Cloning, expression and functional characterization of the full-length murine ADAMTS13

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A + mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73592/1/j.1538-7836.2005.01246.x.pd

Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events

ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura

The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP

A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications

Vectors Based on Modified Vaccinia Ankara Expressing Influenza H5N1 Hemagglutinin Induce Substantial Cross-Clade Protective Immunity

New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine

A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza