Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

The interplay between serine proteases and caspase-1 regulates the autophagy-mediated secretion of Interleukin-1 beta in human neutrophils.

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti.

BACKGROUND: Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies-CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE-based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. METHODS: Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. RESULTS: We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. CONCLUSIONS: Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity.

Staphylococcus aureus Depends on Eap Proteins for Preventing Degradation of Its Phenol-Soluble Modulin Toxins by Neutrophil Serine Proteases.

Neutrophil granulocytes act as a first line of defense against pathogenic staphylococci. However, Staphylococcus aureus has a remarkable capacity to survive neutrophil killing, which distinguishes it from the less-pathogenic Staphylococcus epidermidis. Both species release phenol-soluble modulin (PSM) toxins, which activate the neutrophil formyl-peptide receptor 2 (FPR2) to promote neutrophil influx and phagocytosis, and which disrupt neutrophils or their phagosomal membranes at high concentrations. We show here that the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin G and proteinase 3, which are released into the extracellular space or the phagosome upon neutrophil FPR2 stimulation, effectively degrade PSMs thereby preventing their capacity to activate and destroy neutrophils. Notably, S. aureus, but not S. epidermidis, secretes potent NSP-inhibitory proteins, Eap, EapH1, EapH2, which prevented the degradation of PSMs by NSPs. Accordingly, a S. aureus mutant lacking all three NSP inhibitory proteins was less effective in activating and destroying neutrophils and it survived less well in the presence of neutrophils than the parental strain. We show that Eap proteins promote pathology via PSM-mediated FPR2 activation since murine intraperitoneal infection with the S. aureus parental but not with the NSP inhibitors mutant strain, led to a significantly higher bacterial load in the peritoneum and kidneys of mFpr2-/- compared to wild-type mice. These data demonstrate that NSPs can very effectively detoxify some of the most potent staphylococcal toxins and that the prominent human pathogen S. aureus has developed efficient inhibitors to preserve PSM functions. Preventing PSM degradation during infection represents an important survival strategy to ensure FPR2 activation.

Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis.

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40-60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.

The immune protection induced by a serine protease from the Trichinella spiralis adult against Trichinella spiralis infection in pigs.

Trichinellosis is a major foodborne parasitosis caused by Trichinella spiralis. In the present study, a serine protease gene from an adult T. spiralis (Ts-Adsp) cDNA library was cloned, expressed in Escherichia coli and purified by Ni-affinity chromatography. Previous studies of our laboratory have found that mice vaccinated with recombinant Ts-Adsp protein (rTs-Adsp) exhibited partial protection against T. spiralis infection. In this study, the protective effect of rTs-Adsp against T. spiralis infection in pigs was further explored. The cell-mediated and humoral immune responses induced by rTs-Adsp were measured, including the dynamic trends of specific antibody levels (IgG, IgG1, IgG2a and IgM), as well as the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) in the serum. Moreover, the changes in T lymphocytes, B lymphocytes, and neutrophils were measured to evaluate cellular immune responses in pigs vaccinated with rTs-Adsp. The results indicated that a Th1-Th2 mixed immune response with Th1 predominant was induced by rTs-Adsp after vaccination. Flow cytometric analysis showed that the proportions of CD4+ T cells, B cells, and neutrophils in the immunized groups were significantly increased. Furthermore, pigs vaccinated with rTs-Adsp exhibited a 50.9% reduction in the muscle larvae burden, compare with pigs from the PBS group five weeks after challenged. Our results suggested that rTs-Adsp elicited partial protection and it could be a potential target molecule for preventing and controlling Trichinella transmission from pigs to human.

Immune Protection of a Helminth Protein in the DSS-Induced Colitis Model in Mice.

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer, and it has the potential to diminish the quality of life. Recent clinical and experimental evidence demonstrate protective aspects of parasitic helminth infection against IBD. Reports have highlighted the potential use of helminths and their byproducts as potential treatment for IBD. In the current study, we studied the effect of a newborn larvae-specific serine protease from Trichinella spiralis (TsSp) on the host immune and inflammatory responses. A 49-kDa recombinant TsSp (rTsSp) was expressed in Escherichia coli BL21 (DE3) and purified. The cytotoxicity of rTsSp was analyzed. The immune protective effect of rTsSp was studied by using dextran sodium sulfate (DSS)-induced mouse colitis model. The result illustrated that rTsSp has no toxic effects on cells. We further demonstrated that administration of the rTsSp without the additional adjuvant before the induction of DSS-induced colitis reduced the severity of intestinal inflammation and the disease index; it suppressed macrophage infiltration, reduced TNF-α secretion, and induced IL-10 expression. Our findings suggest therapeutic potential of rTsSp on colitis by altering the effect of macrophages. Data also suggest immunotherapy with rTsSp holds promise for use as an additional strategy to positively modulate inflammatory processes involved in IBD.

Allergic Reactions to Serine Protease-Like Proteins of Staphylococcus aureus.

In cystic fibrosis (CF) infectious and allergic airway inflammation cause pulmonary exacerbations that destroy the lungs. Staphylococcus aureus is a common long-term colonizer and cause of recurrent airway infections in CF. The pathogen is also associated with respiratory allergy; especially the staphylococcal serine protease-like proteins (Spls) can induce type 2 immune responses in humans and mice. We measured the serum IgE levels specific to 7 proteases of S. aureus by ELISA, targeting 5 Spls (76 CF patients and 46 controls) and the staphopains A and B (16 CF patients and 46 controls). Then we compared cytokine release and phenotype of T cells that had been stimulated with Spls between 5 CF patients and 5 controls. CF patients had strongly increased serum IgE binding to all Spls but not to the staphopains. Compared to healthy controls, their Spl-stimulated T cells released more type 2 cytokines (IL-4, IL-5, IL-13) and more IL-6 with no difference in the secretion of type 1- or type 3 cytokines (IFNγ, IL-17A, IL-17F). IL-10 production was low in CF T cells. The phenotype of the Spl-exposed T cells shifted towards a Th2 or Th17 profile in CF but to a Th1 profile in controls. Sensitization to S. aureus Spls is common in CF. This discovery could explain episodes of allergic inflammation of hitherto unknown causation in CF and extend the diagnostic and therapeutic portfolio.

Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3.

Neisseria meningitidis (meningococcus) is a common bacterial colonizer of the human nasopharynx but can occasionally cause very severe systemic infections with rapid onset. Meningococci are able to degrade IgA encountered during colonization of mucosal membranes using their IgA1-specific serine protease. During systemic infection, specific IgG can induce complement-mediated lysis of the bacterium. However, meningococcal immune evasion mechanisms in thwarting IgG remain undescribed. In this study, we report for the first time that the meningococcal IgA1-specific serine protease is able to degrade IgG3 in addition to IgA. The IgG3 heavy chain is specifically cleaved in the lower hinge region thereby separating the antigen binding part from its effector binding part. Through molecular characterization, we demonstrate that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Epidemiological analysis of 7581 clinical meningococcal isolates shows a significant higher proportion of cleavage type 1 among isolates from invasive cases compared to carrier cases, regardless of serogroup. Notably, serogroup W cc11 which is an increasing cause of invasive meningococcal disease globally harbors almost exclusively cleavage type 1 protease. Our study also shows an increasing prevalence of meningococcal isolates encoding IgA1P cleavage type 1 compared to cleavage type 2 during the observed decade (2010-2019). Altogether, our work describes a novel mechanism of IgG3 degradation by meningococci and its association to invasive meningococcal disease.

Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Molecular characterization of a Trichinella spiralis serine proteinase.

The aim of this study was to investigate the biological characteristics and functions of a Trichinella spiralis serine proteinase (TsSerp) during larval invasion and development in the host. The full-length TsSerp cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and western blotting analyses showed that TsSerp was a secretory protein that was highly expressed at the T. spiralis intestinal infective larva and muscle larva stages and primarily located at the cuticle, stichosome and intrauterine embryos of the parasite. rTsSerp promoted the larval invasion of intestinal epithelial cells (IECs) and the enteric mucosa, whereas an anti-rTsSerp antibody impeded larval invasion; the promotion and obstruction roles were dose-dependently related to rTsSerp and the anti-rTsSerp antibodies, respectively. Vaccination of mice with rTsSerp elicited a remarkable humoral immune response (high levels of serum IgG, IgG1/IgG2a, IgE and IgM), and it also triggered both systemic (spleen) and local intestinal mucosal mesenteric lymph node (MLN) cellular immune responses, as demonstrated by a significant elevation in Th1 cytokines (IFN-γ) and Th2 cytokines (IL-4) after the spleen and MLN cells from vaccinated mice were stimulated with rTsSerp. Anti-TsSerp antibodies participated in the killing and destruction of newborn larvae via ADCC. The mice vaccinated with rTsSerp exhibited a 48.7% reduction in intestinal adult worms and a 52.5% reduction in muscle larvae. These results indicated that TsSerp participates in T. spiralis invasion and development in the host and might be considered a potential candidate target antigen to develop oral polyvalent preventive vaccines against Trichinella infection.

Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta.

Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR176*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH112*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.

The immune protection induced by a serine protease from the Trichinella spiralis adult administered as DNA and protein vaccine.

Trichinellosis is caused by Trichinella spiralis (T. spiralis), which is an important public health problem. In this study, a gene encoding a serine protease from adult worms of T. spiralis (Ts-Adsp) was screened from a cDNA library of adult worms and was cloned and expressed in a prokaryotic expression system. The gene Ts-Adsp was subcloned into the eukaryotic expression vector pcDNA3.1(+), which was named pcDNA3.1(+)-Adsp. Previous studies have found that recombinant Ts-Adsp protein (rTs-Adsp) can elicit partial protection against T. spiralis infection in mice. Our aim was to explore the protective effect of combining a DNA vaccine with the rTs-Adsp protein against T. spiralis. One week after the last vaccination, the serum and spleen were obtained. The levels of IgG, IgG1 and IgG2a and cytokine production in serum and spleen cells were analyzed. The results showed that the levels of humoral and cell-mediated immune responses increased in the pcDNA3.1(+)-Adsp/rTs-Adsp group mice and demonstrated that a Th1/Th2 mixed immune response was induced by pcDNA3.1(+)-Adsp/rTs-Adsp after vaccination. Moreover, mice vaccinated with pcDNA3.1(+)-Adsp/rTs-Adsp displayed a 69.50% reduction in muscle larvae burden. This study suggested that mixed immunity could improve the muscle larvae reduction rate.

An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection.

A novel serine protease contains two ShK-domain was found from the Chinese mitten crab Eriocheir sinensis (EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed that EsShK-SP was expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasma eriocheiris, the expression of EsShK-SP was significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC and S. eriocheiris stimulated the hemocytes of E. sinensis, the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number of S. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number of S. eriocheiris in the EsShK-SP silenced group was significantly increased compared to the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role during E. sinensis against S. eriocheiris through regulation of the proPO system.

In silico design of a vaccine candidate based on autotransporters and HSP against the causal agent of shigellosis, Shigella flexneri.

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.

NETosis occurs independently of neutrophil serine proteases.

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.

Immunogenic Properties of Recombinant Enzymes from Bothrops Ammodytoides Towards the Generation of Neutralizing Antibodies against Its Own Venom.

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.

The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs.

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network.

Functional selection of protease inhibitory antibodies.

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli-an antibody clone, a protease of interest, and a ß-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified ß-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), ß-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aß40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

Intranasal immunization with recombinant Trichinella spiralis serine protease elicits protective immunity in BALB/c mice.

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection.