Proceedings of the 2019 Viral Clearance Symposium, Session 1: Viral Clearance Strategies and Case Studies.

Session 1 of the 2019 Viral Clearance Symposium proposed strategies to use prior knowledge for demonstrating robust viral clearance for biotechnologically produced recombinant proteins, such as monoclonal antibodies, and introduced methods for virus reduction applicable to recombinant adeno-associated virus (rAAV) vectors. Proposals for generic virus clearance claims were made for detergent inactivation with Triton CG-110, low pH inactivation, anion-exchange membrane chromatography, and virus filtration, and acceptance of these proposals were discussed. Furthermore, the development of an ASTM standard for retrovirus removal by small virus filters was presented, and it was found that updating and refining of conditions is still warranted.

Proceedings of the 2019 Viral Clearance Symposium, Session 2: New Modalities in Chromatography and Adsorptive Filters.

In Session 2 of the 2019 Viral Clearance Symposium, new outcomes of viral clearance performance of polishing chromatography and depth filters utilized in the purification processes of biotechnologically produced recombinant proteins such as monoclonal antibodies were presented. It was shown that flow through both multimodal anion-exchange and anion-exchange chromatography are effective tools for removal of retroviruses and, under defined conditions, also for parvoviruses. Cation-exchange chromatography, when operated in bind/elute mode, hydrophobic interaction chromatography, and depth filtration can also be potent for virus removal, but are less robust for parvoviruses.

Proceedings of the 2017 Viral Clearance Symposium, Session 4: Submission Strategies.

Appropriate performance of virus validation studies and testing of unprocessed bulk harvests for retrovirus particle count are procedures in the demonstration of an acceptable level of viral safety for cell-derived biotechnology products. Product-specific validation studies on virus reduction with two model viruses [usually murine leukemia virus (MuLV) and a parvovirus] performed in duplicate runs are standard for clinical trial applications. For the retroviral safety margin, a 6 log reduction is normally expected.

Interleukin-1β receptor expressed by modified vaccinia virus Ankara interferes with interleukin-1β activity produced in various virus-infected antigen-presenting cells.

Background: Modified vaccinia virus Ankara (MVA) is a highly attenuated virus and a promising vaccine vector with potent immune stimulating properties. Deletion of the gene encoding the viral interleukin-1beta receptor (vIL-1βR) in MVA (MVAΔIL-1βR) was previously shown to enhance memory T cell function. Here, we investigated the influence of vIL-1βR on blocking interleukin-1beta (IL-1β) upon MVA infection in various antigen presenting cells of murine and human origin, and analyzed whether inflammasome function contributes to IL-1β production in different cell types.

Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III.

A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA.

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection.

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection.

Evaluation of a modified vaccinia virus Ankara (MVA)-based candidate pandemic influenza A/H1N1 vaccine in the ferret model.

The zoonotic transmissions of highly pathogenic avian influenza viruses of the H5N1 subtype that have occurred since 1997 have sparked the development of novel influenza vaccines. The advent of reverse genetics technology, cell-culture production techniques and novel adjuvants has improved the vaccine strain preparation, production process and immunogenicity of the vaccines, respectively, and has accelerated the availability of pandemic influenza vaccines. However, there is still room for improvement, and alternative vaccine preparations can be explored, such as viral vectors.

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.

MVA-based H5N1 vaccine affords cross-clade protection in mice against influenza A/H5N1 viruses at low doses and after single immunization.

Human infections with highly pathogenic avian influenza viruses of the H5N1 subtype, frequently reported since 2003, result in high morbidity and mortality. It is feared that these viruses become pandemic, therefore the development of safe and effective vaccines is desirable. MVA-based H5N1 vaccines already proved to be effective when two immunizations with high doses were used.

Viral host-range factor C7 or K1 is essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase R-mediated phosphorylation of eukaryotic translation initiation factor 2alpha.

Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which inhibits cellular and viral protein synthesis. In turn, VACV has evolved the capacity to antagonize this antiviral response by expressing the viral host-range proteins K3 and E3. This study revealed that the host-range genes K1L and C7L also prevent eIF2alpha phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines.

The orthopoxvirus 68-kilodalton ankyrin-like protein is essential for DNA replication and complete gene expression of modified vaccinia virus Ankara in nonpermissive human and murine cells.

Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus.

Vaccinia virus-mediated inhibition of type I interferon responses is a multifactorial process involving the soluble type I interferon receptor B18 and intracellular components.

Poxviruses such as virulent vaccinia virus (VACV) strain Western Reserve encode a broad range of immune modulators that interfere with host responses to infection. Upon more than 570 in vitro passages in chicken embryo fibroblasts (CEF), chorioallantois VACV Ankara (CVA) accumulated mutations that resulted in highly attenuated modified vaccinia virus Ankara (MVA). MVA infection of mice and of dendritic cells (DC) induced significant type I interferon (IFN) responses, whereas infection with VACV alone or in combination with MVA did not.

Postexposure immunization with modified vaccinia virus Ankara or conventional Lister vaccine provides solid protection in a murine model of human smallpox.

Background: Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.

The highly conserved orthopoxvirus 68k ankyrin-like protein is part of a cellular SCF ubiquitin ligase complex.

The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase complex.

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.

The kinase insert domain-containing receptor is an angiogenesis-associated antigen recognized by human cytotoxic T lymphocytes.

Antigen-specific cancer immunotherapy directed toward tumor-nourishing angiogenic blood vessels holds the promise of high efficacy, low toxicity, and ease of application. To evaluate whether the human angiogenic kinase insert domain-containing receptor (KDR) can serve as a target for cellular immunotherapy, 19 peptide sequences with HLA-A*0201 motifs were selected by computer-based algorithms. Five peptides (KDR82-90, KDR288-297, KDR766-774, KDR1093-1101, KDR1035-1044) stimulated specific cytotoxic T lymphocytes (CTLs) from peripheral-blood mononuclear cells (PBMCs) of 3 HLA-A*0201 donors.

Application of chimeric feline foamy virus-based retroviral vectors for the induction of antiviral immunity in cats.

In order to define the potential and applicability of replication-competent foamy virus-based vaccine vectors, recombinant feline foamy virus (FFV) vectors encoding defined segments of the feline calicivirus (FCV) capsid protein E domain were constructed. In cell cultures, these FFV-FCV vectors efficiently transduced and expressed a hybrid fusion protein consisting of the essential FFV Bet protein and the attached FCV E domains. The stability of the vectors in vitro was inversely correlated to the size of the heterologous insert.

Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis.

Previous studies have shown that foamy virus (FV) particle budding, especially the involvement of the viral env glycoprotein is different from that of other (ortho) retroviruses: the N-terminal Env leader protein Elp is a constituent of released FV particles. A defined sequence in Elp required for particle budding binds to the MA domain of Gag. To extend these findings, we show that feline FV Elp is a membrane-anchored protein with the N-terminus located inside the particle.

Construction and functional characterization of feline foamy virus-based retroviral vectors.

Replication-competent feline foamy or spuma virus (FFV) vectors were constructed and functionally tested. The unmodified FFV vector genome expressed by the strong human cytomegalovirus immediate early promoter encodes FFV particles that were replication-competent in cell cultures. Virus derived from the cloned FFV DNA replicated and persisted in experimentally infected cats similar to the FFV isolate FUV.