Search

With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic-co-glycolic acid) (PLGA) particles containing antigens and toll-like receptor ligands induces vigorous antitumor immune responses in vivo. Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8 T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy. +, Nature Communications, 12 (1), ISSN:2041-1723

Toll-like receptor (TLR) 3 agonists emerged as attractive candidates for vaccination strategies against tumors and pathogens. An important mechanism of action of such agonists is based on the activation of TLR3-expressing dendritic cells (DCs), which display a unique capacity to induce and stimulate T-cell responses. In this context, it has been demonstrated that targeting of TLR3 by double-stranded RNA such as poly(I:C) results in potent activation of DCs. Major disadvantages of poly(I:C) comprise its undefined chemical structure and very poor homogeneity, with subsequent unpredictable pharmacokinetics and high toxicity. In the present study, we evaluated the physicochemical properties and biological activity of the novel TLR3 agonist RGC100. RGC100 has a defined chemical structure, with a defined length (100 bp) and molecular weight (64.9 KDa) and a good solubility. RGC100 is stable in serum and activates myeloid DCs through TLR3 targeting, as evidenced by gene silencing experiments. Activation of mouse and human myeloid CD1c+DCs by RGC100 leads to secretion of several proinflammatory cytokines. In addition, RGC100 improves the ability of CD1c+DCs to stimulate T-cell proliferation. Due to its physicochemical properties and its immunostimulatory properties, RGC100 may represent a promising adjuvant for prophylactic and therapeutic vaccination strategies.

Caliciviridae are human or non-human pathogenic viruses with a high diversity. Some members of the Caliciviridae, i.e. human pathogenic norovirus or rabbit hemorrhagic disease virus (RHDV), are worldwide emerging pathogens. The norovirus is the major cause of viral gastroenteritis worldwide, accounting for about 85% of the outbreaks in Europe between 1995 and 2000. In the United States, 25 million cases of infection are reported each year. Since its emergence in 1984 as an agent of fatal hemorrhagic diseases in rabbits, RHDV has killed millions of rabbits and has been dispersed to all of the inhabitable continents. In view of their successful and apparently increasing emergence, the development of antiviral strategies to control infections due to these viral pathogens has now become an important issue in medicine and veterinary medicine. Antiviral strategies have to be based on an understanding of the epidemiology, transmission, clinical symptoms, viral replication and immunity to infection resulting from infection by these viruses. Here, we provide an overview of the mechanisms underlying calicivirus infection, focusing on the molecular aspects of replication in the host cell. Recent experimental data generated through an international collaboration on structural biology, virology and drug design within the European consortium VIZIER is also presented. Based on this analysis, we propose antiviral strategies that may significantly impact on the epidemiological characteristics of these highly successful viral pathogens.

In immunocompromised patients, the diagnosis of infections with herpesviruses and adenoviruses relies mainly on PCR amplification of viral genomic DNA from clinical samples. In the case of co-infections with two or more viruses, single amplification of viral DNA from clinical samples has proven to be time-consuming and expensive, hampering the efficient diagnosis and therapy of viral co-infections. In this study, a diagnostic DNA-microarray allowing simultaneous detection of herpes simplex virus types 1 and 2 (HSV 1/2), varicella zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 types A and B (HHV-6 A/B), and adenovirus in clinical samples was developed and validated. The assay displays a high analytical sensitivity (10 genome equivalents (GE)/reaction) and specificity, being cost-effective and time-saving. Because the DNA-microarray uses the same analytical conditions as real-time quantitative PCR, it can be used as a screening device for multiple viral infections, followed by selective viral load measurement depending on the clinical context. Those features make the DNA-microarray an attractive device for the management of viral infections in immunocompromised patients.

We report on a stem cell-transplanted patient with B cell chronic lymphatic leukemia who presented with a subacute onset of focal neurological deficits, gait abnormalities, emotional lability and dementia. Progressive multifocal leukoencephalopathy was diagnosed by magnetic resonance imaging (MRI) of the brain and detection of JC virus genome in the cerebrospinal fluid. Cidofovir and the 5HT2A receptor antagonist chlorpromazine were subsequently administered. A follow-up MRI of the brain 2 weeks after initiation of the antiviral therapy displayed progress of the demyelination, and the patient died 3 months after onset of the neurological symptoms. This report highlights the need for the development of novel and potent strategies for treatment of progressive multifocal leukoencephalopathy. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The aim of this study was to investigate the clinical impact of reactivation of human herpes virus-6 (HHV-6) and HHV-7 infections in stem cell transplantation recipients, and to examine a possible increase in virulence of the two roseoloviruses when a reactivation of CMV (HHV-5) simultaneously occurs. For this purpose, quantitative real-time PCR systems were developed to assess the viral load of CMV, HHV-6, or HHV-7 in the plasma of haematopoetic stem cell recipients. One hundred and ninety-eight plasma samples from 37 patients who underwent allogeneic stem cell transplantation were tested for CMV, HHV-6, and HHV-7 by a 5'-exonuclease (TaqMan) quantitative real-time PCR. The CMV load obtained by the real-time PCR assay was compared retrospectively with results generated previously with a commercially available test (COBAS AMPLICOR CMV MONITOR Test, Roche). The results suggest that CMV and HHV-6 may be associated with post-transplantation end-organ disease, while HHV-7 reactivation had no impact on the patients included in this study. No evidence for a potential interaction of the roseoloviruses and CMV infections was found.

Norovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

By screening a transcriptome database for expressed sequence tags that are specifically expressed in mammary gland and breast carcinoma, we identified a new human cytochrome P450 (CYP), termed CYP4Z1. The cDNA was cloned from the breast carcinoma line SK-BR-3 and codes for a protein of 505 amino acids. Moreover, a transcribed pseudogene CYP4Z2P that codes for a truncated CYP protein (340 amino acids) with 96% identity to CYP4Z1 was found in SK-BR-3. CYP4Z1 and CYP4Z2P genes consisting of 12 exons are localized in head-to-head orientation on chromosome 1p33. Tissue-specific expression was investigated using real-time reverse transcription PCR with normalized cDNA from 18 different human tissues. CYP4Z1 mRNA was preferentially detected in breast carcinoma tissue and mammary gland, whereas only marginal expression was found in all other tested tissues. Investigation of cDNA pairs from tumor/normal tissues obtained from 241 patients, including 50 breast carcinomas, confirmed the breast-restricted expression and showed a clear overexpression in 52% of breast cancer samples. The expression profile of CYP4Z2P was similar to that of CYP4Z1 with preference in breast carcinoma and mammary gland but a lower expression level in general. Immunoblot analyses with a specific antiserum for CYP4Z1 clearly demonstrated protein expression in mammary gland and breast carcinoma tissue specimens as well as in CYP4Z1-transduced cell lines. Confocal laser-scanning microscopy of MCF-7 cells transfected with a fluorescent fusion protein CYP4Z1-enhanced green fluorescent protein and a subcellular fractionation showed localization to the endoplasmic reticulum as an integral membrane protein concordant for microsomal CYP enzymes.

The enzyme‐linked immunospot (ELISPOT) assay has become a widely employed method for quantification of antigen‐reactive T lymphocytes. In recent years, various types of antigen‐presenting cells (APCs) have been tested as stimulator cells in ELISPOT protocols to achieve a highly sensitive and rapid assay which is not impaired by a marked nonspecific cytokine release. However, the currently available APCs still have disadvantages, such as significant background reactivities, limited sensitivity, and time‐consuming preparation procedures. Recently, we succeeded in defining a novel subpopulation of circulating dendritic cells (DCs) that can easily be prepared from human blood. These M‐DC8+ DCs proved to be very effective in the induction of antigen‐specific T cell responses. In the present study we provide evidence that M‐DC8+ DCs are particularly well suited as APCs for the detection of antigen‐specific CD8+ T cells after challenge with viral or tumor peptides in ELISPOT assays. In addition, protein‐loaded M‐DC8+ DCs proved to be quite efficient in the presentation of MHC class II‐bound peptides, thus allowing the determination of frequencies of antigen‐reactive CD4+ T cells. The use of M‐DC8+ DCs as stimulator cells can improve the ELISPOT assay by combining high sensitivity, rapidity, and low background reactivity. J. Clin. Lab. Anal. 16:30–36, 2002. © 2002 Wiley‐Liss, Inc.

Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3–7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

International audience; Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA2'OMe-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA2'OMe-RNA74, by ligation of two RNA fragments. Then, we use GpppA2'OMe-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA74 in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.

Imiquimod and resiquimod represent Toll-like receptor (TLR) 7 and 8 agonists, which emerged as attractive candidates for tumor therapy. To elucidate immune cells, which mainly contribute to TLR7/8-mediated antitumoral activity, we investigated the impact of imiquimod and resiquimod on native human 6-sulfo LacNAc (slan) dendritic cells (DCs). We found that both TLR7/8 agonists significantly improve the release of various proinflammatory cytokines by slanDCs and promote their tumor-directed cytotoxic activity. Furthermore, resiquimod efficiently augmented the ability of slanDCs to stimulate T cells and natural killer cells. These results indicate that imiquimod and resiquimod trigger various immunostimulatory properties of slanDCs, which may contribute to their antitumor effects.

Objective There is a broad therapeutic potential for the application of small interfering RNAs (siRNAs). However, one has to ensure that siRNAs act specifically, only targeting the expression of one gene. Off-target effects raised by the sense strand have to be eliminated. Methods and results We examined a particular bidirectional siRNA molecule, able to knockdown intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor receptor-1 (TNFR-1) by the sense or antisense strand, respectively. Transfection of human venous endothelial cells with an unmodified siRNA molecule led to equal silencing of ICAM-1 and TNFR-1. In contrast, modified siRNA was able to knockdown ICAM-1 and TNFR-1 separately, with only the antisense strand. Discussion We found the modified siRNAs to inhibit off-target effects originated by the sense strand. Our approach demonstrates one possibility to modify siRNAs before starting a clinical approach to eliminate off-target effects.

We report on the case of a healthy young boy who developed a fulminant myocarditis due to acute coinfection with erythrovirus (parvovirus B19) and human herpesvirus 6 (HHV-6) in the absence of an antiviral immune response. We suggest that the HHV-6-induced immunosuppression enhanced dissemination of parvovirus B19, which led to fatal myocarditis.

The p53 tumor suppressor gene encodes a 53-kDa nuclear phosphoprotein that is thought to protect cells against the accumulation of genetic alterations (1). The p53 phosphoprotein is involved in cycle arrest, apoptosis, inhibition of tumor growth, and preservation of genetic stability (2). Abnormalities of the p53 gene are reported to be the most common genetic alterations in human cancer (3)(4). Mutated p53 gene encodes for mutant p53 proteins that may serve as targets of the host immune system as tumor-specific antigens (5). However, accumulation of the p53 protein in the cell is considered the main cause of anti-p53 antibody production (6). The presence of anti-p53 antibodies has been demonstrated in sera of patients with various cancers (7)(8)(9), including lung (10), breast (11)(12), liver (13), colorectal (14), and prostate cancer, and blood cell malignancies (15). Anti-p53 antibodies initially were detected by immunoblot and immunoprecipitation with extracts of transformed cells as a source of antigen. In recent years, various ELISAs have been described that use mutant (8) or wild-type (16) p53 as antigen, solid-phase or sandwich methods, and prokaryotically or eukaryotically expressed p53 proteins. Our purpose was to evaluate the potential of the tests to provide correct diagnostic classification (17). We studied three commercial ELISAs for anti-p53 antibodies by use of ROC curve analysis. We selected 72 patients presenting with suspicion of malignancy (unexplained body weight loss and chronic fatigue associated with chronic rectal hemorrhage and/or intermittent obstipation, hemoptysis, and/or pathognomonic aspect of tumoral development on chest radiographs). Ethics approval by the Ethics Commission of the University of Dresden (Germany) according to the Helsinki Declaration of 1975 as revised in 1996 as well as informed consent from the study subjects was obtained. Blood samples from patients with suspicion of lung cancer and/or other …

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 A resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface beta-ribbons together with a "featureless" shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.

Coutard, B. et al., Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules. © 2007 Elsevier B.V. All rights reserved., This work was supported by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework Programme (FP6)

In order to assess the risk of transmission of viral diseases during floods, the viral burden in flooded areas of the city of Dresden (Germany) in August 2002 was investigated. Water samples were collected from 9 sampling sites and tested for the presence of 11 enteric viral pathogens. As a control, water samples from the same sites were analyzed in seasonal intervals over the following year. A total of 36 samples were collected, 92% (33/36) being positive for at least one virus. Adenovirus type 40/41 was the most frequently detected (53%), followed by astrovirus (50%) and enterovirus (50%). In all samples, low levels of bacteriophages were detected with no specificity as to sampling site and season, indicating a moderate river contamination with wastewater. A striking association between water temperature and viral genome detection was observed, as illustrated in August 2002 (mean water temperature of 17.8°, 8 sites positive for 17 viruses), in comparison to November 2002 (mean water temperature of 7.6°, 9 sites positive for 45 viruses). Importantly, hepatitis A viral RNA was not detected in the flooded area. In conclusion, our results indicate no increased risk for transmission of viral diseases through water contact in flooded areas.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at -80 degrees C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

Differentiation of genetically modified CD34(+) hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34(+) cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.