‐infected patients compared with controls, both after 1 and 5 years of HAART (P < 0·001), despite fully suppressed HIV‐RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T_regs were elevated significantly in HIV‐infected patients (P < 0·001) and were associated with thymic output measured as the T_REC frequency in CD4⁺ cells (P = 0·038). In summary, T_reg levels in HIV‐infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T_regs may contribute to higher T_reg levels in HIV‐infection.     

Partial and transient modulation of the CD3–T‐cell receptor complex,elicited by low‐dose regimens of monoclonal anti‐CD3, is sufficient to induce disease remission in non‐obese diabetic mice

It has been established that a total of 250 μg of monoclonal anti‐mouse CD3 F(ab′)₂ fragments, administered daily (50 μg per dose), induces remission of diabetes in the non‐obese diabetic (NOD) mouse model of autoimmune diabetes by preventing β cells from undergoing further autoimmune attack. We evaluated lower‐dose regimens of monoclonal anti‐CD3 F(ab′)₂ in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3–T‐cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4⁺, CD8⁺ and CD4⁺ FoxP3⁺ T cells. Four doses of 2 μg (total dose 8 μg) induced 53% remission of diabetes, similarly to the 250 μg dose regimen, whereas four doses of 1 μg induced only 16% remission. While the 250 μg dose regimen produced nearly complete and sustained modulation of the CD3 –TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4⁺ and CD8⁺ T cells decreased, whereas the proportions of CD4⁺ FoxP3⁺ T cells increased; these effects were transient. Mice with greater residual β‐cell function, estimated using blood glucose and C‐peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti‐CD3 that produced only partial and transient modulation of the CD3–TCR complex induced remission rates comparable to higher doses of monoclonal anti‐CD3. Accordingly, in a clinical setting, lower‐dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti‐CD3, potentially including reductions in cytokine release‐related syndromes and maintenance of pathogen‐specific immunosurveillance during treatment.     

Type 1 diabetes associated HLA‐DQ2 and DQ8 molecules are relatively resistant to HLA‐DM mediated release of invariant chain‐derived CLIP peptides

HLA‐DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4⁺ T cells. Individuals expressing HLA‐DQ2 or DQ8, and DQ2/8 trans‐dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM‐mediated editing of CLIP was further confirmed by HPLC‐MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D‐associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D‐susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2‐CLIP complexes are highly resistant to DM editing, whereas DQ8‐CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM‐binding region. Our findings show that T1D‐susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D‐susceptible DQ molecules to DM editing and preferential presentation of T1D‐associated autoantigenic peptides may contribute to the pathogenesis of T1D.     

Isoniazid‐induced control of Mycobacterium tuberculosis by primary human cells requires interleukin‐1 receptor and tumor necrosis factor

Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL‐1β and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T‐cell‐mediated Mtb killing, nor production of IL‐1β and TNF. However, blockade of both IL‐1R1 and TNF signaling rescued INH‐induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase‐peroxidase KatG and involved a host PI3K‐dependent pathway. Finally, experiments using coinfected (KatG‐mutated and H37Rv strains) cells suggested that active INH does not directly enhance host‐mediated killing of Mtb. Our results thus indicate that Mtb‐stimulated host IL‐1 and TNF have potential roles in TB chemotherapy.     

Prevalence of and risk factors for penile infection by high‐risk human papillomavirus among men infected with HIV

This study aimed to determine the prevalence of and risk factors for high‐risk human papillomavirus (HPV) genital infection and precursor lesions of penile cancer among patients infected with human immunodeficiency virus (HIV). In total, 276 men with a mean age of 34.6 years were included. All participants were subjected to peniscopic examination under magnification, collection of genital exfoliated cells for detecting HPV types using Hybrid Capture, and biopsy surgery of clinically observable lesions and aceto‐white areas for histopathological studies. The prevalence of high‐risk HPV types was 43%. Peniscopicy showed clinically visible lesions or aceto‐white areas in 75/276 participants (27%), of which genital warts were the most common (22/75; 29%). HIV‐positive (HIV⁺) men with CD4⁺ T‐cell counts <200 cells/mm³ showed a higher prevalence of penile lesions. Multivariate logistic regression was applied to identify independent risk factors for high‐risk HPV types. The results showed that high‐risk HPV was associated with lower education level (OR = 1.89, 95% CI: 1.15–3.13), illicit drug use (OR = 1.80, 95% CI: 1.03–3.14), mulatto ethnicity (OR = 2.51, 95% CI: 1.38–4.54), heterosexual orientation (OR = 2.12, 95% CI: 1.30–3.47) symptomatic AIDS (OR = 2.80, 95% CI: 1.65–4.77), AIDS‐associated opportunistic infections (OR = 2.92, 95% CI: 1.78–4.78), on HAART (OR = 2.91, 95% CI: 1.78–4.77), and CD4⁺ T‐cell count <200 cells/mm³ (OR = 3.31, 95% CI: 1.84–5.96). Immunocompromised men were more susceptible to developing penile lesions associated with high‐risk HPV types. J. Med. Virol. 85:413–418, 2013. © 2013 Wiley Periodicals, Inc.