Allergen-specific immunotherapy and evidence: A European regulatory perspective.

Allergen immunotherapy (AIT) has been performed for 112 years. In this article we summarize regulatory standards and challenges based on scientific evidence on AIT. Most crucial and timely aspects concerning AIT are addressed from the regulatory perspective of the authors as employees of a national competent authority in Europe: (1) product specificity; (2) clinical efficacy; (3) treatment for adults and children (needs for extrapolation); (4) allergen exposure chambers; (5) biomarkers; (6) standardization; (7) real-world evidence; (8) independent official batch release (benefit and challenges); (9) harmonization on the EU level. The Paul-Ehrlich-Institut (PEI), the Federal Institute for Vaccines and Biomedicines, in Langen near Frankfurt/Main in Germany, examines and evaluates the benefits and risks of AIT products within the course of clinical development, marketing authorization, and subsequently throughout their entire life cycle to ensure high-quality, safe, and effective AIT products.

Interference with SAMHD1 Restores Late Gene Expression of Modified Vaccinia Virus Ankara in Human Dendritic Cells and Abrogates Type I Interferon Expression.

Attenuated poxviruses like modified vaccinia virus Ankara (MVA) are promising vectors for vaccines against infectious diseases and cancer. However, host innate immune responses interfere with the viral life cycle and also influence the immunogenicity of vaccine vectors. Sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a phosphohydrolase and reduces cellular deoxynucleoside triphosphate (dNTP) concentrations, which impairs poxviral DNA replication in human dendritic cells (DCs). Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) encode an accessory protein called viral protein X (Vpx) that promotes proteasomal degradation of SAMHD1, leading to a rapid increase in cellular dNTP concentrations. To study the function of SAMHD1 during MVA infection of human DCs, the SIV vpx gene was introduced into the MVA genome (resulting in recombinant MVA-vpx). Infection of human DCs with MVA-vpx led to SAMHD1 protein degradation and enabled MVA-vpx to replicate its DNA genome and to express genes controlled by late promoters. Late gene expression by MVA-vpx might improve its vaccine vector properties; however, type I interferon expression was unexpectedly blocked by Vpx-expressing MVA. MVA-vpx can be used as a tool to study poxvirus-host interactions and vector safety.IMPORTANCE SAMHD1 is a phosphohydrolase and reduces cellular dNTP concentrations, which impairs poxviral DNA replication. The simian SIV accessory protein Vpx promotes degradation of SAMHD1, leading to increased cellular dNTP concentrations. Vpx addition enables poxviral DNA replication in human dendritic cells (DCs), as well as the expression of viral late proteins, which is normally blocked. SAMHD1 function during modified vaccinia virus Ankara (MVA) infection of human DCs was studied with recombinant MVA-vpx expressing Vpx. Infection of human DCs with MVA-vpx decreased SAMHD1 protein amounts, enabling MVA DNA replication and expression of late viral genes. Unexpectedly, type I interferon expression was blocked after MVA-vpx infection. MVA-vpx might be a good tool to study SAMHD1 depletion during poxviral infections and to provide insights into poxvirus-host interactions.

Suramin is a potent inhibitor of Chikungunya and Ebola virus cell entry.

BACKGROUND: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions and currently causes large outbreaks in the Caribbean and Latin America. Ebola virus (EBOV) is a member of the filovirus family. It causes the Ebola virus disease (EDV), formerly known as Ebola hemorrhagic fever in humans and has a mortality rate of up to 70 %. The last outbreak in Western Africa was the largest in history and has caused approximately 25,000 cases and 10,000 deaths. For both viral infections no specific treatment or licensed vaccine is currently available. The bis-hexasulfonated naphthylurea, suramin, is used as a treatment for trypanosome-caused African river blindness. As a competitive inhibitor of heparin, suramin has been described to have anti-viral activity. METHODS: We tested the activity of suramin during CHIKV or Ebola virus infection, using CHIKV and Ebola envelope glycoprotein pseudotyped lentiviral vectors and wild-type CHIKV and Ebola virus. RESULTS: Suramin efficiently inhibited CHIKV and Ebola envelope-mediated gene transfer while vesicular stomatitis virus G protein pseudotyped vectors were only marginally affected. In addition, suramin was able to inhibit wild-type CHIKV and Ebola virus replication in vitro. Inhibition occurred at early time points during CHIKV infection. CONCLUSION: Suramin, also known as Germanin or Bayer-205, is a market-authorized drug, however shows significant side effects, which probably prevents its use as a CHIKV drug, but due to the high lethality of Ebola virus infections, suramin might be valuable against Ebola infections.

Identification of entry inhibitors of Ebola virus pseudotyped vectors from a myxobacterial compound library.

Myxobacteria produce secondary metabolites many of which were described to have various biological effects including anti-fungal, anti-bacterial and anti-viral activity. The majority of these metabolites are novel scaffolds with unique modes-of-action and hence might be potential leads for drug discovery. Here, we tested a myxobacterial natural product library for compounds with inhibitory activity against Ebola virus (EBOV). The assay was performed with a surrogate system using Ebola envelope glycoprotein (GP) pseudotyped lentiviral vectors. EBOV specificity was proven by counter-screening with vesicular stomatitis virus G protein pseudotyped vectors. Two compounds were identified that preferentially inhibited EBOV GP mediated cell entry: Chondramides that act on the actin skeleton but might be too toxic and noricumazole A, a potassium channel inhibitor, which might constitute a novel pathway to inhibit Ebola virus cell entry.

Curcumin and Boswellia serrata gum resin extract inhibit chikungunya and vesicular stomatitis virus infections in vitro.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever, and severe arthritis that can persist for years. CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions including Europe and the United States of America. CHIKV has recently caused large outbreaks in Latin America. No treatment or licensed CHIKV vaccine exists. Traditional medicines are known to have anti-viral effects; therefore, we examined whether curcumin or Boswellia serrata gum resin extract have antiviral activity against CHIKV. Both compounds blocked entry of CHIKV Env-pseudotyped lentiviral vectors and inhibited CHIKV infection in vitro. In addition, vesicular stomatitis virus vector particles and viral infections were also inhibited to the same extent, indicating a broad antiviral activity. Although the bioavailability of these compounds is rather poor, they might be used as a lead structure to develop more effective antiviral drugs or might be used topically to prevent CHIKV spread in the skin after mosquito bites.

The green tea catechin, epigallocatechin gallate inhibits chikungunya virus infection.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever and severe arthritis that can persist for years. CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions, including Europe and the United States of America and might cause new, large outbreaks there. No treatment or licensed CHIKV vaccine exists. Epigallocatechin-3-gallate (EGCG), the major component of green tea, has, among other beneficial properties, antiviral activities. Therefore, we examined if EGCG has antiviral activity against CHIKV. EGCG inhibited CHIKV infection in vitro, blocked entry of CHIKV Env-pseudotyped lentiviral vectors and inhibited CHIKV attachment to target cells. Thus EGCG might be used as a lead structure to develop more effective antiviral drugs.

Latest animal models for anti-HIV drug discovery.

INTRODUCTION: HIV research is limited by the fact that lentiviruses are highly species specific. The need for appropriate models to promote research has led to the development of many elaborate surrogate animal models. AREAS COVERED: This review looks at the history of animal models for HIV research. Although natural animal lentivirus infections and chimeric viruses such as chimera between HIV and simian immunodeficiency virus and simian-tropic HIV are briefly discussed, the main focus is on small animal models, including the complex design of the 'humanized' mouse. The review also traces the historic evolution and milestones as well as depicting current models and future prospects for HIV research. EXPERT OPINION: HIV research is a complex and challenging task that is highly manpower-, money- and time-consuming. Besides factors such as hypervariability and latency, the lack of appropriate animal models that exhibit and recapitulate the entire infectious process of HIV, is one of the reasons behind the failure to eliminate the lentivirus from the human population. This obstacle has led to the exploitation and further development of many sophisticated surrogate animal models for HIV research. While there is no animal model that perfectly mirrors and mimics HIV infections in humans, there are a variety of host species and viruses that complement each other. Combining the insights from each model, and critically comparing the results obtained with data from human clinical trials should help expand our understanding of HIV pathogenesis and drive future drug development.

Vaccination directed against the human endogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system.

BACKGROUND: Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. RESULTS: To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. CONCLUSION: The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.

Vaccination directed against the human endogenous retrovirus-K envelope protein inhibits tumor growth in a murine model system.

Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer.

Expression of human endogenous retrovirus-K coincides with that of micro-RNA-663 and -638 in germ-cell tumor cells.

BACKGROUND/AIM: The cell line GH was established from germ-cell tumor tissue; human endogenous retrovirus-K (HERV-K) expression was detectable after prolonged culture of the cells, particularly in cells that formed domes and vesicles. In addition, keeping GH cells in culture at high cell densities increased HERV-K expression. Here, we studied whether this inducible HERV-K expression is accompanied by differences in microRNA (miRNA) expression patterns of GH cells. MATERIALS AND METHODS: The global miRNA expression pattern of GH cell samples (HERV-K high versus low) was analyzed by miRNA arrays. RESULTS: Two miRNAs were found to be differentially regulated and to exhibit expression parallel to that of HERV-K. The identified miRNAs-663 and -638, have been reported to be involved in multiple processes, including cellular senescence. However, induction of HERV-K expression did not change the cellular senescence status of GH cells. CONCLUSION: The expression of these two miRNAs might be useful as novel diagnostic and prognostic markers in patients with tumors.

The cellular antiviral restriction factor tetherin does not inhibit poxviral replication.

Interferon-stimulated genes fulfill innate antiviral effector functions. Among them, tetherin (THN) blocks the release of many enveloped viruses from infected cells. Vaccinia virus (VACV) encodes immune modulators interfering with antiviral host responses. Therefore, it was tempting to study a potential VACV-THN interaction. Remarkably, THN expression did not inhibit VACV release and replication. VACV infection did not diminish THN surface levels or impair its function on retroviral release. This suggests that THN is unable to restrict VACV replication.

Selective gene silencing by viral delivery of short hairpin RNA.

RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells.

Functional F11L and K1L genes in modified vaccinia virus Ankara restore virus-induced cell motility but not growth in human and murine cells.

Modified vaccinia virus Ankara (MVA) was generated by serial passaging in chicken embryo fibroblasts. During this attenuation, MVA lost the capacity to productively grow in human and most other mammalian cell lines, as well as acquiring a multitude of deletions and mutations in the MVA genome. This means that the precise molecular basis for the MVA host-range restriction is still unknown. The vaccinia virus (VACV) genes F11L and K1L are mutated or truncated in MVA. F11L was previously implicated in VACV-induced cell motility and virion maturation. Here, we demonstrate that the restoration of F11L gene expression in MVA rescued virus-induced cell motility, but had no impact on MVA virion maturation and host-range restriction. Additional insertion of the K1L gene, which restores MVA replication in RK-13 cells, was not sufficient to extend MVA growth capacity to other mammalian cells.

Cutaneous T-cell lymphoma cells are sensitive to rapamycin.

Cutaneous T-cell lymphomas (CTCL) are characterised by clonal expansion of helper T lymphocytes that infiltrate the skin. Only a small number of cell lines exist to study cellular pathways leading to T-cell transformation and to identify new targets for intervention. We wanted to investigate the inhibition of mTOR as a possible therapeutic target in CTCL. Primary cells of patients with Sézary syndrome (SS) and conventional CTCL cell lines were analysed. Constitutive activation of mTOR was found, and concomitantly, we could show that rapamycin, a specific inhibitor of mTOR, inhibits CTCL cell growth in vitro by induction of cell cycle arrest. Using a previously established animal model for CTCL, we additionally observed upon rapamycin treatment tumor growth inhibition in vivo. In summary, primary cells from patients with SS as well as CTCL cell lines allowed us to identify mTOR as an important target for intervention.

Vaccinia virus double-stranded RNA-binding protein E3 does not interfere with siRNA-mediated gene silencing in mammalian cells.

Vaccinia virus (VACV) evolved several strategies to evade antiviral cellular defence. The vaccinia virus E3 protein for example binds and sequesters double stranded RNA (dsRNA) and counteracts interferon action. We were interested to find out whether and to what extend E3 interferes with RNA silencing mediated by short interfering RNA (siRNA) in mammalian cells. We could show that the expression of a VACV-encoded marker gene can be efficiently inhibited by siRNA independently of the presence of the E3 protein. In addition, expression of E3 had no impact on RNA polymerase III promoter-derived shRNA-induced silencing of a cellular gene in human cells. Both VACV early and late gene expression could be inhibited by siRNA. Furthermore, downregulation of the expression of the E3L gene itself by siRNA in VACV infected cells produced the previously described phenotype of a knock-out virus, which illustrates the power of siRNA for vaccinia virus gene function analysis.

From actually toxic to highly specific--novel drugs against poxviruses.

The potential use of variola virus, the causative agent of smallpox, as a bioweapon and the endemic presence of monkeypox virus in Africa demonstrate the need for better therapies for orthopoxvirus infections. Chemotherapeutic approaches to control viral infections have been less successful than those targeting bacterial infections. While bacteria commonly reproduce themselves outside of cells and have metabolic functions against which antibiotics can be directed, viruses replicate in the host cells using the cells' metabolic pathways. This makes it very difficult to selectively target the virus without damaging the host. Therefore, the development of antiviral drugs against poxviruses has initially focused on unique properties of the viral replication cycle or of viral proteins that can be selectively targeted. However, recent advances in molecular biology have provided insights into host factors that represent novel drug targets. The latest anti-poxvirus drugs are kinase inhibitors, which were originally developed to treat cancer progression but in addition block egress of poxviruses from infected cells. This review will summarize the current understanding of anti-poxvirus drugs and will give an overview of the development of the latest second generation poxvirus drugs.

Stable integration of a functional shRNA expression cassette into the murine leukemia virus genome.

Short hairpin RNA (shRNA) can be stably expressed in cells to down-modulate gene expression. While retroviral and lentiviral vectors can be used to deliver shRNAs, the restricted viral titer is the major limitation for efficient gene transfer, which is especially important for cancer gene therapy. We were interested in using replicating murine leukemia virus (MLV) to enhance the shRNA transfer. Although stem loop structures could potentially interfere with the retroviral life cycle, we were able to demonstrate that the insertion of shRNA expression cassettes into MLV did not interfere significantly with viral fitness. The virus was genetically stable and able to silence target gene expression. Our results show that replicating MLVs are excellent tools for delivering shRNAs efficiently throughout the culture and have the potential to be used for gene function elucidation or even for cancer gene therapy.

Murine leukemia virus (MLV) replication monitored with fluorescent proteins.

BACKGROUND: Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. RESULTS: We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. CONCLUSIONS: Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.