ABSTRACT
BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.
Subject(s)
ADAMTS13 Protein/antagonists & inhibitors , Autoantibodies , Purpura, Thrombotic Thrombocytopenic/immunology , Purpura, Thrombotic Thrombocytopenic/therapy , ADAMTS13 Protein/immunology , Animals , Autoantibodies/genetics , Cloning, Molecular , DNA, Complementary/administration & dosage , Humans , Mice , Models, Animal , Molecular Targeted Therapy/methods , Single-Chain Antibodies/genetics , Single-Chain Antibodies/toxicity , von Willebrand Factor/metabolismABSTRACT
BACKGROUND: Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand factor-cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular or genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. STUDY DESIGN AND METHODS: Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal immunoglobulin (Ig)G in patient plasma. RESULTS: Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed both to the amino terminal domains and to those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were noninhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. CONCLUSIONS: Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal models of acquired TTP. Shared idiotypes of inhibitory clones with circulating IgG from multiple patients suggest common features of pathogenic autoantibodies that could be exploited for developing more targeted therapies.
Subject(s)
ADAMTS13 Protein/immunology , Autoantibodies/isolation & purification , Purpura, Thrombotic Thrombocytopenic/immunology , Adult , Antibody Specificity , Cell Surface Display Techniques , Child , Cloning, Molecular , Cross Reactions/immunology , Epitope Mapping , Humans , Immunoglobulin G/blood , Middle AgedABSTRACT
Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005-07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts.
