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Viral SERPINS—A Family of Highly Potent Immune-Modulating Therapeutic Proteins


Abstract and Figures

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2–10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics.
Content may be subject to copyright.
Citation: Varkoly, K.; Beladi, R.;
Hamada, M.; McFadden, G.; Irving, J.;
Lucas, A.R. Viral SERPINS—A
Family of Highly Potent
Immune-Modulating Therapeutic
Proteins. Biomolecules 2023,13, 1393.
Academic Editor: Sihong Song
Received: 28 June 2023
Revised: 3 August 2023
Accepted: 6 September 2023
Published: 15 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
Viral SERPINS—A Family of Highly Potent
Immune-Modulating Therapeutic Proteins
Kyle Varkoly 1, Roxana Beladi 2, Mostafa Hamada 3,4, Grant McFadden 4, James Irving 5
and Alexandra R. Lucas 4, 6, *
1Department of Internal Medicine, McLaren Macomb Hospital, Michigan State University College of Human
Medicine, 1000 Harrington St., Mt Clemens, MI 48043, USA;
2Department of Neurological Surgery, Ascension Providence Hospital,
Michigan State University College of Human Medicine, 16001 W Nine Mile Rd., Southfield, MI 48075, USA;
3College of Medicine, Kansas City University, 1750 Independence Ave, Kansas City, MO 64106, USA;
4Center for Immunotherapy Vaccines and Virotherapy, Biodesign Institute, Arizona State University,
727 E Tyler St., Tempe, AZ 85287, USA;
5UCL Respiratory and the Institute of Structural and Molecular Biology, University College London,
5 University Street, London WC1E 6JF, UK
6Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, 727 E Tyler St.,
Tempe, AZ 85287, USA
*Correspondence:; Tel.: +1-352-672-2301
Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate
serine proteases in the central coagulation and immune pathways, representing 2–10% of circulating
proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct
thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune
responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved
with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian
proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine
proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-,
and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at
micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal
side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-
tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven
effective in a wide range of animal models and in one clinical trial in patients with unstable coronary
disease. Here, we outline the known viral serpins and review prior studies with viral serpins,
considering their potential for application as new sources for immune-, coagulation-, and apoptosis-
modulating therapeutics.
serpin; virus; immune-modulating; coagulation; apoptosis; poxvirus; plant virus;
baculovirus; biologic
1. Introduction
1.1. Serpins—Serine Protease Inhibitors
Serpins comprise a large family of protease inhibitors, which is ubiquitous in higher
eukaryotes, and they have a shared tertiary structure that adopts a unique inhibitory
conformation when bound to targeted proteases (Figure 1). Most serpins are serine protease
inhibitors, these are not enzymes but rather are suicide inhibitors of selectively targeted
proteases. Currently, over 6000 serpins are known in all kingdoms of life, with 37 found in
humans [
]. They represent roughly 2–10% of proteins in human blood and are the third
most common protein family, regulating central pathways in coagulation, apoptosis (cell
Biomolecules 2023,13, 1393.
Biomolecules 2023,13, 1393 2 of 23
death), and immune responses, as well as maintaining normal lung, immune and neuronal
health [
]. Viral serpins have evolved to inhibit selected targets within mammalian
coagulation, inflammation, and apoptotic protease-dependent pathways. However, the
timing and original genes that have been co-opted by viruses, and whether additional
lateral transfer events have occurred among the many kingdoms of life, remains a matter
of speculation [2].
Biomolecules 2023, 13, x FOR PEER REVIEW 2 of 25
neuronal health [2,3]. Viral serpins have evolved to inhibit selected targets within mam-
malian coagulation, inammation, and apoptotic protease-dependent pathways. How-
ever, the timing and original genes that have been co-opted by viruses, and whether ad-
ditional lateral transfer events have occurred among the many kingdoms of life, remains
a maer of speculation [2].
Figure 1. Proposed mechanisms of Serp-1 with prior preclinical and clinical studies examining the
potential for virus-derived serpin protein therapeutics. Serp-1 is a multifunctional serine protease
inhibitor, a serpin. The reactive center loop, RCL, of a serpin is cleaved by an active protease with
subsequent formation of a covalent bond, forming an inactive serpin–protease complex, with the
cleaved RCL forming an additional strand in the A beta sheet of the serpin, a form of suicide inhibi-
tion. Serpins are inhibitors, not proteases, with the capacity to (1) focus inhibition at sites of protease
activity, (2) bind multiple targets, and (3) be modied to allow ne tuning of inhibitory functions.
Serp-1 has potential as a therapeutic for a wide array of inammatory and coagulopathic disorders.
Orange—postulated ecacy, blue—prior preclinical and/or clinical ecacy.
Biologically, most serpins function as serine protease inhibitors, some function as
‘cross-class’ cysteine protease inhibitors, and others as non-inhibitory hormone and
Figure 1.
Proposed mechanisms of Serp-1 with prior preclinical and clinical studies examining the
potential for virus-derived serpin protein therapeutics. Serp-1 is a multifunctional serine protease
inhibitor, a serpin. The reactive center loop, RCL, of a serpin is cleaved by an active protease with
subsequent formation of a covalent bond, forming an inactive serpin–protease complex, with the
cleaved RCL forming an additional strand in the A beta sheet of the serpin, a form of suicide inhibition.
Serpins are inhibitors, not proteases, with the capacity to (1) focus inhibition at sites of protease
activity, (2) bind multiple targets, and (3) be modified to allow fine tuning of inhibitory functions.
Serp-1 has potential as a therapeutic for a wide array of inflammatory and coagulopathic disorders.
Orange—postulated efficacy, blue—prior preclinical and/or clinical efficacy.
Biomolecules 2023,13, 1393 3 of 23
Biologically, most serpins function as serine protease inhibitors, some function as
‘cross-class’ cysteine protease inhibitors, and others as non-inhibitory hormone and protein
transporters. The inhibitory mechanism of serpins has been extensively investigated [
Serpins are folded into a high-energy structure with an exposed region known as the
reactive center loop (RCL) that contains a bait recognition sequence which is specific to a
target protease. Upon cleavage in the vicinity of this sequence, this energy is expended
by incorporating the RCL as an additional
-strand inserted into the central A
(Figure 1). In this way, serpins utilize some of the energy accumulated during their synthesis
to exert a biological function. For inhibitory serpins, the result is a covalent inhibitory
serpin–protease complex, in which the activity of both the serpin and the protease is lost,
thus often termed suicide inhibition. This mechanism gives rise to an important property
of serpins, unlike non-covalent competitive protease inhibitors, they effectively do not have
a dissociation rate, and therefore do not need to be maintained at a concentration above
a dissociation constant to continue to inhibit the bound pool of proteases. Some serpins,
such as the mammalian plasminogen activator inhibitor-1 (PAI-1, SERPINE1) that inhibits
tissue- and urokinase-type plasminogen activators (tPA and uPA, respectively), can also
exist in a third state where fluidity of the RCL allows partial insertion into the
-sheet A in
the absence of cleavage, resulting in a latent, inactive, low-energy state [
]. The process of
RCL presentation or insertion is, in some cases, modulated by a ligand, such as heparin,
which increases the activity of antithrombin III from 100- to 1000-fold (AT, SERPINC1),
making it a more potent inhibitor of clotting factors. Similarly vitronectin stabilizes PAI-1
against the latent transition [5].
The impact of serpins as regulatory proteins that maintain normal physiological
functions is highlighted by the profound effects of genetic mutations in serpins, which
cause a class of disease known as a serpinopathy. These mutations lead, in some cases, to a
loss of inhibitory function and, in other cases, a gain of function. In some of these conditions,
the deposition of serpin aggregates and protein misfolding is associated with tissue damage
and leads to a deficiency state which prevents the effective inhibition of target proteases.
For neuroserpin (SERPINI1) and alpha 1 antitrypsin (A1AT, SERPINA1), this aggregation
is mediated by a repeating intermolecular contact that generates linear polymers, termed
‘beads-on-a-string’ morphology [
]. In A1AT deficiency, polymer formation in the liver
causes liver damage (cirrhosis) as well as a lack of effective inhibition of lung neutrophil
elastase, causing severe emphysema (lung damage) [
]. For neuroserpin (SERPINI1),
polymer formation in the central nervous system is the basis for a hereditary dementia
termed Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). Notably,
the onset and severity of neurodegenerative disease was associated with the rate and
magnitude of neuronal protein aggregation [7,10,11].
As polymers share a similar increase in stability to the RCL-inserted, protease-cleaved
form of the protein, and polymerization is prevented by pre-incubation with a peptide
mimetic of the RCL, it was proposed that polymers form by insertion of the RCL from
one serpin into the
-sheet A of an adjacent serpin. This is often referred to as the ‘loop-
sheet model’ [
]. The feasibility of an alternative mechanism in which polymerization
is mediated by a domain swap of the entire 4 kDa C-terminus of the protein has been
demonstrated crystallographically using a double cysteine mutant of alpha-1-antitrypsin
supported by disulfide trapping experiments [
]. Subsequent structural characterization of
polymers extracted from patient livers strongly supports this as the form that is present
in vivo [7,9].
Other serpinopathies have been described that are linked to a variety of clinical
disorders which are associated with deficiency or dysfunction of the serpin, including
antithrombin (AT), which results in excess thrombosis, and C1 esterase inhibitor (C1Inh,
SERPING1), which is associated with severe angioedema [
]. These mutations demon-
strate the importance of normal serpin function in maintaining a normal physiological
balance in the coagulation, neurologic, and immune pathways.
Biomolecules 2023,13, 1393 4 of 23
1.2. Serpin Regulation of Coagulation and Immune Responses
Serpins function to regulate cardiovascular, hematological, and immunological re-
sponses [Figure 1]. The thrombotic proteases, as components of intrinsic and extrinsic
coagulation cascades, produce a series of clotting factor protease activations and fibrin
deposition, both of which form clots. These thrombotic cascades have reciprocal interaction
and activation with the innate immune response pathways. The thrombolytic enzymes
activate plasmin to allow for the breakdown of fibrin in clots, termed fibrinolysis. The
thrombolytic proteases can also drive immune cell activation and invasion at sites of en-
dothelial damage, again with the reciprocal activation of immune responses. In particular,
the urokinase-type plasminogen activator (uPA), although an activator of plasmin, is con-
sidered to be predominantly a meditator of inflammatory response pathways. uPA binds
to the uPA receptor (uPAR), allowing local plasminogen activation and forming plasmin
that, in turn, activates matrix-degrading enzymes. uPA that is bound to the uPAR activates
plasmin, which then activates pro-forms of matrix metalloproteinases (MMPs). MMPs,
in turn, break down the extracellular matrix, releasing growth factors and increasing im-
mune cell invasion at sites of tissue injury. The thrombotic and thrombolytic cascades thus
have ongoing interactions with immune response pathways, where activated coagulation
pathways induce inflammatory cell activation and vice versa. This interaction of activated
proteases and receptors in coagulation and immune pathways is regulated by serpins,
providing opportunities for clinical intervention. For example, heparin, which increases
the efficacy of clotting factor inhibition by AT, is one of the most frequently used drugs that
is given to patients to reduce excess clotting associated with heart attacks, atrial fibrillation
with emboli, pulmonary emboli, and deep venous thrombosis [13].
The capacity of serpins to target a wide range of proteases provides a unique and po-
tentially beneficial method for the use of serpins as therapeutics. Indeed, some serpins are
already used as biological therapeutics to treat patients with genetically dysfunctional ser-
pins through augmentation therapy including A1AT and C1Inh. Further, there is currently
active development of modified serpins to target excess bleeding in haemophilia. The use
of AT as a potential treatment for severe bacterial sepsis with disseminated intravascular
coagulation (DIC) has also been assessed [2,1316].
Myxomavirus (MyxV) is a poxvirus that infects rabbits, but the MyxV serpin can
recognize protease targets outside the rabbit, including proteases in mice and humans.
A serpin called Serp-1, that is derived from MyxV, is the most extensively studied virus-
derived serpin for use as an anti-inflammatory therapeutic. The areas studied for possible
therapeutic benefits are broad due to the potential of serpins for modifying vascular
immuno-coagulopathic responses to injury. Preclinical models of disease that have been ex-
amined for treatment with viral serpins include vasculitis, unstable angina and myocardial
infarction, restenosis after percutaneous coronary intervention, transplant vasculitis and
rejection, arthritis, muscular dystrophy, spinal cord injury, wound healing, inflammatory
bowel disease, severe colitis, uveitis, diabetic retinopathy, macular degeneration, lupus asso-
ciated diffuse alveolar lung hemorrhage (DAH), viral acute respiratory distress syndrome
(ARDS) with lung infection and coagulopathy, giant cell arteritis, and other acute and
chronic inflammatory immune and coagulation disorders [2,1741] [Figure 1and Table 1].
Here, we focus on serpins that are encoded and expressed by viruses and their potential
applications as a new class of protein therapeutic. Viral serpins derived from Myxoma
poxviruses in particular have been extensively studied in translational and clinical research,
with an emphasis on potential therapeutic applications. The current work indicates that
these serpins have great promise as potent, anti-inflammatory immune-modulating agents,
agents which have demonstrated marked efficacy and minimal adverse effects. The broad
efficacy of virus-derived biologics indicates that viral serpins may provide a rich and
extensive platform for the discovery of new approaches to treating disease.
Biomolecules 2023,13, 1393 5 of 23
Table 1.
Virus-derived Serpins–Prior Pre-clinical and Clinical analyses of Virus-derived Serpins as
Therapeutics for Inflammatory Disease. Diseases that have been assessed and efficacy are listed.
Viral Serpin Inflammatory Disorder Target/Outcomes Subjects Studied Reference
Atherosclerotic Plaque Acute coronary
syndromes with stent implant
(1) Phase II Clinical Trial, Mechanism
extensively studied/Reduced
markers of cardiac damage, MACE
= 0, No neutralizing antibodies
(2) Preclinical–Reduced
intimal hyperplasia
(1) Human Clinical Trial,
Randomized dose
escalating trial at 7
sites Canada and US
(2) Preclinical–Rabbits–
Reduced intimal hyperplasia
Angioplasty injury and intimal
plaque restenosis
Efficacy and Mechanism
extensively studied
Rabbits, Rats, Mice,
Swine/Reduced intimal
hyperplasia and
inflammation in rodents
and rabbits
Aortic Allograft Transplant Established uPAR as central for Serp-1
therapeutic efficacy Mouse and rat [21,33,42]
MHV68—Mouse Herpes virus aortitis
and lung inflammation
Serp-1 reduced mortality, whereas NSP
did not
Reduction in monocytes and
pro-inflammatory cytokines, reduced
lung hemorrhage, NSP not effective
Mice [26,43]
Carotid compression—
Atherosclerotic Plaque
Reduction in carotid arterial
inflammation and plaque Mice [22]
Giant Cell Arteritis
Human Temporal artery biopsy implants
Reduction in monocytes and
pro-inflammatory cytokines SCID Mice [25]
Ebola infection Reduced liver damage and
improved survival Mice [26]
Diffuse Alveolar Hemorrhage (DAH) in
Pristane induced Systemic
Lupus Erythematosus
Reduced uPAR and Complement and
reduced macrophage infiltrates Mice [17,44]
SARS-CoV-2 Reduction in M1 macrophage recruitment
in cardiac and pulmonary tissue
Mouse adapted SARS models
in C57Bl/6 and BALB/c mice [39]
Colitis model Serp-1 reduced mortality Mice In preparation
Collagen induced arthritis Reduced joint swelling without
antibodies to Serp-1 treatment Rats [28]
Retinal Inflammation—Uveitis
Corneal wound healing
Retinal-Intra-vitreal injection of AAV
vector expressing Serp-1
Mice [29,45]
Periodontal bacteria with associated
increased atherosclerotic plaque
Decreased pro-inflammatory markers
and reduced plaque Mice [30]
Scar Reduction in Wound Healing
Accelerated wound healing-Improved
collagen formation in wound bed
Effects blocked by uPAR antibody
Mice [31]
Transplant Rejection
Rat and Mouse renal, aortic and
heterotopic heart allografts
Rat to mouse cardiac xenografts
Human, Rat, Mice [32,33,46,47]
Effects blocked by uPAR antibody in
wound healing
Reduced availability of VEGF
Chicken angiogenesis CAM
Mouse wound healing model
Pancreatic Cancer subcutaneous implants
Reduced tumor weight for pancreatic cell
line implant and reduced
macrophage infiltration
Human to Mice cell
xenografts in SCID mice [35]
Spinal Cord Injury
Balloon angioplasty crush injury
Improved motor function, reduced
inflammation and improved
neuronal growth
Local infusion
Local infusion [36,37]
Duchenne Muscular Dystrophy
Reduced leukocyte invasion, Improved
myofibril organization
Serp-1, PEGSerp-1
Double knock out DMD Mice [38]
Biomolecules 2023,13, 1393 6 of 23
Table 1. Cont.
Viral Serpin Inflammatory Disorder Target/Outcomes Subjects Studied Reference
Angioplasty injury and
intimal hyperplasia
Reduced inflammation and
intimal hyperplasia Mice [23,40]
Carotid cuff compression injury in
hyperlipidemic mouse models
Reduction in aortic Atherosclerotic
Plaque Development
ApoEnull Mice carotid
cuff compression [40]
Aortic Transplant
Reduced intimal plaque and
Inhibition of Granzyme-B
mediated apoptosis
Mouse aortic Allografts (1)
WT C57Bl/6 to BALB/c and
(2) Granzyme B KO
donor allografts
Liver Transplant Improved survival, reduced
hepatocyte necrosis Mice [41]
SPI-1 Chemotherapy Multi-pathway inhibitor of apoptosis In vitro [48]
Viral Host Defense and Vaccination Mice, in vitro [48]
SPI-2 and CrmA
Multi-pathway inhibitor of apoptosis
In vitro [39,40,48]
Neurodegeneration Mice, in vitro [49]
Fulminant Liver Failure Mice, in vitro [50,51]
Autoimmune Hepatitis Reduction in inflammatory cell (CD11),
inhibitor of apoptosis Mice, in vitro [50,51]
Injury/Chemotherapeutic Cardiotoxicity Multi-pathway inhibitor of apoptosis Mice, in vitro [52,53]
SPI-3 Hemophilia Inhibits uPA, plasmin, and tPA Mice, in vitro–
proposed function [54,55]
2. Virus-Derived Serpins
Viral serpins operate as extracellular and intracellular agents, both of which have
demonstrated efficacy in animal models, exerting effects on immunologic, thrombotic,
and apoptotic processes (Figure 2). These virus-derived proteins provide potential new
treatments with advantages that include: (1) efficacy at very low doses (micrograms of drug
per kilogram of patient), (2) the ability to target sites of protease activation, e.g., focused
efficacy, (3) minimal side effects and low antigenicity at administered doses, and (4) the
capacity for modification to alter protease target specificity [2].
The known viral serpins that have been the most extensively studied are derived from
poxviridae, but serpin sequences are also reported in herpesviruses and insect viruses.
The majority of viral serpin therapeutic studies have been performed with myxomavirus,
vaccinia, or cowpox viral serpins. Myxomavirus is a poxvirus that is lethal in European
rabbits and was introduced to cull the rabbit population in Australia. Vaccinia virus is a
vaccine strain that is used for developing vaccines for smallpox virus and, more recently,
as an oncolytic virus for cancer treatments. Vaccinia was originally thought to be derived
from cowpox, but more recent work suggests that the vaccinia virus is most likely to have
been derived from a virus we now call horsepox. These virus-derived serpins have been
studied as potential therapeutics in isolation from other viral components, as described in
the following sections.
2.1. Poxvirus Serpins
Myxomavirus encodes and expresses two distinct serpins, Serp-1, which is secreted by
an infected cell, and Serp-2, which operates intracellularly, albeit displaying extracellular
anti-inflammatory activities. Serp-1 binds and inhibits extracellular thrombolytic and
thrombotic proteases as well as complement proteases [
], while Serp-2 inhibits
intracellular and cell surface apoptosis pathway proteases, granzyme B, a serine protease,
and caspases 1 and 8, which are cysteine proteases [
]. CrmA and Spi2 are
intracellular serpins derived from cowpox and vaccinia virus and also target the apoptotic
pathways. Other poxviral serpins include the Spi1 and Spi3 serpins, both of which are
intracellular [48].
Biomolecules 2023,13, 1393 7 of 23
Biomolecules 2023, 13, x FOR PEER REVIEW 8 of 25
Serp-1 regulates the excess activation of immune responses through the inhibition of
coagulation and complement proteases. Serp-1 binds thrombolytic and thrombotic prote-
ases (tPA, uPA, plasmin, fXa, and thrombin (in the presence of heparin, as well as comple-
ment proteases, as demonstrated by immunoprecipitation and mass spectrometry [17,57].
Serp-1, as in the case for the mammalian serpins PAI-1, alpha 2 anti-plasmin (A2AP), pro-
tease nexin-1 (PN-1), and A1AT, binds and inhibits more than one serine protease. Serp-1
modies leukocyte adhesion, activation, gene expression, and calcium homeostasis
[18,19]. Additionally, Serp-1 has potent anti-inammatory activity through its inhibition
of the uPA/uPA receptor (uPA/uPAR) and complement receptors, and associated eects
on monocyte and endothelial cell responses [18,19,21,58].
In the following sections, we will review preclinical and clinical studies of diseases
for which Serp-1 treatment has been examined which have supported its therapeutic
safety and ecacy (Figure 2).
Figure 2. Immune-modulating mechanisms of myxoma poxvirus-derived serpins. Serp-1 operates
predominantly extracellularly to inhibit thrombotic, thrombolytic, and complement proteases,
reducing leukocyte recruitment. Serp-2 and CrmA operate predominantly intracellularly to inhibit
Figure 2.
Immune-modulating mechanisms of myxoma poxvirus-derived serpins. Serp-1 operates
predominantly extracellularly to inhibit thrombotic, thrombolytic, and complement proteases, reduc-
ing leukocyte recruitment. Serp-2 and CrmA operate predominantly intracellularly to inhibit a variety
of pro-inflammatory and pro-apoptotic factors. However, Serp-2 has immune-modulating functions
when given systemically, via an extracellular or systemic infusion. SPI-1and SPI-2/ CrmA are multi-
pathway inhibitors of apoptosis. SPI-3 targets coagulation factors extracellularly. A1AT—alpha 1
anti-trypsin, A2AP—alpha 2 antiplasmin, AT—anti-thrombin, C1Inh—C1 complement esterase in-
hibitor 1, CRM—Cytokine response modifier, IL-1b—interleukin 1beta, PAI-1—plasminogen activator
inhibitor -1, SPI—serine protease inhibitor, tP—tissue type plasminogen activator, uPA—urokinase-
type plasminogen activator, II—factor II, X—factor X.
2-1A). Serp-1
Serp-1 has been extensively investigated over the past 30 years as a new class of
virus-derived immune-modulating therapeutic. Serp-1 has developed a high efficacy
during evolution, protecting MyxV from attack and clearance by host inflammatory cells
at picomolar concentrations when secreted by virus-infected cells. When the Serp-1 gene
is deleted in myxomavirus, this lethal European rabbit infection becomes benign and
Biomolecules 2023,13, 1393 8 of 23
the blockade of rabbit immune cell responses, that are produced in response to myxoma
infection, is lost [
]. Purified Serp-1 protein has proven effective as a therapeutic
at reducing damaging inflammation in a wide array of animal models of disease when
delivered systemically at picogram to microgram doses (Figures 1and 2and Table 1), and
shows promise as a potential treatment for other disorders [1740].
Serp-1 regulates the excess activation of immune responses through the inhibition
of coagulation and complement proteases. Serp-1 binds thrombolytic and thrombotic
proteases (tPA, uPA, plasmin, fXa, and thrombin (in the presence of heparin, as well as
complement proteases, as demonstrated by immunoprecipitation and mass spectrome-
try [
]. Serp-1, as in the case for the mammalian serpins PAI-1, alpha 2 anti-plasmin
(A2AP), protease nexin-1 (PN-1), and A1AT, binds and inhibits more than one serine
protease. Serp-1 modifies leukocyte adhesion, activation, gene expression, and calcium
homeostasis [
]. Additionally, Serp-1 has potent anti-inflammatory activity through its
inhibition of the uPA/uPA receptor (uPA/uPAR) and complement receptors, and associated
effects on monocyte and endothelial cell responses [18,19,21,58].
In the following sections, we will review preclinical and clinical studies of diseases for
which Serp-1 treatment has been examined which have supported its therapeutic safety
and efficacy (Figure 2).
2.1A)-1. Acute and Chronic Inflammatory Diseases
2.1A)-1-1. Atherosclerotic Disease
2.1A)-1-1-1. Percutaneous Coronary Intervention (PCI) and Restenosis in Unstable
Atherosclerotic Plaque—preclinical testing
Inflammatory macrophage- and T-cell-mediated atherosclerotic plaque rupture in-
duces the exposure of the inner atheroma layers, specifically fat (lipids) and collagen
layers, and this in turn causes platelet activation and local thrombosis. Inflammatory
plaque rupture in coronary arteries and carotid and/or cerebral arteries is associated with
acute thrombotic arterial occlusion causing myocardial infarction (MI) and cerebrovascu-
lar accidents (CVA or stroke). Percutaneous coronary intervention (PCI) also can cause
endothelial damage, dissection and acute thrombosis or more simple intimal hyperplasia
with restenosis and/ or thrombosis [20,24].
Purified Serp-1 protein was first investigated as a potential therapeutic in a hyperlipi-
demic rabbit model for the accelerated atherosclerotic plaque that is seen after angioplasty,
termed restenosis. Serp-1 is a secreted 55kDa glycoprotein that binds and inhibits tissue
and urokinase-type plasminogen activator (tPA, uPA), plasmin, factor X, and thrombin as
well as complement proteases. Single picogram to nanogram doses were given by local in-
fusions (Wolinsky perforated balloon catheters) immediately after angioplasty. Significantly
reduced intimal hyperplasia and inflammation were demonstrated at 30-days follow-up.
Subsequent studies examined systemic infusions and stent implants [
]. This was the first
report on the use of a viral anti-inflammatory serpin as an immune-modulating treatment.
At low-dose infusions, only the primary site demonstrated plaque improvement, whereas,
at high-dose infusions, more systemic reductions in plaque development were detected.
Associated with the reduced plaque after treatment, there was reduced mononuclear cell
infiltration in the arterial intimal layer when compared to control saline or inactive Serp-1
SAA (a Serp-1 protein where the Arg and Asn at the P1-P1
RCL scissile bond are replaced
by Ala). Overall, the marked reduction at 30-days follow-up after single picogram doses
were given at the time of angioplasty injury demonstrated marked efficacy. No adverse
effects were seen in this study. PCI with stent implant was also assessed in rabbits, and
demonstrated efficacy with preventative treatments given for three days post stent implant.
This study demonstrated early promise for Serp-1 as a virus-derived immune modulating
protein, potentially over other current standard therapeutics that are given after PCI, where
restenosis can occur in up to 30% of lesions [20,24].
Serp-1 further demonstrated promise in angioplasty and aortic transplant models
in rodent models. One study investigated the effects of Serp-1 following rat iliofemoral
Biomolecules 2023,13, 1393 9 of 23
angioplasty and injury. A reduction in intimal hyperplasia was seen in animals that
were treated with wild-type Serp-1 when compared to treatment with a range of Serp-1
reactive center loop mutants [
]. Inflammatory plaque formation was also examined in
mouse aortic allograft transplants. In this transplant vasculopathy model, Serp-1 treatment
improved inflammation and intimal hyperplasia. Serp-1 blocked intimal hyperplasia
after aortic allograft transplant in donor aortic transplants derived from PAI-1-deficient
aortic donor allografts (C57Bl/6 to BALB/c mice) [
]. However, Serp-1 treatment was
ineffective after donor aortic transplants in uPAR-deficient mouse aortic donor allografts.
Serp-1, but not a reactive center loop mutant, up-regulated PAI-1 serpin expression in
human endothelial cells. The treatment of endothelial cells with an inactivating antibody
to uPAR and vitronectin blocked these observed Serp-1-induced changes. This indicated
that Serp-1 treatment is not dependent upon mammalian PAI-1 expression. These early
studies supported the idea that Serp-1 reduced vascular inflammatory responses to injury
through native uPA/uPAR receptor interactions and potentially via uPAR lipid raft protein
interactions, as with vitronectin [1921].
In experiments by Ilze Bot and Erik Biessen, Serp-1 treatment was investigated in a
hyperlipidemic ApoE
mouse model after carotid cuff compression injury. Continuous
infusions of Serp-1 by osmotic pump over 30 days, again, significantly reduced arterial
(carotid) inflammation and plaque growth at the site of carotid compression. This study
further demonstrated safety and efficacy for continuous infusions of Serp-1 [22].
To better elucidate the mechanisms behind Serp-1-mediated decreases in arterial
plaque growth, the signaling pathways were investigated.
In vitro
analysis of Serp-1 re-
vealed decreased monocyte activation, as well as decreased membrane fluidity. In these
studies, Serp-1 was noted to bind to the uPA receptor (uPAR) and to increase beta actin
(filamin expression) expression and decrease CD18 (beta integrin) expression [
]. De-
creased membrane fluidity is seen in a variety of disease states including hypercholes-
terolemia, hyperlipidemia, and atherosclerotic heart disease. Both Serp-1 and Serp-2
decreased the expression of WNT2 as well, which is an important signaling molecule in
cell-to-cell communication. Decreased expression of the atherogenic molecules CD5, SELE,
and VCAM1 was also seen when monocytes were treated with Serp-1, providing another
molecular pathway for atherosclerotic plaque reduction with this therapeutic [23].
2.1A)-1-1-2. Periodontal Disease and Associated Atherosclerotic Plaque Development
The oral microbiome is closely associated with atherosclerosis and heart health. In a
mouse study with aortic injury and balloon angioplasty, mice infected with Porphyromonas
gingivalis had increased intimal plaque following balloon angioplasty. When treated with
Serp-1, reductions in this coronary intimal plaque were observed. Toll-like receptor 4
and myeloid differentiation primary response 88 were also decreased following Serp-1
treatment, suggesting a potential anti-inflammatory mechanism for Serp-1 treatment [30].
2.1A)-1-1-3. Acute Coronary Syndrome (ACS)—Clinical Trial
Based on the prior preclinical studies which demonstrated Serp-1-mediated reductions
in atheroma after aortic and carotid injury, and following a safety assessment in rat and
primate models, Serp-1 was investigated in a randomized, blinded, dose-escalating study
in patients with unstable coronary syndromes receiving coronary stent implants [
Acute or unstable coronary syndrome (ACS) is caused by inflammatory macrophage
infiltration into artherogenic plaque in the coronary arterial intimal layer that leads to
eventual plaque rupture and the superimposed activation of thrombosis. This causes either
total arterial thrombotic occlusion with heart attack, stroke, or peripheral vascular occlusion
or intermittent occlusion termed unstable angina (also termed non ST elevation myocardial
infarction or NSTEMI). In the Phase 2A trial that was undertaken at seven sites in the US
and Canada, Serp-1 was infused at doses of 0, 5, and 15
g/kg daily for three days by
intravenous bolus together with standard of care and with comparison to patients receiving
only standard of care with no Serp-1 protein treatment. Treatment with the higher dose of
Serp-1 significantly reduced troponin I and CK-MB, markers of heart damage, when given
Biomolecules 2023,13, 1393 10 of 23
immediately after coronary stent implant in patients with unstable angina and non-ST
elevation MI, (i.e., in patients with ACS). There were no adverse events, and the major
adverse cardiovascular event (MACE) rate of death, myocardial infarction, or coronary
revascularization at the higher dose of Serp-1 was zero; further, no neutralizing antibodies
were detected. There was no significant decrease in plaque growth seen upon conducting a
follow-up intravascular ultrasound. The reduction in myocardial damage supported the
further assessment of Serp-1 in ACS patients undergoing stent deployment [24].
Inflammatory Vasculitis Syndromes—Giant Cell Arteritis and Takayasu Disease
Given the efficacy of Serp-1 treatment in atherosclerotic plaque and intimal hyperpla-
sia, Serp-1 treatment was also examined in models for inflammatory vascular disease.
2.1A)-1-2-1. Giant Cell Arteritis
Giant cell arteritis and Takayasu’s disease are two large-vessel inflammatory vasculitic
syndromes (IVS). IVS are associated with sudden onset blindness, aneurysm formation,
and diffuse vascular occlusions. High-dose pulsed steroids, the chemotherapeutic cy-
clophosphamide, aspirin, and steroid-sparing drugs such as the interleukin 6 receptor
(IL 6R) inhibitor Tocilizumab are currently the mainstays of treatment for IVS. However
recurrent disease is frequent and additional therapies are needed. Temporal artery (TA)
biopsies from human patients with suspected giant cell arteritis (GCA) were obtained for
the diagnosis of temporal arteritis and GCA. In a blinded analysis, full-thickness sections
of the TA biopsy specimens were implanted into the aorta of immunodeficient SCID mice.
The mice implanted with human TA biopsy specimens that were diagnosed as positive for
giant cell arteritis had significantly more inflammation than those with negative implants,
confirming the model. Peripheral blood mononuclear cells were infused to more closely
simulate the inflammation in GCA. Serp-1 reduced inflammatory cell invasion, including a
reduction in Th1, Th17, T-reg, and, more importantly, the cytokine interleukin 1-
This study demonstrated that unmodified Serp-1 may provide a new immune-modulating
biologic for the treatment of IVS where current treatments have failed, with limited side
effects [25].
2.1A)-1-2-2. Mouse Gamma Herpes Viral Infection (MHV68)
Serp-1 treatment was also assessed in a mouse gamma herpesvirus herpesvirus
(MHV68) model for inflammatory vasculitis. Vascular inflammation is seen in some severe
viral infections, as has been reported in the recent COVID19 pandemic [
]. Sepsis can
induce a severe imbalance in the coagulation pathways wherein excess clot formation in
bacterial and viral sepsis can lead to the depletion of proteases, inducing thrombosis. The
depletion of coagulation factors can then cause subsequent bleeding. This imbalance in
the clotting and clot-dissolving cascades is termed DIC. In severe bacterial, fungal, or viral
infections with sepsis, treatments are often ineffective and have a high rate of mortality.
In prior work, AT and heparin treatments in bacterial sepsis with DIC were assessed.
While early studies showed some promise, a larger study indicated only an equivocal
benefit [
]. Given the capacity for Serp-1 to inhibit thrombotic, thrombolytic, and
complement proteasesa, and that it functions as an inhibitor that reduces intimal plaque
and arterial inflammation, Serp-1 treatment was postulated to hone to sites of protease
activation in MHV68 virus-induced inflammatory vascular disease, sepsis, and DIC. Serp-1
treatment was compared with neuroserpin (NSP) treatment in this model, as NSP inhibits
only the thrombolytic proteases tPA and uPA, and does not bind or inhibit fXa or thrombin.
In this study, Serp-1 improved survival, with 60% of mice surviving at day 150, versus 0%
in the saline-treated and NSP-treated mice. Serp-1 reduced the viral load, lung hemorrhage,
and aortic and lung inflammation [
]. Additionally, NSP suppressed splenic cell responses
during MHV68 infection, while Serp-1 increased the amount of CD11c+ monocytes and
dendritic cells and reduced the amount of resident tissue macrophages. Serp-1 treatment
also modulated the gene expression for selected coagulation and inflammatory responses
when compared to NSP. Beneficial effects of Serp-1 were also seen in mouse-adapted Zaire
Biomolecules 2023,13, 1393 11 of 23
ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis.
In recent work with a mouse-adapted SARS coronavirus infection, treatment with PEGy-
lated Serp-1 (PEGSerp-1), again, significantly improved clinical scores and outcomes, with
reduced lung consolidation and inflammation [
]. Thus, in both DNA and RNA viruses,
Serp-1 has demonstrated therapeutic efficacy in mice models for deadly viral infections [
The treatment of MHV-68 infections in interferon gamma receptor knockout mice
was further tested as a model for lethal vasculitis, DIC, colitis, and lung hemorrhage in
a study investigating the gut microbiome. The suppression of gut bacteria by the oral
administration of an antibiotic cocktail worsened MHV-68 infection and blocked Serp-1
treatment efficacy, indicating that the Serp-1 therapeutic efficacy in this MHV68 model was
dependent upon an intact gut microbiota [27].
2.1A)-1-3. Autoimmune Disorders
2.1A)-1-3-1. Transplant Rejection
While acute transplant rejection is well-controlled with current immunosuppressants,
chronic transplant vasculopathy and rejection remain a barrier to long-term transplant
function. Vascular rejection is closely associated with chronic transplant vascular inflamma-
tion, occlusions, rejection, and scarring. Acute and hyperacute rejection also limit allograft
transplant outcomes as well as function and survival after xenotransplant. A reduction in
chronic rejection and vasculitis as well as the improvement of xenograft transplant out-
comes would be paradigm-shifting in improving long-term outcomes, particularly given
the limited supply of organs that are available for transplantation. There are, however,
barriers which persist, from immunologic to physiological barriers, not to mention zoonosis
and ethical considerations in developing functional xenotransplants [21,32,33].
Given the efficacy of Serp-1 treatment in reducing vascular inflammation and in
atherosclerosis, Serp-1 treatment was assessed in rat and mouse aortic and rat renal allograft
transplant models for transplant rejection and transplant vasculitis. Serp-1 or control
infusions were given by venous bolus infusions immediately following aortic allograft
transplant. Both intimal plaque and early macrophage, and lymphocyte invasion in the
intimal, medial, and adventitial layers, were reduced with Serp-1, and it also attenuated the
depletion of medial smooth muscle cells [
]. Zhong and Wang demonstrated that vascular
inflammation and organ scarring were significantly reduced in renal allografts [
]. Serp-1
also was effective at improving cardiac heterotopic allografts in mice and rats to mouse
xenotransplants. However, the survival of the xenografts was not prolonged [32,47].
In the mouse aortic allograft transplant model, the role of the uPA/ uPAR complex
and the mammalian PAI-1 serpin in Serp-1-mediated reductions in vasculitis were analyzed
using donor-transplanted aortic segments from mouse models that were deficient in uPAR
or PAI-1 knock out mice. Serp-1 blocked plaque growth after aortic isograft transplant and
after wire-induced injury in PAI-1-deficient mice, indicating that PAI-1 expression is not
required for Serp-1 to block the development of vasculopathy. However, Serp-1 did not
inhibit plaque growth in uPAR-deficient aortic allografts, indicating that Serp-1 required
uPAR expression in the donor graft to reduce aortic allograft inflammation and intimal
hyperplasia [21].
2.1A)-1-3-2. Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a severe and chronic autoimmune and inflammatory
joint disease, one of the most common in the world. Although the complex inflammatory
pathways of RA are not completely understood, the rodent collagen-induced arthritis
model has been well-established to study antigen-induced autoimmune-based disease.
Rats that were administered Serp-1 at 50
g/kg via intravenous (IV) injections had reduced
clinical arthritis, with reduced joint swelling when it was given at the time of inducing the
disease [
]. The clinical severity was significantly lower and bony erosions on blinded
radiographs were also reduced. However, the treatment was less effective when given later,
after initiating joint inflammation. Delayed-type hypersensitivity reactions were lower in
the Serp-1 treatment group, while no antibodies to Type II collagen were detected. Minimal
Biomolecules 2023,13, 1393 12 of 23
histopathological synovial changes were detectable in the recipient rats and no neutralizing
antibodies to Serp-1 were developed. These results indicate promise for a therapeutic
potential of Serp-1 in arthritis [28].
2.1A)-1-4. Systemic Lupus Erythematosus and Diffuse Alveolar Hemorrhage (DAH)
Diffuse alveolar hemorrhage (DAH) is a rare and highly lethal complication in sys-
temic lupus erythematosus (SLE), occurring in 1–5% of cases but with a mortality up to
. Current treatments are limited, although some newer treatment approaches such
as infusion of the clotting factor VII are under investigation [
]. WT Serp-1 and a
PEGylated variant, PEGSerp-1, that has an increased half-life from 20 min to 8 h, were
assessed for treatment in a mouse model of pristane-induced DAH. PEGSerp-1 markedly
reduced DAH in the pristane model in the mouse DAH models, with significant associated
reductions in macrophage invasion and detectable uPAR and complement membrane at-
tack complex, and reduced macrophage invasion, as determined by immunohistochemical
analysis (IHC). Of even greater interest, both WT Serp-1 and PEGSerp-1 were effective
when given prophylactically immediately after a pristane injection was given to induce
]. However, PEGSerp-1 also significantly improved pristane-induced DAH
when given as a delayed treatment, starting seven days after inducing DAH. The capacity
to improve outcomes in the SLE DAH model would be of greater potential benefit in a
clinical setting [17].
2.1A)-1-5. Neurological, Musculoskeletal and Ophthalmological Disorders
2.1A)-1-5-1. Spinal Cord Injury
Spinal cord injury (SCI) initiates a severe pro-inflammatory response in the dura, result-
ing in a debilitating loss of function as well as increased mortality. The ensuing hemorrhage
and necrosis from the initial injury is complex with associated severe inflammatory destruc-
tion. Kwiecien et al. assessed Serp-1 infusion in a balloon crush injury model of the distal
thoracic spine in a rat model. Thirty-three mature male rats sustained an epidural crush
SCI and then received a subdural infusion of Serp-1 or a second Myxomavirus-derived
chemokine modulating protein, M-T7, in comparison to steroid infusion (dexamethasone,
DEX) or saline [
]. The infusions were given locally at the site of SCI for seven days via
osmotic pump, with a separate group of rats receiving intraperitoneal infusion. The clinical
endpoint monitoring included bodyweight, hemorrhagic cystitis, and bilateral pinch re-
sponse. The spinal cord was sectioned and the macrophage infiltrates were measured at
the site of crush injury. Rats infused with DEX had side effects of DEX toxicity, including
dermal atrophy and body weight loss. The rats infused with Serp-1 and M-T7 had no such
toxicity. Serp-1 improved the clinical bilateral pinch withdrawal responses. The subdural
infusions of all treatments had reduced CD68+ macrophage numbers. This study indicates
Serp-10s efficacy in a rat SCI model [36].
The effects of Serp-1 on rat SCI were validated in an additional study on 58 rats
which determined the optimal dose (0.2 mg/week) with comparison to a saline control.
Motor function was improved with Serp-1 infusion, together with reduced phagocytic
macrophages at the site of SCI injury in rats infused with from 8
g to 200
g of Serp-
1. Large amounts of myelin-rich necrotic debris and red blood cells were detected be-
tween infiltrating macrophages, supporting the inhibition of active macrophage phagocy-
tosis. The macrophage counts in the Serp-1-treated rats were reduced to approximately
half the macrophage counts in the saline-treated rats at eight weeks, supporting an anti-
inflammatory effect of Serp-1 treatment for SC crush injuries [37].
2.1A)-1-5-2. Duchenne Muscular Dystrophy
Duchene muscular dystrophy (DMD) is an x-linked recessive disease which causes
progressive weakness and muscle wasting in early childhood. Over 1000 variants of DMD
have been reported. Newer gene therapy approaches have been developed for severe
DMD variants but they are limited to a small percentage of genetic mutations. However,
inflammation and fibrosis cause ongoing severe diaphragm and cardiac muscle damage
Biomolecules 2023,13, 1393 13 of 23
with respiratory and cardiac failure. Treatment is often limited to high-dose steroids
with limited efficacy and associated side effects. Studies which investigated wild type
Serp-1 and PEG Serp-1 treatment demonstrated a reduction in M1 macrophages with
reduced diaphragm fibrosis and increased myofibril diameters [
]. Thus, PEGSerp-1 has
the potential to be used as a therapeutic approach in DMD pathology and potentially
improve muscle regeneration [
]. Studies are in progress to assess AAVSerp-1 as a gene
therapy approach to provide a sustained benefit with reduced damaging inflammation in
DMD patients.
2.1A)-1-5-3. Retinal Inflammation
Retinal inflammation is associated with vision loss in uveitis, age-related macular
degeneration, and diabetic retinopathy. As a new approach to treatment, Lewin, Ildefonso
et al. developed an AAV2 expression vector. AAVSerp-1, with expression confirmed by
Western blot, was used to prevent endotoxin-induced uveitis in a mouse model. In a pilot
study, AAV2 Serp-1 reduced endotoxin-induced uveitis in mouse models. Corneal injury
was also examined by Zhu et al., who demonstrated improved healing with Serp-1 given
topically together with IP injections. This allows for future studies investigating intra-
vitreal injection of this AAV vector
in vivo
with potential clinical applications in ocular
inflammatory diseases or as a treatment approach for chronic disorders such as DMD [
2.1A)-1-6. Wound Healing
Serp-1 has been investigated in early wound healing models. The chronic impairment
of wound healing is a major health problem in diabetics, with marked increases in risk for
morbidity with infection, amputation, and mortality. Serp-1 was applied topically at low
doses or via release from a chitosan-collagen hydrogel at sites of 3.5 mm punch biopsy
after 15 days of treatment in a mouse model [
]. Topical Serp-1 treatment significantly
accelerated wound healing, with repeated dosing at a lower dose proving more effective
than single high dosing. Continuous low-level Serp-1 release from the chitosan collagen
hydrogel similarly improved wound healing. These effects were blocked by uPAR anti-
body, again indicating the uPA/uPAR as a key site for Serp-1-mediated anti-inflammatory
activity. The Serp-1-treated wounds had elevated arginase-1, expressing M2-polarized
macrophages, which indicated a relative increase in anti-inflammatory M2 when compared
to pro-inflammatory M1 responses, with associated periwound angiogenesis. Improved
collagen maturation and organization were also demonstrated with more normal skin
architecture at the wound site. Serp-1 was deemed as a potential therapeutic for reducing
scarring in deep wounds [
]. Topical Serp-1 treatment also proved effective at improving
wound healing after alkali-induced corneal injury in mice [29,45]
2.1A)-1-7. Cancer Therapeutics
Angiogenesis is a critical factor in the development of a broad range of chronic dis-
eases such as malignant tumors, as well as a natural defense against vascular occlusions in
arthritis, wound healing, and cardiovascular disease. Myxomavirus is also under investi-
gation as an oncolytic virotherapy for solid tumors. To assess the potential for Serp-1 to
reduce the blood supply to tumors, Serp-1 was initially investigated by Richardson and
Hatton in an angiogenic chicken chorioallantoic membrane model (CAM). Serp-1 inhibited
endogenous angiogenesis in a dose-dependent fashion through significantly inhibiting
gene expression of laminin and reducing the expression of VEGF. Serp-1 did not affect
the CAM following the rapid growth phase. This was not seen after treatment with the
Serp-1SAA reactive center loop mutant that lacks inhibitory activity for tPA, uPA, plasmin,
and fX. This study highlighted how Serp-1 modulates the angiogenic process by specifically
targeting endothelial cells and reducing the availability of VEGF [34].
Serp-1 treatment was also assessed in a model for subcutaneous implant of pancreatic
tumor cells in mice. Inflammation in pancreatic cancer is associated with tumor-associated
macrophage and myeloid-derived suppressor cell (MDSC) activity, which correlates with
cancer progression. Human cancer cells were engrafted subcutaneously into SCID mice,
Biomolecules 2023,13, 1393 14 of 23
and were subsequently treated with Serp-1, NSP, or M-T7. Serp-1 and NSP both inhibited
the growth of the pancreatic cell line Hs776t four weeks following implantation. Serp-1 also
inhibited the growth of a second pancreatic cell line, MIA PaCa-2. The inhibition of tumor
growth by Serp-1 was associated with a significant decrease in splenocyte MDSC counts
as assessed by flow cytometry, without reductions in other splenocyte subpopulations.
Serp-1 and NSP both also reduced tumor-associated macrophage infiltration [
]. This
study suggested that Serp-1 may contribute to the oncolytic tumor suppressing activity
of myxomavirus.
2.1A)-1-8. Severe Acute Respiratory Distress syndromes, SARS-CoV-2 Infection
Severe acute respiratory distress syndrome (ARDS) during coronavirus-2 (SARS-CoV-
2) infection is associated with cytokine storm, uncontrolled inflammation, and systemic
thrombosis with a high mortality in both those with and without significant comorbidities.
Mortality is as high as 25–40% once in the ICU setting. Antivirals have been used clinically
in early COVID-19 infection to reduce the infection severity in the early phase of viral
infection; however, treatments are limited for the later immune-thrombotic syndromes
and cytokine storm. In a prior work with the MHV68 mouse herpesviral infections, lung
hemorrhage as well as vasculitis were improved with Serp-1 treatments [
]. To this end,
purified PEGSerp-1 was tested in two mouse-adapted SARS-CoV-2 models: the MA10
SARS-CoV-2 infection in BALB/c mice and the MA30 SARS-CoV-2 infection in C57Bl/6
mice [
]. PEGSerp-1 given prophylactically at the time of the initial infections significantly
reduced lung consolidation, with associated reductions in M1 macrophage invasion in both
the lung and heart. When given as a delayed treatment in the MA30SARS infection model,
Serp-1 again improved the outcomes at a lower dose. Detectable uPAR and complement
membrane attack complex (MAC) were closely associated with reductions in clinical symp-
toms, weight loss lung consolidation, and macrophage invasion in both the MA10 and
MA30 models. Of interest, the detectable uPAR on IHC staining was significantly reduced
in effective PEGSerp-1 doses with delayed treatments while C5b9 complement membrane
attack complex (MAC) detection was reduced at all Serp-1 doses. This would suggest,
again, a central role of the uPAR in Serp-1-mediated immune modulation. This study with
PEGSerp-1 treatment in mouse models of SARS-CoV-2 provides another example of im-
proved outcomes in mouse models of severe vasculitis and lung infection, with therapeutic
potential for treatment during the later phases of viral infections [39].
2-1B). Serp-2 and CrmA
Myxomavirus Serp-2 is an intracellular cross-class serpin with distinct functions that
differ from the Serp-1 protein discussed above. Cytokine response modifier A (CrmA) is
also a cross-class serpin that is expressed by cowpox virus. CrmA, also termed Spi2, shares
similar cross-class inhibitory functions to Serp-2 [
]. Both block the serine protease
granzyme B as well as the cysteine proteases caspase 1 (interleukin converting enzyme)
and caspase 8 (Figure 2). Granzyme B and caspases 1 and 8 drive apoptosis, a form of
programmed cell death that cells can use to limit viral replication: a form of auto-destruct
sequence. Viral anti-apoptotic functions are projected to protect viruses, allowing viral
replication to proceed without inducing cell death [40,41].
In vitro
, Serp-2 and CrmA both significantly reduce camptothecin (CPT)-induced
elevations in caspase 3 and caspase 7 in monocytes. However, only Serp-2 reduces caspase
3 activity in Jurkat T-cells when compared to controls. In T-cells treated with cytotoxic T-cell
medium (CTLm), Serp-2 attenuated CTL-mediated increases in Granzyme B/ Caspase
8 and Caspase 3/7. CrmA did not attenuate Granzyme B/Caspase 8 and Caspase 3/7
expression. Serp-2 further demonstrated a greater affinity for binding to T-cells than CrmA
upon flow cytometric analysis [
]. Of interest, CrmA has a greater inhibitory activity
in vitro
than Serp-2 for these targeted proteases, underscoring the differences in the
in vitro
and in vivo analyses of these unique virus-derived serpins.
Biomolecules 2023,13, 1393 15 of 23
Similar to studies with Serp-1, Serp-2 and CrmA have been tested as immune-modulating
proteins in mouse models of transplant and carotid cuff compression injury. These studies are
described below.
2-1B)-1. Atherosclerotic Plaque and Restenosis following Angioplasty Injury
In mouse models of atherosclerotic plaque growth and intimal hyperplasia after arte-
rial injury (termed restenosis), both Serp-2 and CrmA were assessed for their efficacy in
reducing plaque growth. In balloon angioplasty and aortic transplant models, Serp-2, but
not CrmA nor Serp-2 RCL mutants, demonstrated statistically significant reductions in ar-
terial plaque. Serp-2 treatment was also investigated by Bot and Biessen in hyperlipidemic
mice following carotid cuff compression injury. In this model, yet again, Serp-2
demonstrated reduced plaque growth and macrophage invasion. Of interest, the systemic
increase in aortic plaque, that was seen proximal to the carotid injury in the hyperlipidemic
mouse model, demonstrated an attenuated inflammatory response with greater
reductions in plaque in the aorta proximal to the site of carotid cuff compression injury, in-
dicating a systemic reduction in plaque growth. CrmA, again, did not reduce inflammation
nor plaque, further demonstrating the specific immune modulating effects of Serp-2 [40].
To examine the mechanisms for Serp-2-mediated reductions in arterial plaque growth,
the signaling pathways were investigated. The
in vitro
analysis of Serp-2 and its effect
on monocytes revealed decreased monocyte activation, as well as decreased membrane
fluidity. Changes in membrane fluidity are seen in a variety of pathogenic states including
hypercholesterolemia, hyperlipidemia, and atherosclerotic heart disease. Signaling path-
way activation was also reduced by Serp-2, with detected decreases in WNT2, which is an
important signaling molecule for cell-to-cell communication. The decreased atherogenic
molecule expression of CD5, SELE, and VCAM1 was seen when monocytes were treated
with Serp-2, to a greater extent than was seen with Serp-1, providing another molecular
pathway for atherosclerotic plaque reduction [23,41].
2-1B)-1-2. Transplant Rejection
2-1B)-1-2-1. Intimal Plaque Hyperplasia in Aortic Transplant
In aortic allograft transplants, Serp-2 reduced inflammation and intimal plaque de-
velopment. Granzyme B deficiency in donor aortic allografts attenuated Serp-2 efficacy,
reducing the Serp-2-mediated reduction in aortic plaque and intimal hyperplasia (thicken-
ing), and indicating Granzyme B as a central target for Serp-2-mediated anti-inflammatory
and anti-atherogenic activity. In this aortic allograft model, Serp-2 but not CrmA reduced
monocyte apoptosis without affecting the T-cells. Thus, in conclusion, multiple
in vitro
in vivo
studies have demonstrated that Serp-2 has the potential to inhibit arterial vascular
disease progression through the immune-modulating inhibition of Granzyme B-dependent
apoptosis [40].
2-1B)-1-2-2. Ischemia-Reperfusion Injury in Liver Transplant
Following liver transplantation, ischemia-reperfusion injury causes acute transplant
rejection and allograft transplant loss. To model this, mice sustained 90 min of hepatic
injury and were then treated with Serp-2 with comparison to a control. Serp-2 improved
the survival along with a statistically significant reduction in liver enzyme (ALT) levels,
infarct scar thickness, and hepatocyte necrosis [41].
2-1C). Orthopoxviral Serpins Spi1, Spi2 and Spi3
Orthopoxviruses include smallpox, cowpox, vaccinia, horsepox, camelpox, and mon-
keypox (now called Mpox). There is extensive shared activity and shared gene sequences
between the cowpox virus and vaccinia virus genes referred to as CrmA, CrmB, and CrmC
or Spi1, Spi2, and Spi3. The terms SPI1, 2, and 3 were initialy coined to stand for Serine
Protease Inhibitors, but with ongoing research there has been some overlap in terminology
for the term Spi used to refer to mammalian cellular serpins. These cowpox and vaccinia
serpins are often referred to as interchangeable. Spi2 and CrmA share protease targets in the
Biomolecules 2023,13, 1393 16 of 23
apoptotic pathways while Serp-1 and Spi3 share targets in the thrombotic and thrombolytic
pathways [
]. There have been some interesting preclinical studies on these serpins;
however, work with these serpin classes as potential protein treatments and remains in the
early stages. These studies are described below.
2-1C)-1. Spi1 and Spi2
Cancer: Potential for Oncolytic Therapeutic
SPI-2 in combination with SPI-1 protects cells from alloreactive cytotoxic T lymphocyte
(CTL)-induced killing via perforin and death receptor-dependent pathways
in vitro
. Spi-2,
also termed CrmA, is a potent inhibitor of apoptosis [
]. Inhibitors of apoptosis can be
considered as potential therapeutics for cancer therapy. Through antagonizing inhibitors
of apoptosis, it is proposed immune cell responses to tumors can be improved. A similar
approach designed to reduce immune cell apoptosis has been investigated as an oral
apoptosis protein inhibitor in patients with advanced solid tumors [59].
Viral Infections
As noted, poxviral serpins can inhibit apoptosis during viral infection
in vitro
. Through
the inhibition of apoptosis, Spi1 has potential as an antiviral therapeutic for poxviral infec-
tions by prolonging the viral antigen presentation to host dendritic cells in the immune
response [
]. The inactivation of immunomodulating genes such as B13R (encoding Spi1)
or B22R (Spi2) has been proposed as an approach to enhance the safety of vaccinia virus
vaccines while maintaining a high level of immunogenicity in general vaccination [60].
2.1C)-2. Spi2/CrmA
CrmA is a member of the Ov-serpins clade, which lack typical, cleavable hydrophobic
signal sequences, resulting in inefficient (and often undetectable) translocation and secre-
tion. CrmA, similar to Serp-2, is a predominantly cytoplasmic serpin [Figure 2]. In the
mouse aortic allograft transplant studies, both Serp-2 and CrmA were assessed for efficacy
in reducing transplanted aortic inflammation. In this model, Serp-2 did significantly reduce
transplant vasculitis, inflammation, and plaque growth; however, CrmA was not effective
despite having greater
in vitro
inhibitory activity [
]. Thus, the question arises as to
whether other protease targets may be involved in the efficacy demonstrated for Serp-2 in
these transplant vascular models.
As noted above, Spi2, when transfected into cells, inhibits apoptosis through the
targeting of Granzyme B (GrzmB) and caspases [
]. CrmA targets the serine protease
GrzmB as well as the cysteine proteases caspases 1 and 8. SPI-2, in combination with
SPI-1, protects cells from alloreactive CTL-induced killing via perforin and death receptor-
dependent pathways
in vitro
. Serp-2 and CrmA thus bind and inhibit similar classes of
serine and cysteine proteases, specifically Granzyme B and caspases 1 and 8 [
]. Turner
and Moyer et al. examined the effects of swapping the RCL sequences of Serp-2 with CrmA.
However, CrmA sequences, inserted into the Serp-2 protein, did not retain the same level
of anti-apoptotic activity as seen with the wild type serpins [6062].
Sindbis virus is an arthropodal (insect) virus of genome alphavirus. Sindbis is trans-
mitted between vertebrate (bird) and invertebrate (mosquito) vectors. The virus itself
induces classical features of apoptosis.
In vivo
, treatment with CrmA serpin from the
cowpox virus inhibited sindbis virus-induced cell death (apoptosis) rather than impairing
viral replication, improving the survival in infected mice. This is an early study of the use
of a cowpox virus-derived inhibitor of caspase (CrmA) demonstrating improved outcomes
during infection by an unrelated Sindbis virus [63].
Apoptosis can alter neuroexcitability in neurodegeneration disorders.
In vitro
have demonstrated that CrmA treatment was able to rescue neurons, reducing toxicity
following a necrotic infection, even in conditions of low caspase activation and morpho-
logical apoptosis. CrmA apparently reduced the drop in mitochondrial potential and the
Biomolecules 2023,13, 1393 17 of 23
reduction in ATP. This work suggests a potential therapeutic application for the cowpox
virus-derived CrmA [49].
Viral Infections
Cytotoxic T lymphocytes (CTL) induce cell death after viral infection by the secretion
of perforin/granzymes and Fas cell surface antigen. Proteinase activity is important in
these CTL-mediated cell death pathways. SPI-2 inhibits the proteolytic activity of caspase
1 (Interleukin 1 beta, IL-1b, converting enzyme) and granzyme B. Cells infected with the
orthopoxviruses cowpox and rabbitpox are resistant to cytolysis via these mechanisms.
Mutation of the SPI-2 gene prevents virus inhibition of Fas-mediated cytolysis, allowing
cell death to proceed and reduce viral replication. The mutation of both SPI-2 and SPI-1 is
reported to completely reverse or block viral cytolysis.
The viral inhibition of perforin/granzyme-mediated cell killing was unaffected by
mutation of SPI-2, and perforin-/granzyme-mediated killing was reduced when SPI-1
genes were inactivated. Thus, SPI-1 and SPI-2 together inhibit both cytolysis pathways,
and also a common pathway enzyme, IL-1b convertase, which is associated with both
perforin/granzyme and Fas-cell surface killing [50].
Fulminant and Autoimmune Hepatitis
Fulminant hepatitis can occur with severe viral infections, as well with chemical
toxicity with a rapid progression of liver failure within days to weeks, causing massive liver
parenchymal necrosis. Fas-mediated cytolysis and perforin-/granzyme-mediated killing
have a role in this hyperimmune response in hepatitis. CrmA inhibits both Granzyme B
and caspase activation. The injection of anti-Fas antibody into mice leads to death due
to liver cell apoptosis. In mice with Adenovirus expression of higher levels of CrmA,
apoptosis and hepatitis were dramatically reduced, with increased survival. Additionally,
in vitro
analysis, active Caspase 3 was inhibited by the transfection of CrmA into mouse
hepatocytes. Thus, CrmA may block immune-mediated apoptosis, as seen in autoimmune
hepatitis [
]. Concanavalin-A (Con A)-induced hepatitis is a model for studying and
mimicking human autoimmune hepatitis, with a massive hepatocyte apoptosis and CD11b+
leukocyte infiltration. This liver damage was also dramatically reduced in mice that
expressed the CrmA gene, without effecting CD4+ cell responses. Mouse survival was also
increased to 100% when compared to the control group [50,51].
Detection of Resurgent Variola (Smallpox) Pandemic Infections
One recent analysis of smallpox gene expression has projected that the detection of
the expression of three smallpox genes may allow for the detection of Variola variants that
are predictive of risk for recurrent smallpox epidemics. One gene that appears to have
diagnostic and predictive potential is the CrmB serpin gene as expressed by variola [64].
Ischemia Reperfusion following Myocardial Infarction
Ischemia reperfusion injuries are also closely associated with apoptosis. To deliver
extracellular CrmA into eukaryotic cells, a TAT transduction domain from HIV was fused
onto the N-terminus of CrmA, allowing intracellular translocation. In this work, CrmA
inhibited the intrinsic apoptotic pathway, inhibiting Caspase-8, Caspase-9, and Caspase-3
in vitro [52].
In vivo
, 90% of mice survived a lethal dose of antibody to Fas when treated with
CrmA [
]. To examine the extrinsic pathway
in vivo
, mice were treated with a lethal dose
of doxorubicin in the control group, with another group receiving CrmA. A total of 40%
of those who received TAT-crmA via intraperitoneal injection survived, whereas those
without died within 31 days. TAT-crmA was also found to reduce myocardial infarct size by
40% in treated mice while preserving left ventricular systolic function. This study provides
preclinical evidence of the potential use of CrmA in treating ischemia reperfusion injury in
the setting of a myocardial infarction (MI) [
]. The temporal effects of CrmA were also
examined in Doxorubicin-induced apoptotic myocardial injury and cardiomyopathy. The
early, six-day survival was increased to 81% in CrmA-treated mice vs 38% in control mice.
Biomolecules 2023,13, 1393 18 of 23
This effect disappeared by day 12, with similar levels of apoptosis and survival having
been observed [53].
2.1C)-2-3. Spi3
2.1C)-2-3-1. Inflammation and Vascular Permeability
Spi3 is a cowpox viral protein. Spi3 complexes with and inhibits a variety of throm-
bolytics, including uPA, plasmin, and tPA, similarly to the Myxomaviral Serp-1 [Figure 2]
or the mammalian serpins PAI-1 and NSP. Through the inhibition of thrombolytic pro-
teases, Spi3 has potential for both research and the treatment of bleeding disorders such as
hemophilia and vascular inflammation and permeability, albeit not yet proven. Hemagglu-
tinin has been shown to retain Spi3 in the plasma membrane [54,55].
Both Spi3 (from cowpox virus) and Serp-1 (from myxomavirus) exhibit anti-inflammatory
activity, inhibiting uPA, plasmin, and tPA. Both have arginine at the P1 site in the P1P1
bond in the RCL. Both also have the capacity to complex with thrombin or factor Xa. However,
they are not functionally equivalent. Unexpectedly, when cowpox virus expresses Spi3 under
the myxomavirus Serp-1 promoter, there is greater Spi3 secretion; thus, either the cowpox virus
inhibits Spi3 through another mechanism, or myxoma enhances the secretion of Spi3 [65].
2-2). Herpesviral serpins
ORF1 (Open reading frame 1) in the murine gammaherpesvirus 68 (MHV68) encodes
for a protein that contains amino acid sequences which are similar in homology to the
poxvirus Serp-1 protein. The potential for therapeutic clinical applications of ORF1 has
yet to be determined; however, this does present an example of a serpin-like molecule
in Herpesviruses [
]. Herpes simplex virus-1 (HSV-1) DNA is immunostimulatory both
in vivo
in vitro
, promoting T helper (Th1) cell responses [
]. Given the immunomod-
ulatory action of many serpins, it is reasonable to predict that other viral serpins will be
discovered with the capacity to combat antiviral immune responses. As has already been
studied, the Myxomaviral Serp-1 protein improved survival and reduced lung and vascular
inflammation in MHV68 mouse gamma herpesviral infection in interferon gamma receptor-
deficient mouse models. These new herpes virus-derived serpins may also provide a new
source of serpins with the potential for therapeutic approaches for diseases with chronic
inflammatory responses, and perhaps keratitis.
2-3). Plant Viral Serpins
Plant viruses are believed to be safe for administration in humans, being from differ-
ent kingdoms. Plant viruses are used to safely present epitopes to create novel vaccines.
Recently, several plant viruses have demonstrated immunogenicity, inducing IgG titers in
mice. Tobacco Mosaic Virus (TMV) and spherical particles from TMV induced immuno-
genicity with low self IgG titers. Thus, viruses from plants can be investigated as a potential
safe adjuvant for novel vaccines. Furthermore, this study demonstrates that plant viruses
have an immunogenic effect [68].
One such immunogenic effect may involve immunomodulating serpins. Several plant
serpins have been identified following viral infection. Their function remains obscure. Rice-
stripe virus is one of the most destructive viruses of rice, and its microarray expression has
identified a Serpin named Serpin-5, a possible pathogen resistance protein. Its biological
significance remains unknown [69].
2-4). Arthropod Serpins and Athropod-Viral Serpins—Baculovirus Serpins
2.2. Nucleopolyhedroviral Serpins
Baculoviruses are large DNA viruses that predominantly infect Lepidoptera (moths
and butterflies). The Hesp018 protein is a functional cross-class serpin with inhibitory
activity against serine and cysteine proteinases. Hesp018 is the first viral serpin homologue
to be characterized outside of the chordopoxviruses and is encoded as a baculovirus
gene [
]. Serpin 4 from baculovirus inhibits PPO activation and melanization in the
Biomolecules 2023,13, 1393 19 of 23
Asian Corn Borer, Ostrinia furnacalis (Guenee), forming covalent complexes with serine
proteases. The baculoviral Serpin 4, again, demonstrates the conservation of a Serpin
suicide-inhibitory mechanism of action [70].
Baculovirus is a virus which commonly attacks the insect H. armigera. Melaniza-
tion is an insect defense mechanism that is regulated by serpins. Proteolytic activation
of prophenoloxidase (PPO), as for the RSV (Rice-stripe virus), kills baculovirus. In H.
armigera, baculovirus downregulates the protein levels in the PPO cascade and innate
immune responses, but upregulates serpin5 and serpin9 expression in the hemolymph of
insects. This is similar to the effects of the RSV on host-insect serpin pathways, a secondary
effect of viruses on the insect expression of serpins. The inhibition of these serpins increases
baculoviral infection. This suggests that the insect virus baculovirus has evolved a mecha-
nism to combat the insect host immune response to the virus, again illustrating that serpins
are important for viral immunomodulation in the host [
]. This response represents a
virus-induced alteration in the host serpin response where the virus uses the host serpins
as part of a defense mechanism.
2.3. Rice Stripe Virus Serpins (RSV)
As noted, a serpin sequence has been identified in the Rice-stripe virus, RSV. Further-
more, viruses are reported to hijack host-insect serpins. Rice gall dwarf virus hijacks the
sperm protein HongrES1 in order to facilitate its spread, helping another virus to spread
in the host, the symbiotic virus Recilia dorsalis filamentous virus. The activation of this
serpin also facilitates decreased melanization and allows arbovirus host to spread. Thus,
different viruses have been shown to act cooperatively through different kingdoms to help
each other spread via these highly conserved serpin pathways [69].
RSV, a highly pathogenic virus for rice plants, is transmitted to the rice plant in a small
planthopper insect. In one study, the transcription of seven serpins, termed Lsserpin1-7,
has been detected in the insect. Phenoloxidase (PO) activity is activated by serine proteases
in the insect vector and is one of the immune responses that are mounted by the host
insect against viral infections. PPO activity is suppressed by the upregulation of these
insect serpins during RSV infection, and the suppression of PPO can be up to 60% after
upregulation (increased expression) of these serpins. The knock out of several of these
serpins resulted in dramatically increased PO inhibitor activity, increasing innate immunity
in the insect hemolymph [
]. Thus, RSV can induce the increased expression of serpins
in the plant hopper insect, representing another virus-mediated activity that is related to
altered insect host serpin expression.
3. Modifying Viral Serpins—New Therapeutic Constructs
Of great interest, recent work has illustrated the capacity for mammalian serpins
to be modified such that these serpins can provide the selective or targeted blockade of
coagulation pathways. The RCL of a human variant of A1AT, called A1AT Pittsburg,
has been mutated to a KRK sequence, converting it into a specific inhibitor of activated
protein C, termed SerpinPC. The results of a Phase 2 clinical trial presented at a hematology
congress demonstrated that SerpinPC was well-tolerated and reduced bleeding in persons
with severe haemophilia [
]. These serpins are in development as new approaches to
reduce the excess activation of serpin pathways that can increase bleeding in hemophilia.
Thus, viral serpins, as for mammalian serpins, can be modified to alter protease target
specificity. The MyxV Serp-1 gene sequence has been mutated in prior work wherein Serp-1
anti-inflammatory activity was either lost or led to excess inflammation with aneurysm
development [
]. Several RCL peptides derived from the Serp-1 RCL sequence have
also been developed and tested in mouse models of vasculitis [
]. While wild type (WT)
Serp-1 lost efficacy in a MHV68 model of vascular disease and lung hemorrhage after
antibiotic suppression of the gut microbiome, one such Serp-1 RCL peptide retained anti-
inflammatory activity in this model [
]. The modulation of pathways targeted by these
RCL mutants of Serp-1 and/or the RCL peptides may provide interesting approaches to
Biomolecules 2023,13, 1393 20 of 23
understanding protease pathways, as well as interaction with the microbiota that are central
to disease development. Serpins may thus provide a mechanism utilized by viruses to alter
the gut microbiome and their effects on host immune responses.
4. Conclusions
Given the widespread distribution of serpins in biology, reflecting the adaptability
of the serpin protein structure and mechanisms of inhibition to different physiological
contexts, there is great interest in their potential therapeutic benefit when used as drugs.
Viral serpins have been shown to modulate the immune response with a therapeutic benefit
in over 30 different diseases of inflammation with minimal if any adverse effects. This
review highlights the known viral serpins and disease models that have been studied
to date. Given what is presently known about serpins and their widespread studied
therapeutic benefits, it is reasonable to continue translational and clinical research on serpin
therapy on these disease states. Given the extensive variety of serine proteases that are
inhibited by viral serpins, dedicated modifications of these serpins that are designed to
target specific target proteases may provide new targeted treatment approaches, similar to
those that have recently been developed for modified serpin treatment in hemophilia [
One might also consider viruses as a rich source of immune-modulating therapeutics for
many other classes of proteins that target different immune pathways. In addition to
serpins, chemokine modulators, growth factors, and viral cytokines such as vIL-10 have
been studied as virus-derived immune-coagulopathic and apoptosis-modulating biologics,
representing a new class of virus-derived therapeutics [43,58,7274].
The funding provided was for the basic research in the referenced studies. To A.R.L.—NIH
We would like to thank the following scientists for their original contributions
to the viral serpin fields, their ongoing collaborative work in this field and their work in proofing this
review—Liqiang Zhang and Jordan R. Yaron at the Biodesign Institute, Arizona State University 727
E Tyler Street Tempe, AZ, Liying Liu and Erbin Dai at Beth Israel Deaconess Medical Center, Harvard,
330 Brookline Avenue, Boston, MA 02215, and Hao Chen at Department of Hepatobiliary Surgery,
Chinese People’s Liberation Army General Hospital, Haidian, Beijing, China.
Conflicts of Interest:
Dr. Lucas is the founding scientist for Serpass Biologics, however this is a new
Biotech startup and has not provided funding for the research reported here.
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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.
Full-text available
Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies.
Full-text available
Hereditary angioedema with C1 Inhibitor deficiency (C1-INH-HAE) is caused by a constellation of variants of the SERPING1 gene (n = 809; 1,494 pedigrees), accounting for 86.8% of HAE families, showing a pronounced mutagenic liability of SERPING1 and pertaining to 5.6% de novo variants. C1-INH is the major control serpin of the kallikrein-kinin system (KKS). In addition, C1-INH controls complement C1 and plasminogen activation, both systems contributing to inflammation. Recognizing the failed control of C1s protease or KKS provides the diagnosis of C1-INH-HAE. SERPING1 variants usually behave in an autosomal-dominant character with an incomplete penetrance and a low prevalence. A great majority of variants (809/893; 90.5%) that were introduced into online database have been considered as pathogenic/likely pathogenic. Haploinsufficiency is a common feature in C1-INH-HAE where a dominant-negative variant product impacts the wild-type allele and renders it inactive. Small (36.2%) and large (8.3%) deletions/duplications are common, with exon 4 as the most affected one. Point substitutions with missense variants (32.2%) are of interest for the serpin structure-function relationship. Canonical splice sites can be affected by variants within introns and exons also (14.3%). For noncanonical sequences, exon skipping has been confirmed by splicing analyses of patients' blood-derived RNAs (n = 25). Exonic variants (n = 6) can affect exon splicing. Rare deep-intron variants (n = 6), putatively acting as pseudo-exon activating mutations, have been characterized as pathogenic. Some variants have been characterized as benign/likely benign/of uncertain significance (n = 74). This category includes some homozygous (n = 10) or compound heterozygous variants (n = 11). They are presenting with minor allele frequency (MAF) below 0.00002 (i.e., lower than C1-INH-HAE frequency), and may be quantitatively unable to cause haploinsufficiency. Rare Drouet et al. SERPING1 Variants and C-INH Expression benign variants could contribute as disease modifiers. Gonadal mosaicism in C1-INH-HAE is rare and must be distinguished from a de novo variant. Situations with paternal or maternal disomy have been recorded (n = 3). Genotypes must be interpreted with biological investigation fitting with C1-INH expression and typing. Any SERPING1 variant reminiscent of the dysfunctional phenotype of serpin with multimerization or latency should be identified as serpinopathy.
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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.
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Serine proteases drive important physiological processes such as coagulation, fibrinolysis, inflammation and angiogenesis. These proteases are controlled by serine protease inhibitors (SERPINs) that neutralize their activity. Currently, over 1,500 SERPINs are known in nature, but only 37 SERPINs are found in humans. Thirty of these are functional protease inhibitors. The inhibitory potential of SERPINs is in perfect balance with the proteolytic activities of its targets to enable physiological protease activity. Hence, SERPIN deficiency (either qualitative or quantitative) can lead to disease. Several SERPIN resupplementation strategies have been developed to treat SERPIN deficiencies, including concentrates derived from plasma and recombinant SERPINs. SERPINs usually inhibit multiple proteases, but only in their active state. Over the past decades, considerable insights have been acquired in the identification of SERPIN biological functions, their inhibitory mechanisms and specificity determinants. This paves the way for the development of therapeutic SERPINs. Through rational design, the inhibitory properties (selectivity and inhibitory potential) of SERPINs can be reformed and optimized. This review explores the current state of SERPIN engineering with a focus on reactive center loop modifications and backbone stabilization. We will discuss the lessons learned from these recombinant SERPINs and explore novel techniques and strategies that will be essential for the creation and application of the future generation of therapeutic SERPINs.