The serpin saga; development of a new class of virus derived anti-inflammatory protein immunotherapeutics.

Serine proteinase inhibitors, also called serpins, are an ancient grouping of proteins found in primitive organisms from bacteria, protozoa and horseshoe crabs and thus likely present at the time of the dinosaurs, up to all mammals living today. The innate or inflammatory immune system is also an ancient metazoan regulatory system, providing the first line of defense against infection or injury. The innate inflammatory defense response evolved long before acquired, antibody dependent immunity. Viruses have developed highly effective stratagems that undermine and block a wide variety of host inflammatory and immune responses. Some of the most potent of these immune modifying strategies utilize serpins that have also been developed over millions of years, including the hijacking by some viruses for defense against host immune attacks. Serpins represent up to 2-10 percent of circulating plasma proteins, regulating actions as wide ranging as thrombosis, inflammation, blood pressure control and even hormone transport. Targeting serpin-regulated immune or inflammatory pathways makes evolutionary sense for viral defense and many of these virus-derived inhibitory proteins have proven to be highly effective, working at very low concentrations--even down to the femptomolar to picomolar range. We are studying these viral anti-inflammatory proteins as a new class of immunomodulatory therapeutic agents derived from their native viral source. One such viral serpin, Serp-1 is now in clinical trial (conducted by VIRON Therapeutics, Inc.) for acute unstable coronary syndromes (unstable angina and small heart attacks), representing a 'first in class' therapeutic study. Several other viral serpins are also currently under investigation as anti-inflammatory or anti-immune therapeutics. This chapter describes these original studies and the ongoing analysis of viral serpins as a new class of virus-derived immunotherapeutic.

Serpins, the vasculature, and viral therapeutics.

Serine protease inhibitors, termed serpins, are key regulators of numerous biological pathways that initiate inflammation, coagulation, angiogenesis, apoptosis, extra-cellular matrix composition and complement activation responses. Viruses have encoded serpins to guard themselves from host immune attack. The myxoma virus which infects rabbits secretes a highly potent anti-inflammatory serpin, Serp-1, which targets thrombolytic and thrombotic proteases as a means to fend off coagulation and inflammatory reactions to viral infection. These reactions act as a defense, produced by the host, to counter viral infection and invasion. When infused in animals after vascular injury, Serp-1 elicits exceptional anti-inflammatory activity, whereas the mammalian serpin, plasminogen activator inhibitor-1 (PAI-1), which also targets thrombotic and thrombolytic proteases can induce a pro-thrombotic response. During arterial injury, PAI-1 is highly expressed and increased PAI-1 concentration can result in acute thrombosis after aortic transplant in mouse models. The reactive center loop amino acid sequence is a fingerprint for serpin function and this function is highly sequence specific such that modification in this sequence can markedly alter activity. For instance, the alteration of the serpin reactive site loop P1-P1I amino acid sequence nullified the anti-inflammatory activity of Serp-1 and modification of P2-P7 initiated a pro-inflammatory response with vascular remodeling with aneurysm formation. Furthermore Serp-1 has demonstrated the capacity to utilize a mammalian serine protease receptor, the urokinase-type plasminogen activator receptor (uPAR), to alter cellular signaling in part through the actin binding protein cytoskeletal system (via filamin B). In this review, the molecular mechanisms relating inflammation and coagulation pathways to atherosclerosis and how the viral serpin, Serp-1, modifies these pathways in order to exhibit this profound anti-inflammatory activity without associated adverse thrombosis are discussed. Viral and vascular serpins targeting the thrombolytic cascade represent a potential new and untapped therapeutic resource.

Myxoma viral serpin, Serp-1, a unique interceptor of coagulation and innate immune pathways.

Serpins maintain haemostasis through regulation of serine proteinases in the thrombotic and thrombolytic pathways. Viruses encode serpins that can alter thrombotic and thrombolytic responses producing, in some cases, disseminated intravascular coagulation (DIC). However, it has not been precisely defined how viral serpins induce these profound responses. The rabbit myxoma viral serpin, Serp-1 inhibits urokinase- and tissue-type plasminogen activators (uPA and tPA), plasmin and factor Xa in vitro and exhibits remarkable anti-inflammatory activity in various animal models. The effects of Serp-1 on activation of human platelets, endothelial cells, monocytes and T cells that mediate thrombosis and innate immune responses were therefore examined. We found that Serp-1 attenuated platelet and mononuclear cell adhesion to fibronectin and collagen. Serp-1 similarly inhibited monocyte migration into the peritoneum. Serp-1 inhibition of monocyte migration was lost in uPA receptor (uPAR) deficient mice. Serp-1 bound to the plasma membrane surface and altered uPA activation of endothelial cells (p=0.001), thrombin activation of platelets (p=0.021) and phorbol ester activation of endothelial (p=0.047), monocyte (p=0.011) and Jurkat T cells (p=0.012) as measured by intracellular calcium. Modulation of cellular activation was confirmed by membrane fluidity analysis. Microarray analysis of Serp-1 treated endothelial cells revealed alterations in Inositol 1,4,5-triphosphate receptor type II (ITPR2) a calcium-regulating gene. This study demonstrates the unique capacity of a viral serpin, Serp-1 to modify adhesion, activation, gene expression and calcium homeostasis in a wide range of cells that regulate coagulation and inflammation. Endothelial cells potentially represent a pivotal regulatory point for Serp-1 anti-inflammatory activity.

Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines in vitro.

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.

Myxoma viral serpin, Serp-1, inhibits human monocyte adhesion through regulation of actin-binding protein filamin B.

Serp-1 is a secreted myxoma viral serine protease inhibitor (serpin) with proven, highly effective, anti-inflammatory defensive activity during host cell infection, as well as potent immunomodulatory activity in a wide range of animal disease models. Serp-1 binds urokinase-type plasminogen activator (uPA) and the tissue-type PA, plasmin, and factor Xa, requiring uPA receptor (uPAR) for anti-inflammatory activity. To define Serp-1-mediated effects on inflammatory cell activation, we examined the association of Serp-1 with monocytes and T cells, effects on cellular migration, and the role of uPAR-linked integrins and actin-binding proteins in Serp-1 cellular responses. Our results show that Serp-1 associates directly with activated monocytes and T lymphocytes, in part through interaction with uPAR (P<0.001). Serp-1, but not mammalian serpin PA inhibitor-1 (PAI-1), attenuated cellular adhesion to the extracellular matrix. Serp-1 and PAI-1 reduced human monocyte and T cell adhesion (P<0.001) and migration across endothelial monolayers in vitro (P<0.001) and into mouse ascites in vivo (P<0.001). Serp-1 and an inactive Serp-1 mutant Serp-1(SAA) bound equally to human monocytes and T cells, but a highly proinflammatory mutant, Serp-1(Ala(6)), bound less well to monocytes. Serp-1 treatment of monocytes increased expression of filamin B actin-binding protein and reduced CD18 (beta-integrin) expression (P<0.001) in a uPAR-dependent response. Filamin colocalized and co-immunoprecipitated with uPAR, and short interference RNA knock-down of filamin blocked Serp-1 inhibition of monocyte adhesion. We report here that the highly potent, anti-inflammatory activity of Serp-1 is mediated through modification of uPAR-linked beta-integrin and filamin in monocytes, identifying this interaction as a central regulatory axis for inflammation.

Identification of myxomaviral serpin reactive site loop sequences that regulate innate immune responses.

The thrombolytic serine protease cascade is intricately involved in activation of innate immune responses. The urokinase-type plasminogen activator and receptor form complexes that aid inflammatory cell invasion at sites of arterial injury. Plasminogen activator inhibitor-1 is a mammalian serpin that binds and regulates the urokinase receptor complex. Serp-1, a myxomaviral serpin, also targets the urokinase receptor, displaying profound anti-inflammatory and anti-atherogenic activity in a wide range of animal models. Serp-1 reactive center site mutations, mimicking known mammalian and viral serpins, were constructed in order to define sequences responsible for regulation of inflammation. Thrombosis, inflammation, and plaque growth were assessed after treatment with Serp-1, Serp-1 chimeras, plasminogen activator inhibitor-1, or unrelated viral serpins in plasminogen activator inhibitor or urokinase receptor-deficient mouse aortic transplants. Altering the P1-P1' Arg-Asn sequence compromised Serp-1 protease-inhibitory activity and anti-inflammatory activity in animal models; P1-P1' Ala-Ala mutants were inactive, P1 Met increased remodeling, and P1' Thr increased thrombosis. Substitution of Serp-1 P2-P7 with Ala6 allowed for inhibition of urokinase but lost plasmin inhibition, unexpectedly inducing a diametrically opposed, proinflammatory response with mononuclear cell activation, thrombosis, and aneurysm formation (p < 0.03). Other serpins did not reproduce Serp-1 activity; plasminogen activator inhibitor-1 increased thrombosis (p < 0.0001), and unrelated viral serpin, CrmA, increased inflammation. Deficiency of urokinase receptor in mouse transplants blocked Serp-1 and chimera activity, in some cases increasing inflammation. In summary, 1) Serp-1 anti-inflammatory activity is highly dependent upon the reactive center loop sequence, and 2) plasmin inhibition is central to anti-inflammatory activity.