Giant cell arteritis (GCA) and Takayasu's disease are inflammatory vasculitic syndromes (IVS) causing sudden blindness and widespread arterial obstruction and aneurysm formation. Glucocorticoids and aspirin are mainstays of treatment, predominantly targeting T cells. Serp-1, a Myxomavirus-derived serpin, blocks macrophage and T cells in a wide range of animal models. Serp-1 also reduced markers of myocardial injury in a Phase IIa clinical trial for unstable coronary disease. In recent work, we detected improved survival and decreased arterial inflammation in a mouse Herpesvirus model of IVS. Here we examine Serp-1 treatment of human temporal artery (TA) biopsies from patients with suspected TA GCA arteritis after implant (TAI) into the aorta of immunodeficient SCID (severe combined immunodeficiency) mice. TAI positive for arteritis (GCApos) had significantly increased inflammation and plaque when compared to negative TAI (GCAneg). Serp-1 significantly reduced intimal inflammation and CD11b+ cell infiltrates in TAI, with reduced splenocyte Th1, Th17, and Treg. Splenocytes from mice with GCApos grafts had increased gene expression for interleukin-1beta (IL-1β), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1β expression. In conclusion, GCApos TAI xenografts in mice provide a viable disease model and have increased intimal inflammation as expected and Serp-1 significantly reduces vascular inflammatory lesions with reduced IL-1β.

Background Glucocorticoids are a principal treatment for giant cell arteritis (GCA), with some studies reporting predominate effect on T helper17 (Th17) cell activity in GCA, with lesser effects on other inflammatory cells. Serp-1 is a 55kDa myxomaviral serpin that modifies macrophage, Th1, and Th17 responses in aortic transplants in animal models. Objectives We have analyzed the effects of a virus-derived anti-inflammatory ser ine p rotease in hibitor ( serpin ) on human temporal artery (TA) biopsies isolated from patients with suspected GCA. Methods Using a newly developed “Aortic Window Xenopatch” model, human TA biopsy sections were divided implanted as full thickness grafts into the abdominal aorta of SCID mice in parallel and tested for response to Serp-1, with and without human PBMC infusion (N = 32). Results TA sections positive for arteritis (GCA pos ) displayed significantly increased inflammatory plaque when compared to negative sections (GCA neg ). Serp-1 reduced plaque in GCA pos sections (<0.01) with concommitant reduction in Th1, and Th17 splenocytes on flow cytometry (P < 0.01), with decreased IFNγ and IL-17 gene expression by RT-PCR array analysis. Serp-1 also reduced arterial CD11b + cells after PBMC infusion with reduced TNF-α expression in spleen, but not CD3 + , CCR6 + , nor CD86 + . Splenocytes from mice with GCA pos grafts had increased interleukin-1beta (Il-1β), IL-17, and CD25 expression, while IL-1β was significantly reduced by Serp-1.Serp-1 also markedly reducedt gene expression for the FII, FXa-PAR2, tPA, uPA in splenocytes (P <0.05). Conclusions Treatment with a virus-derived serpin, significantly reduces inflammation and plaque thickness in human GCA pos TA xenograft implants with associated reductions in macrophage and Th1 and Th17 responses in mice.

Background Lethal viral infections produce widespread inflammation with vascular leaking, clotting and bleeding (DIC), and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by a cytokine storm and also induce systemic inflammation. Normal host ser ine p roteases in hibitor ( serpin ) regulation is lost. Serp-1 is a potent myxomavirus anti-inflammatory protein, which inhibits factor X (fX), tPA and uPA in vitro . Serp-1 reduces arterial inflammation and atherogenesis in many animal models and has been successfully tested in patients with unstable plaque and stent implants. Methods and results Purified Serp-1 protein treatment significantly improved survival in lethal murine Gammaherpesvirus-68 (MHV68) infection in interferon gamma receptor (IFNγR) knock-out mice (N = 72 mice; P < 0.02) when given for 10 up to 30 days after infection. Treatment with a mammalian serpin, neuroserpin, that inhibits only tPA and uPA, was ineffective (N = 30 mice; P = NS). Serp-1 reduced viral antigen, lung hemorrhage, and aortic, lung and colon inflammation in MHV68 infected mice. Neuroserpin suppressed a wide swath of immune cell responses, while Serp-1 selectively increased CD11c+ splenocytes (macrophage and dendritic cells) and reduced CD11b+ tissue macrophage. Serp-1 significantly increased the IL-10 gene expression in aorta and plasma protein, whereas neuroserpin decreased IL-10. Serp-1 mediated survival in MHV68 infected mice was lost after treatment with IL-10 receptor antibody immediately post infection (N = 12 mice; P =0.014). Conclusions Serp-1, a unique myxomavirus-deried serpin, improves survival after lethal mouse herpesviral infection, reduces viral load, hemorrhage and arterial inflammation. Serp-1 provides a potential new therapeutic approach for lethal viral sepsis and DIC through altered FX and IL-10.

ABSTRACT Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal ser ine p rotease in hibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c + splenocytes (macrophage and dendritic cells) and reduced CD11b + tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.

Vasculitic syndromes cause devastating arterial occlusion with associated end organ ischemia and significant morbidity. Treatment for vasculitic syndromes remains very limited. Gamma 68 herpesvirus (g68HV) infection in interferon gamma receptor deficient (IFNγR -/- ) mice provides a good working model of artery inflammation, inducing early death with associated inflammatory cell invasion. Myxomavirus serpin, Serp-1, inhibits tPA, uPA, plasmin (thrombolytic) and fXa (thrombotic) proteases, reducing inflammation in a wide range of animal models whereas mammalian neuroserpin (NSP) inhibits tPA and uPA and reduced myocardial damage in clinical trial of unstable angina and stent implant. To assess the capacity of serpins to block inflammatory vasculopathy development, γ68HV infected IFNγR -/- mice were treated with either Serp-1 (100ng/g/100μl), NSP (100ng/g/100μl) or saline (100ul) .Serp-1 treatment given by daily intraperitoneal injections (i.p.) for the first 10 days post infection (6 - 12.5 X 106 PFU), significantly prolonged survival in mice (IFNγR -/- ) (P ≤ 0.048). Saline treated γ68HV infected mice survived up to 42 days, whereas Serp-1 treated mice survived up to 55 days (N=60 mice, 6-12 mice per group). NSP treatment (100ng/gm i.p. X 10 days) produced an opposite trend toward increased mortality (P ≤ 0.079; N=16). In a subsequent study, prolonged daily treatment with Serp-1 (100ng/gm i.p.) given for the first 30 days post infection, markedly prolonged survival in γ68HV infected INFγR -/- mice for up to 150 days (P ≤ 0.032, N =10). Treatment with Serp-1, that was started 3 weeks post γ68HV infection, produced a non-significant trend toward improved survival (P=0.685; N = 12 mice), whereas treatment begun at 1 week post infection again significantly improved survival (P ≤ 0.020; N=12 mice). Serp-1 treatment significantly reduced inflammatory cell invasion in the aortic adventitia ( p < 0.010) and lung ( p < 0.047), NSP significantly increased inflammatory cell invasion when compared to Serp-1 treatment in infected mice in the lung interstitial layer (p ≤ 0.005). At 10 days follow up the plasma cytokine interleukin-1beta (IL-1β) was significantly reduced by Serp-1 treatment (p ≤ 0.049), Treatment with the NSP reduced plasma cytokine TNFα ( p ≤ 0.022), IFNγ (p ≤ 0.022), and IL-4 (p ≤ 0.022) in γ68HV infected IFNγR -/- mice (N=14).Gene expression was significantly increased in Serp-1 treated mice for regulatory serpins in the thrombolytic cascade, specifically PAI-1 (p ≤ 0.020) and unexpectedly NSP (p ≤ 0.032), gene expression was significantly reduced for E selectin (p ≤ 0.048) and increased for IL-10 (p ≤ 0.031), and addressin (p ≤ 0.002) with Serp-1 treatment at 10 days . Compared to the saline treated mice, NSP treatment lead to decreased arterial IL-17 and IL-10 gene expression (p ≤ 0.006 and 0.041, respectively) and increased PAI-2 (p ≤ 0.021) . When comparing differences in gene expression for Serp-1 and NSP; Serp-1 treatment increased gene expression for IL-10 (p ≤ 0.001), bFGF (p < 0.022), Addressin (p < 0.001), PAI-1 (p < 0.021), and NSP (p < 0.009) and NSP reduced IL-6 (p < 0.016) and PAI-2 (p < 0.016). Conclusion: Serp-1 but not NSP produced an unexpected improved survival after γ68HV infection with associated rebalancing of the coagulation and thrombolytic cascades as well as modulating inflammatory responses. Anti-inflammatory serpin provides a potential new therapeutic approach to virus induced vasculitis.

Background - Giant cell / temporal arteritis (GCTA) is a severe inflammatory vasculitic syndrome (IVS), which causes arterial occlusion and sudden loss of vision. Treatment for IVS remains limited. Intra-aortic full thickness implants of human TA biopsy and left internal mammary artery (LIMA) specimens were surgically engrafted into the abdominal aorta of immunodeficient SCID mice as a new model for analyzing development of vascular inflammation, as well as response to new treatments. Serp-1 is a myxomaviral-derived serine protease inhibitor (serpin) that blocks thrombotic and thrombolytic protease pathways, arterial inflammation, and plaque growth. Serp-1 was recently tested in acute coronary syndromes (ACS), where reduced markers of myocardial damage were detected after stent implant. Objective - We assessed the effects of Serp-1 or saline treatment on inflammatory cell invasion and plaque growth after TA and LIMA engraftment in NOD-SCID mice. Results - Normal arterial (LIMA) specimens were harvested during bypass surgery and implanted into 24 SCID mice. Half the mice had saline (N=12) and half had Serp-1 treatment (N=12). In each group 6 mice had 5x106 peripheral blood mononuclear cells (PBMCs) infusions from the same patient as the donor LIMA graft, given immediately postop by tail vein and 6 had no cell infusion. TA grafts from patients with suspected GCA were implanted into a separate group of 24 SCID mice - 12 mice had saline (100µl) and 12 Serp-1 (100ng/g/100µl) treatment (one i.v. injection followed by daily i.p. injection for 10 days). in each treatment group 4 mice had infusion of non-matched PBMCs. At 4 weeks LIMA (P < 0.034) or TA (P < 0.0001) grafts, had significantly reduced plaque growth with Serp-1 treatment when human PBMC were also infused (p<0.05). In the absence of PBMC, Serp-1 had minimal effect. Mononuclear cell invasion was reduced in Serp-1 treated mice with PBMCs. Flow cytometry did not detect significant changes in splenocytes. Conclusions - Aortic implant of human LIMA or TA xenografts in SCID mice provides a physiological model for investigating inflammatory vasculitis. With PBMC infusion, treatment with the myxomaviral Serp-1, reduced inflammatory cell invasion and plaque growth in arterial xenografts.

Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation. We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.

Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation.We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.