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Serp-1 dose-and schedule-dependently accelerates full-thickness wound healing in mice. (A-C) The full course of wound healing for mice with full-thickness wounds treated with saline alone (N = 13) or Serp-1 at a dose of (A) 2 μg/mouse (Serp-1 [2]; N = 6), (B) 1 μg/mouse (Serp-1 [1]; N = 7), or (C) 1 μg/mouse on days 0 and 3 (Serp-1 [1+1]; N = 5). Dotted horizontal cross lines indicate equivalent healing of Serp-1 mice on day 3 as is achieved by saline alone on day 7.

Serp-1 dose-and schedule-dependently accelerates full-thickness wound healing in mice. (A-C) The full course of wound healing for mice with full-thickness wounds treated with saline alone (N = 13) or Serp-1 at a dose of (A) 2 μg/mouse (Serp-1 [2]; N = 6), (B) 1 μg/mouse (Serp-1 [1]; N = 7), or (C) 1 μg/mouse on days 0 and 3 (Serp-1 [1+1]; N = 5). Dotted horizontal cross lines indicate equivalent healing of Serp-1 mice on day 3 as is achieved by saline alone on day 7.

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Numerous treatments have been developed to promote wound healing based on current understandings of the healing process. Hemorrhaging, clotting, and associated inflammation regulate early wound healing. We investigated treatment with a virus-derived immune modulating serine protease inhibitor (SERPIN), Serp-1, which inhibits thrombolytic proteases...

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... analyzed the effects of Serp-1 on treatment of full thickness wounds in a splinted wound healing model in wild-type C57BL6/J mice, as described in the Experimental Section (Figure 2A,B) [52-54]. The initial dose of Serp-1 applied to the wounds was 2 μg/mouse, based on prior intraperitoneal doses of 100 μg/kg/bodyweight used in other models of vascular inflammatory diseases and based on an average mouse weight of 20 g [43]. ...
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... next tested repeated treatment with the same total amount protein (2 μg) by application of Serp-1 given as a 1.0 μg/20 μL saline/10 mm 2 wound/mouse at day zero and day three, respectively. Wound healing was further enhanced with 40% wound closure at day five ( Figure 2C; p < 0.0001), while there was less than 20% wound closure in saline-treated controls ( Figure 2A) and only 30% in single dose treatment groups ( Figure 2C). Thus, these results demonstrate that the therapeutic efficacy of topical treatment for Serp-1 solution is extended by repeat dosing, further promoting wound healing in the mouse model. ...
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... next tested repeated treatment with the same total amount protein (2 μg) by application of Serp-1 given as a 1.0 μg/20 μL saline/10 mm 2 wound/mouse at day zero and day three, respectively. Wound healing was further enhanced with 40% wound closure at day five ( Figure 2C; p < 0.0001), while there was less than 20% wound closure in saline-treated controls ( Figure 2A) and only 30% in single dose treatment groups ( Figure 2C). Thus, these results demonstrate that the therapeutic efficacy of topical treatment for Serp-1 solution is extended by repeat dosing, further promoting wound healing in the mouse model. ...
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... next tested repeated treatment with the same total amount protein (2 μg) by application of Serp-1 given as a 1.0 μg/20 μL saline/10 mm 2 wound/mouse at day zero and day three, respectively. Wound healing was further enhanced with 40% wound closure at day five ( Figure 2C; p < 0.0001), while there was less than 20% wound closure in saline-treated controls ( Figure 2A) and only 30% in single dose treatment groups ( Figure 2C). Thus, these results demonstrate that the therapeutic efficacy of topical treatment for Serp-1 solution is extended by repeat dosing, further promoting wound healing in the mouse model. ...
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... next examined new vessel growth (vascularization) at seven days post-wounding-an intermediate time during which wounds were observed to begin healing at a rapid rate ( Figure 2C). We analyzed vascularization by immunohistochemistry with antibodies against the endothelial marker CD31 (also called PECAM-1), a marker for endothelial cells commonly used to indicate wound bed blood vessels [68]. ...

Citations

... We examined whether a pegylated version of the Myxoma virus serpin, Serp-1, would ameliorate the chronic inflammatory environment in DMD mdx /Utrn −/− mice. This protein has been shown to induce an anti-inflammatory response in wound healing, transplants, and other acute injuries without any demonstrated increase in adverse effects in multiple animal models and in one Phase IIa clinical trial [39][40][41][42][43][44][45]52,53]. Systemic PEGSerp-1 treatment of DKO mice significantly decreased muscle fibrosis and the number of infiltrating M1 pro-inflammatory macrophages. ...
... Native, unmodified Serp-1, as well as PEGSerp-1, are able to bind and inhibit serine proteases, uPA, tPA, and plasmin, similar to the mammalian serpin, PAI-1. From studies where uPAR has been inactivated, Serp-1 activity, and its ability to promote wound closure and collagen remodeling, is disrupted [52,57,58]. For example, the anti-inflammatory activity of Serp-1 is absent in aortic allograft transplants in uPAR-deficient mice [39]. ...
... PEGSerp-1 modulation of the M1 macrophages is consistent with what has been reported for atherosclerosis, transplant, wound healing, and spinal cord injury [39][40][41][42]. However, an increase in the anti-inflammatory M2 macrophages described in wound healing [52] was not observed in the DMD mdx /Utrn −/− mouse diaphragm. This implies that PEGSerp-1 may not be acting via the same mechanism in skeletal muscle. ...
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Duchenne muscular dystrophy is an X-linked disease afflicting 1 in 3500 males that is characterized by muscle weakness and wasting during early childhood, and loss of ambulation and death by early adulthood. Chronic inflammation due to myofiber instability leads to fibrosis, which is a primary cause of loss of ambulation and cardiorespiratory insufficiency. Current standard of care focuses on reducing inflammation with corticosteroids, which have serious adverse effects. It is imperative to identify alternate immunosuppressants as treatments to reduce fibrosis and mortality. Serp-1, a Myxoma virus-derived 55 kDa secreted glycoprotein, has proven efficacy in a range of animal models of acute inflammation, and its safety and efficacy has been shown in a clinical trial. In this initial study, we examined whether pegylated Serp-1 (PEGSerp-1) treatment would ameliorate chronic inflammation in a mouse model for Duchenne muscular dystrophy. Our data revealed a significant reduction in diaphragm fibrosis and increased myofiber diameter, and significantly decreased pro-inflammatory M1 macrophage infiltration. The M2a macrophage and overall T cell populations showed no change. These data demonstrate that treatment with this new class of poxvirus-derived immune-modulating serpin has potential as a therapeutic approach designed to ameliorate DMD pathology and facilitate muscle regeneration.
... Serp-1 is a myxoma virus derived SERine Protease INhibitor (SERPIN) with previously demonstrated roles in inflammation, cell migration, wound closure, tissue remodeling and fibrosis (9)(10)(11)(12). It is a single-chain glycosylated protein composed of three β-sheets and nine α-helical domains with a strained reactive center loop (RCL) positioned in the carboxy terminus (13). ...
... One group received a control treatment of 10 µL of phosphate-buffered saline (PBS) applied topically for 10 min. The second group not only received a 10 µL (0.1 µg/µl) topical treatment of recombinant Serp-1 protein but also a subcutaneous injection of recombinant Serp-1 protein (0.1 µg/µl) in a dosage equivalent to 100 ng per gram of body weight (recorded every morning) based on our previous publication (10). Treatments were applied twice daily for a total of 10 days following the injury. ...
... Recombinant Serp-1 protein was produced by Chinese hamster ovary (CHO) cell line protein expression system (Viron Therapeutics Inc., London, ON, CA), as described by our previous publication (10). Sequential column chromatographic separation was employed to purify GMP-compliant recombinant Serp-1 protein. ...
Article
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Purpose: Chemical corneal injuries carry a high morbidity and commonly lead to visual impairment. Here, we investigate the role of Serp-1, a serine protease inhibitor, in corneal wound healing. Methods: An alkaline-induced corneal injury was induced in 14 mice. Following injury, five mice received daily topical saline application while nine mice received Serp-1 100 μL topically combined with a daily subcutaneous injection of 100 ng/gram body weight of Serp-1. Corneal damage was monitored daily through fluorescein staining and imaging. Cross sectional corneal H&E staining were obtained. CD31 was used as marker for neovascularization. Results: Serp-1 facilitates corneal wound healing by reducing fibrosis and neovascularization while mitigating inflammatory cell infiltration with no noticeable harm related to its application. Conclusions: Serp-1 effectively mitigates inflammation, decreases fibrosis, and reduce neovascularization in a murine model of corneal injury without affecting other organs. Translational Relavence: Our study provides preclinical data for topical application of Serp-1 to treat corneal wounds.
... While these studies all used an intraperitoneal or intravenous delivery of naked recombinant protein, Serp-1 is also amenable to drug delivery vehicles and is currently the only serpin demonstrated for delivery by such approaches. Serp-1 is capable of sustained delivery in a chitosan-collagen biocompatible hydrogel and has demonstrated therapeutic efficacy in models of full-thickness cutaneous wound healing in mice and in spinal cord injury in rats, with efficacy dependent on engagement of uPAR in the fibrinolytic signaling pathway (212)(213)(214). ...
... Mechanistic studies performed by the authors demonstrate that Ly6E prevents coronavirus entry into host cells by preventing S-protein-mediated membrane fusion. Of interest, the virus derived serpin that we have studied extensively, Serp-1, and also PAI-1 bind and block the uPA/uPAR complex (212,213). With Serp-1 this leads to marked anti-inflammatory function. ...
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The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, “off-the-shelf” system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are the ser ine p rotease in hibitors, termed serpins . Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a “respiratory” coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.
... Skin is a highly vascularized, bio mineralized composite material composed of fibroblasts and collagen surrounded by specialized extracellular matrix with high mechanical strength and structural complexity. [1][2][3][4] Skin tissue defects have become one of the major sources of mortality and morbidity among all age group people worldwide. Although the auto grafts and allograft were regarded as the high attainment for wound repair, the approach was limited because of their unwanted effects (i.e. ...
... We have been developing these naturally evolved viral immune-modulating proteins as a new class of therapeutics. Two of the most effective pathways around which virus-derived immune modulators have been developed are the serpins [160][161][162][163][164][165][166][167][168][169][170][171][172][173][174] and the chemokine modulators [175][176][177][178][179][180][181]. ...
... Several genes identified in Myxomavirus enhance virus pathogenicity and have proven highly effective in targeting key immune response pathways. The immune response pathways targeted by viruses are often central regulatory pathways, allowing the virus to effectively escape the host immune response [63,[160][161][162][163][164][165][166][167][168][169][170][171][172][173][174]. ...
... Serpins expressed by poxviruses target thrombotic and thrombolytic proteases as noted above, as well as apoptotic pathways, often displaying marked efficacy and potency. Myxoma virus encodes two such serpins, Serp-1 and Serp-2 that have been studied in animal models of inflammatory disease [160][161][162][163][164][165][166][167][168][169][170][171][172][173][174]. Serp-1 is a 55 kDa glycosylated secreted serpin that binds tPA, uPA, plasmin, factor X, and thrombin inhibiting immune responses driven by both clots forming and clot-dissolving cascades (Fig. 4). ...
Article
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Progressive neurological damage after brain or spinal cord trauma causes loss of motor function and treatment is very limited. Clotting and hemorrhage occur early after spinal cord (SCI) and traumatic brain injury (TBI), inducing aggressive immune cell activation and progressive neuronal damage. Thrombotic and thrombolytic proteases have direct effects on neurons and glia, both healing and also damaging bidirectional immune cell interactions. Serine proteases in the thrombolytic cascade, tissue- and urokinase-type plasminogen activators (tPA and uPA), as well as the clotting factor thrombin, have varied effects, increasing neuron and glial cell growth and migration (tPA), or conversely causing apoptosis (thrombin) and activating inflammatory cell responses. tPA and uPA activate plasmin and matrix metalloproteinases (MMPs) that break down connective tissue allowing immune cell invasion, promoting neurite outgrowth. Serine proteases also activate chemokines. Chemokines are small proteins that direct immune cell invasion but also mediate neuron and glial cell communication. We are investigating a new class of therapeutics, virus-derived immune modulators; One that targets coagulation pathway serine proteases and a second that inhibits chemokines. We have demonstrated that local infusion of these biologics after SCI reduces inflammation providing early improved motor function. Serp-1 is a Myxomavirus-derived serine protease inhibitor, a serpin, that inhibits both thrombotic and thrombolytic proteases. M-T7 is a virus-derived chemokine modulator. Here we review the roles of thrombotic and thrombolytic serine proteases and chemoattractant proteins, chemokines, as potential therapeutic targets for SCI. We discuss virus-derived immune modulators as treatments to reduce progressive inflammation and ongoing nerve damage after SCI.
... Sections were stained with hematoxylin and eosin (H&E) and by Masson's trichrome using standard procedures, as previously described (21)(22)(23)(24)(25)(26)(27)30). DAH was classified into three degrees of severity according to the percentage of hemorrhage on H&E stained sections as assessed by a blinded histological analysis of DAH score as follows: (1) No hemorrhage (0%); ...
... In prior work, Serp-1 lost its ability to reduce inflammation and to reduce plaque growth in uPAR knock out aortic allograft transplants in mouse models (18). The depletion of uPAR also abolished the function of Serp-1 to promote wound healing (30). We therefor performed IHC staining for uPAR to characterize uPAR expression after pristine induction, with or without serpin treatments. ...
Article
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Diffuse alveolar hemorrhage (DAH) is one of the most serious clinical complications of systemic lupus erythematosus (SLE). The prevalence of DAH is reported to range from 1 to 5%, but while DAH is considered a rare complication there is a reported 50-80% mortality. There is at present no proven effective treatment for DAH and the therapeutics that have been tested have significant side effects. There is a clear necessity to discover new drugs to improve outcomes in DAH. Serine protease inhibitors, serpins, regulate thrombotic and thrombolytic protease cascades. We are investigating a Myxomavirus derived immune modulating serpin, Serp-1, as a new class of immune modulating therapeutics for vasculopathy and lung hemorrhage. Serp-1 has proven efficacy in models of herpes virus-induced arterial inflammation (vasculitis) and lung hemorrhage and has also proved safe in a clinical trial in patients with unstable coronary syndromes and stent implant. Here, we examine Serp-1, both as a native secreted protein expressed by CHO cells and as a polyethylene glycol modified (PEGylated) variant (Serp-1m5), for potential therapy in DAH. DAH was induced by intraperitoneal (IP) injection of pristane in C57BL/6J (B6) mice. Mice were treated with 100 ng/g bodyweight of either Serp-1 as native 55 kDa secreted glycoprotein, or as Serp-1m5, or saline controls after inducing DAH. Treatments were repeated daily for 14 days (6 mice/group). Serp-1 partially and Serp-1m5 significantly reduced pristane-induced DAH when compared with saline as assessed by gross pathology and H&E staining (Serp-1, p = 0.2172; Serp-1m5, p = 0.0252). Both Serp-1m5 and Serp-1 treatment reduced perivascular inflammation and reduced M1 macrophage (Serp-1, p = 0.0350; Serp-1m5, p = 0.0053), hemosiderin-laden macrophage (Serp-1, p = 0.0370; Serp-1m5, p = 0.0424) invasion, and complement C5b/9 staining. Extracellular urokinase-type plasminogen activator Guo et al. Serpin Reduces Lupus Lung Hemorrhage receptor positive (uPAR+) clusters were significantly reduced (Serp-1, p = 0.0172; Serp-1m5, p = 0.0025). Serp-1m5 also increased intact uPAR+ alveoli in the lung (p = 0.0091). In conclusion, Serp-1m5 significantly reduces lung damage and hemorrhage in a pristane model of SLE DAH, providing a new potential therapeutic approach.
... Mice were kept on a standard 12 h light-12 h dark cycle in a specific pathogen-free environment and given food and water ad libitum. Mice were singlehoused after the wounding procedure to prevent interference with wound healing, as previously described [35]. ...
... We performed a splinted, full-thickness wound healing model as previously described [35]. In this model, a silicone splint is used to prohibit the wound contraction of mouse skin around a single, intrascapular full-thickness biopsy punch wound during the first seven days, effectively forcing second-intention healing as occurs in human skin (whereas mouse skin primary heals by contraction). ...
... On day 3 post-wounding, the mice were anesthetized with 1-3% isoflurane, to effect, and 20 μL saline or 20 μL saline containing 1 μg M-T7 was carefully applied topically to the wounds (drop-wise above the wound) by inserting an insulin syringe through the silicon splint, with care not to disrupt the healing wound bed during application. To prevent self-induced secondary skin damage from scratching, mice were again anesthetized with 1-3% isoflurane, to effect, on day 7 post-wounding and the splints were carefully removed with sterile surgical scissors before being returned to single-housed cages as previously described [35]. The experimental design is outlined in Figure 1A. ...
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Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.
... We recently demonstrated that Serp-1 maintained therapeutic efficacy to improve healing in a full-thickness wound mouse model when delivered by a chitosan-collagen hydrogel (CCH) [40]. Chitosan, in a hydrogel combination with the structural stability of collagen, produces a highly biodegradable material with excellent biocompatibility, low antigenicity, and prior success in animal models of SCI [41,42]. Here, we studied whether Serp-1 delivered by chitosan-collagen hydrogel implant provides therapeutic efficacy in a forceps crush-induced spinal cord injury model. ...
... The procedure for preparing the chitosan-collagen hydrogel (CCH) was performed as previously described [42]. Briefly, 15 mg of low molecular weight chitosan (448869l, 75%-85% deacetylated, Sigma Life Science, St. Louis, MO, USA) was swelled in 10 mL of deionized water and gently rotated overnight at 4 • C. The excess water was removed by centrifugation at 1000 ×g for 15 min. ...
... Shortly before application, the lyophilized product was added to Type I collagen solution (C3867, Sigma Life Science, St. Louis, MO, USA) to a total volume of 500 µL to form a chitosan-collagen/Serp-1 gel at a Serp-1 concentration of 0, 10 µg (low dose), and 100 µg (high dose) per 50 µL gel, respectively. Previous characterization by our group demonstrated that this CCH material functions as a sustained release substrate, with in-tact Serp-1 continuing release after at least 4 days post-reconstitution [42]. Other groups have demonstrated similar chitosan-collagen hydrogel materials to release functional protein for 4 weeks [43]. ...
Article
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Spinal cord injury (SCI) results in massive secondary damage characterized by a prolonged inflammation with phagocytic macrophage invasion and tissue destruction. In prior work, sustained subdural infusion of anti-inflammatory compounds reduced neurological deficits and reduced pro-inflammatory cell invasion at the site of injury leading to improved outcomes. We hypothesized that implantation of a hydrogel loaded with an immune modulating biologic drug, Serp-1, for sustained delivery after crush-induced SCI would have an effective anti-inflammatory and neuroprotective effect. Rats with dorsal column SCI crush injury, implanted with physical chitosan-collagen hydrogels (CCH) had severe granulomatous infiltration at the site of the dorsal column injury, which accumulated excess edema at 28 days post-surgery. More pronounced neuroprotective changes were observed with high dose (100 µg/50 µL) Serp-1 CCH implanted rats, but not with low dose (10 µg/50 µL) Serp-1 CCH. Rats treated with Serp-1 CCH implants also had improved motor function up to 20 days with recovery of neurological deficits attributed to inhibition of inflammation-associated tissue damage. In contrast, prolonged low dose Serp-1 infusion with chitosan did not improve recovery. Intralesional implantation of hydrogel for sustained delivery of the Serp-1 immune modulating biologic offers a neuroprotective treatment of acute SCI.
... These prior studies were performed by systemic injection of Serp-1 either by intraperitoneal or intravenous injection or by subdural infusion to the spinal cord in rats. When delivered topically in saline solution or via a chitosan-collagen biocompatible hydrogel sustained-release substrate, Serp-1 accelerated healing in a full-thickness wound healing model in C57BL6/J mice, with improved vascularization and tissue remodeling [52]. These diverse studies attest to the marked capacity and the potential of this protein to function as a biologic therapeutic. ...
... Serp-1 efficacy is abrogated in a uPAR-deficient mouse aortic allograft models [70]. Similarly, Serp-1-mediated promotion of full-thickness wound healing is completely blocked by co-treatment with a neutralizing antibody to uPAR [52]. ...
Article
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Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.