Immune reactivity and host modulatory roles of two novel Haemonchus contortus cathepsin B-like proteases.

BACKGROUND: Haemonchus contortus is a blood-feeding, gastrointestinal nematode (GIN) that causes significant economic losses to the small ruminant industry worldwide. Despite extensive efforts, our understanding of the molecular mechanisms used by GIN to evade host immune responses is limited. Cathepsin B-like proteins (CBPs) are members of the cysteine protease family and are involved in parasite invasion and thus provide viable vaccine candidates. METHODS: In silico comparative analysis was used to identify conserved proteins among a subset of clade V parasitic nematodes with emphasis on blood-feeding worms, among which CBPs appeared prominently. We identified and characterized two novel CBPs designated Hc-CBP-1 and Hc-CBP-2. Rabbit anti-recombinant (r) Hc-CBP-1 and rHc-CBP-2 were used to detect the presence of native proteins in the excretory secretory products (ESP) and in worm tissues of adult H. contortus. Peptide arrays of rHc-CBP-1 and rHc-CBP-2 were screened with the homologous and heterologous anti-sera and with sera from dexamethasone-treated (Dex+) and non-treated (Dex-) H. contortus-infected animals to identify key immunogenic peptides. Gene transcription of Hc-cbp-1 and Hc-cbp-2 was also performed on H. contortus-infected animals treated with Dex+. Finally, the mature recombinant proteins were used to assess their abilities to modulate cell functions. RESULTS: Immunohistochemistry showed that both Hc-CBP-1 and Hc-CBP-2 are present on the brush borders of the intestine; Hc-CBP-2 was also present in the hypodermis of the body wall. Peptide displays screened with rabbit anti-rHc-CBP-1 and anti-rHc-CBP-2 revealed regions within the proteins where dominant and overlapping epitopes prevailed. ELISA results were consistent with only Hc-CBP-1 being present in H. contortus adult ESPs. H. contortus from Dex+ animals exhibited a threefold increase in Hc-cbp-2 transcript while Hc-cbp-1 expression did not change. In contrast, comparisons of immunoreactivities of rHc-CBP-1 and rHc-CBP-2 peptide arrays to sera from Dex+ and Dex- animals primarily showed changes in Hc-CBP-1 binding. Lastly, rHc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers, including TNFα, IL-1, IL-6 and CD86. CONCLUSIONS: These results suggest that as secreted and cryptic proteins, respectively, Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities that have interesting dynamics during infection and may provide viable immune-related targets for attenuating H. contortus infectivity.

Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Recombinant interferon-gamma promotes immunoglobulin G and cytokine memory responses to cathepsin L-like cysteine proteinase of Hyalomma asiaticum and the efficacy of anti-tick.

Among bloodsucking arthropods, hard tick is a vector of transmitting the most diverse human and animal pathogens, leading to an increasing number of manifestations worldwide. The development of the anti-tick vaccine has the potential to be an environmentally friendly and cost-effective option for tick management. We have previously demonstrated the induction of both humoral and cellular response against Hyalomma asiaticum (H. asiaticum) following immunization with recombinant cathepsin L-like cysteine protease from H. asiaticum tick (rHasCPL), and could control tick infestations. Interferon-gamma (IFN-γ), is an immunomodulatory factor that plays an important role in the regulation of adaptive immunity against infection. In the present study, recombinant BALB/c mouse IFN-γ (rMus-IFN-γ) was cloned and expressed using a prokaryotic expression system, and verified by Western blotting and IFN-γ-ELISA kit analysis. Female BALB/c mice (n = 12) were used for immunization using rHasCPL (100 µg) plus IFN-γ as adjuvant (10 µg). In immunized female BALB/c mice, the levels of anti-CPL antibodies as well as cytokines were determined using ELISA analysis. Protective efficacy of immunization was evaluated by larvae H. asiaticum challenge of immunized female BALB/c mice. Using rMus-IFN-γ as an adjuvant to rHasCPL vaccine (CPL + IFN-γ) promoted specific antibody IgG (IgG1 > IgG2a) and increased production of IFN-γ and IL-4 compared to immune rHasCPL group (CPL). The protected rate of immunized mice from tick challenge was significantly higher after immunization with CPL + IFN-γ (85.11 %) than with CPL (63.28 %). Immunization using CPL + IFN-γ promoted the activation of anti-HasCPL humoral and cellular immune responses, and could provide better protection against H. asiaticum infestation. This approach may could help develop a candidate vaccine for control tick infestations.

Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens.

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

TITLE: Caractérisation d'une cystéine protéase des poux rouges de la volaille et son utilisation potentielle comme vaccin pour les poulets. ABSTRACT: Les acariens communément appelés poux rouges de la volaille (PRV, Dermanyssus gallinae) sont des ectoparasites qui affectent négativement les poulets d'élevage, entraînant de graves pertes économiques au niveau mondial. Des acaricides ont été utilisés pour contrôler les PRV dans les poulaillers. Cependant, certains PRV ont développé une résistance aux acaricides, et par conséquent, différentes approches sont nécessaires pour gérer les problèmes qu'ils causent. La vaccination des poulets est l'une des méthodes envisagées pour réduire le nombre de PRV dans les poulaillers. Dans une étude précédente, une cystéine protéase, Deg-CPR-1, a été identifiée comme un vaccin candidat contre les PRV distribués en Europe. Dans cette étude, nous avons étudié les caractéristiques de Deg-CPR-1. L'analyse phylogénétique a révélé que Deg-CPR-1 est étroitement liée aux cystéine protéases digestives d'autres espèces d'acariens, et elle a été classée dans un groupe différent de celui des cathepsines de poulet. La Deg-CPR-1 des PRV au Japon a une substitution d'acide aminé par rapport à celle des PRV en Europe, mais elle a montré une efficacité en tant que vaccin, conformément aux résultats précédents. Deg-CPR-1 a présenté une activité enzymatique de type cathepsine L. De plus, l'ARNm de Deg-CPR-1 était exprimé dans l'intestin moyen et à tous les stades où les PRV se nourrissent de sang. Ces résultats impliquent que Deg-CPR-1 dans l'intestin moyen peut avoir des fonctions importantes dans les processus physiologiques, et que l'inhibition de son expression peut contribuer à l'efficacité d'un vaccin basé sur Deg-CPR-1. Des recherches supplémentaires sont nécessaires pour comprendre pleinement les mécanismes de l'efficacité du vaccin.

A hemocyte-specific cathepsin L-like cysteine protease is involved in response to 20-hydroxyecdysone and microbial pathogens stimulation in silkworm, Bombyx mori.

Cathepsin L protease belongs to the papain-like cysteine proteases family, plays indispensable roles in animals' pathological and physiological processes. However, little is known about Cathepsin L in silkworm, Bombyx mori. Herein, a novel Cathepsin L-like (Cat L-like) was cloned and identified from silkworm by the rapid amplification of cDNA ends (RACE). Cat L-like contains an intact open reading frame (ORF) of 1 668 bp and encodes 556 amino acid residues, consisting of a signal peptide, typical cathepsins' inhibitor_I29, and pept_C1 domain. Cat L-like is specifically and highly expressed in hemocytes. The cathepsin (including Cathepsin L, B, and H) crude extract from hemocytes had typical substrate specific catalytic activities and were sensitive to pH and temperature. Cat L-like up-regulated considerably after 20-hydroxyecdysone (20-E) administration, indicating that Cat L-like may be regulated by insect hormone. The responses of Cat L-like against bacterial infection suggest it may play essential roles in silkworm immunity. Overall, our studies provide a theoretical basis and insights to further investigate the functions of Cat L-like and in insects' innate immunity mechanisms.

Arachidonic Acid Is a Safe and Efficacious Schistosomicide, and an Endoschistosomicide in Natural and Experimental Infections, and Cysteine Peptidase Vaccinated Hosts.

Blood flukes of the genus Schistosoma are covered by a protective heptalaminated, double lipid bilayer surface membrane. Large amounts of sphingomyelin (SM) in the outer leaflet form with surrounding water molecules a tight hydrogen bond barrier, which allows entry of nutrients and prevents access of host immune effectors. Excessive hydrolysis of SM to phosphoryl choline and ceramide via activation of the parasite tegument-associated neutral sphingomyelinase (nSMase) with the polyunsaturated fatty acid, arachidonic acid (ARA) leads to parasite death, via allowing exposure of apical membrane antigens to antibody-dependent cell-mediated cytotoxicity (ADCC), and accumulation of the pro-apoptotic ceramide. Surface membrane nSMase represents, thus, a worm Achilles heel, and ARA a valid schistosomicide. Several experiments conducted in vitro using larval, juvenile, and adult Schistosoma mansoni and Schistosoma haematobium documented ARA schistosomicidal potential. Arachidonic acid schistosomicidal action was shown to be safe and efficacious in mice and hamsters infected with S. mansoni and S. haematobium, respectively, and in children with light S. mansoni infection. A combination of praziquantel and ARA led to outstanding cure rates in children with heavy S. mansoni infection. Additionally, ample evidence was obtained for the powerful ARA ovocidal potential in vivo and in vitro against S. mansoni and S. haematobium liver and intestine eggs. Studies documented ARA as an endogenous schistosomicide in the final mammalian and intermediate snail hosts, and in mice and hamsters, immunized with the cysteine peptidase-based vaccine. These findings together support our advocating the nutrient ARA as the safe and efficacious schistosomicide of the future.

Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design.

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.

Transcriptome Analysis Identifies Immune Markers Related to Visceral Leishmaniasis Establishment in the Experimental Model of BALB/c Mice.

Visceral leishmaniasis (VL) caused by Leishmania donovani and L. infantum is a potentially fatal disease. To date there are no registered vaccines for disease prevention despite the fact that several vaccines are in preclinical development. Thus, new strategies are needed to improve vaccine efficacy based on a better understanding of the mechanisms mediating protective immunity and mechanisms of host immune responses subversion by immunopathogenic components of Leishmania. We found that mice vaccinated with CPA162-189-loaded p8-PLGA nanoparticles, an experimental nanovaccine, induced the differentiation of antigen-specific CD8+ T cells in spleen compared to control mice, characterized by increased dynamics of proliferation and high amounts of IFN-γ production after ex vivo re-stimulation with CPA162-189 antigen. Vaccination with CPA162-189-loaded p8-PLGA nanoparticles resulted in about 80% lower parasite load in spleen and liver at 4 weeks after challenge with L. infantum promastigotes as compared to control mice. However, 16 weeks after infection the parasite load in spleen was comparable in both mouse groups. Decreased protection levels in vaccinated mice were followed by up-regulation of the anti-inflammatory IL-10 production although at lower levels in comparison to control mice. Microarray analysis in spleen tissue at 4 weeks post challenge revealed different immune-related profiles among the two groups. Specifically, vaccinated mice were characterized by similar profile to naïve mice. On the other hand, the transcriptome of the non-vaccinated mice was dominated by increased expression of genes related to interferon type I, granulocyte chemotaxis, and immune cells suppression. This profile was significantly enriched at 16 weeks post challenge, a time-point which is relative to disease establishment, and was common for both groups, further suggesting that type I signaling and granulocyte influx has a significant role in disease establishment, pathogenesis and eventually in decreased vaccine efficacy for stimulating long-term protection. Overall, we put a spotlight on host immune networks during active VL as potential targets to improve and design more effective vaccines against disease.

A highly expressed intestinal cysteine protease of Ancylostoma ceylanicum protects vaccinated hamsters from hookworm infection.

BACKGROUND: Human hookworms (Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum) are intestinal blood-feeding parasites that infect ~500 million people worldwide and are among the leading causes of iron-deficiency anemia in the developing world. Drugs are useful against hookworm infections, but hookworms rapidly reinfect people, and the parasites can develop drug resistance. Therefore, having a hookworm vaccine would be of tremendous benefit. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the vaccine efficacy in outbred Syrian hamsters of three A. ceylanicum hookworm antigen candidates from two classes of proteins previously identified as promising vaccine candidates. These include two intestinally-enriched, putatively secreted cathepsin B cysteine proteases (AceyCP1, AceyCPL) and one small Kunitz-type protease inhibitor (AceySKPI3). Recombinant proteins were produced in Pichia pastoris, and adsorbed to Alhydrogel. Recombinant AceyCPL (rAceyCPL)/Alhydrogel and rAceySKPI3/Alhydrogel induced high serum immunoglobulin G (IgG) titers in 8/8 vaccinates, but were not protective. rAceyCP1/Alhydrogel induced intermediate serum IgG titers in ~60% of vaccinates in two different trials. rAceyCP1 serum IgG responders had highly significantly decreased hookworm burdens, fecal egg counts and clinical pathology compared to Alhydrogel controls and nonresponders. Protection was highly correlated with rAceyCP1 serum IgG titer. Antisera from rAceyCP1 serum IgG responders, but not nonresponders or rAceyCPL/Alhydrogel vaccinates, significantly reduced adult A. ceylanicum motility in vitro. Furthermore, rAceyCP1 serum IgG responders had canonical Th2-specific recall responses (IL4, IL5, IL13) in splenocytes stimulated ex vivo. CONCLUSIONS/SIGNIFICANCE: These findings indicate that rAceyCP1 is a promising vaccine candidate and validates a genomic/transcriptomic approach to human hookworm vaccine discovery.

Protective efficacy of liver fluke DNA vaccines: A systematic review and meta-analysis: Guiding novel vaccine development.

The immunogenicity and efficacy of Fasciola DNA vaccines have not yet been comprehensively summarised in the form of a systematic review and meta-analysis. Though multiple vaccine studies with respect to Fasciola vaccines exist, the variance in the experimental parameters has made comparison difficult. We conducted a bibliographic database search in Scopus, PubMed, Science Direct, Cochrane Library, EMBASE and Web of Science databases, limited to publications from 1998 to 2017. The key words: Liver fluke, Fasciola hepatica, Fasciola gigantica, DNA vaccination, and immunogenicity were used in combination to form search strings. A total of 4760 studies were identified after initial screening, of which 14 qualified for systematic review and 7 for meta-analysis. The mean Odds Ratio (OR) for all studies was 0.565 (95% confidence interval (CI) of 0.293 to 1.087), which means the percentage of protection in terms of decreased fluke burden in animals vaccinated with DNA vaccines was 43.5%. A moderate protective efficacy was observed for cysteine protease and phosphoglycerate kinase vaccine antigen candidates (pooled OR and 95% CI, [0.542; 0.179-1.721] and [0.616; 0.219-1.735], respectively). Vaccine effectiveness was observed in individual studies and cohorts; however, the overall pooled efficacy for all vaccine candidates was found to be non-significant. Despite multiple individual studies showing promising results for various DNA vaccine candidates against fascioliasis, the pooled studies showed the non-significant effect of the vaccine formulations against fluke burden, and displayed minimal protective efficacy against Fasciola infection. Though promising results are observed in isolated studies, further animal trials with standardised experimental parameters are required to develop new vaccine candidates effective against Fasciola.

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance.

Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.

A Preliminary Study of a Lettuce-Based Edible Vaccine Expressing the Cysteine Proteinase of Fasciola hepatica for Fasciolosis Control in Livestock.

Oral vaccination with edible vaccines is one of the most promising approaches in modern vaccinology. Edible vaccines are an alternative to conventional vaccines, which are typically delivered by injection. Here, freeze-dried transgenic lettuce expressing the cysteine proteinase of the trematode Fasciola hepatica (CPFhW) was used to orally vaccinate cattle and sheep against fasciolosis, which is the most important trematode disease due to the parasite's global distribution, wide spectrum of host species and significant economic losses of farmers. In the study, goals such as reducing the intensity of infection, liver damage and F. hepatica fecundity were achieved. Moreover, we demonstrated that the host sex influenced the outcome of infection following vaccination, with female calves and male lambs showing better protection than their counterparts. Since differences occurred following vaccination and infection, different immunization strategies should be considered for different sexes and host species when developing new control methods. The results of the present study highlight the potential of oral vaccination with plant-made and plant-delivered vaccines for F. hepatica infection control.

Recombinant and native TviCATL from Trypanosoma vivax: Enzymatic characterisation and evaluation as a diagnostic target for animal African trypanosomosis.

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat, was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket. Leucine was preferred in P2 and basic and non-bulky, hydrophobic residues accepted in P1 and P3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA.

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism.

Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab')2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

An effector from the Huanglongbing-associated pathogen targets citrus proteases.

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium 'Candidatus Liberibacter asiaticus' (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants.

Protection against Schistosoma haematobium infection in hamsters by immunization with Schistosoma mansoni gut-derived cysteine peptidases, SmCB1 and SmCL3.

We examined the immunogenicity and protective potential of SmCB1 and SmCL3 cysteine peptidases, alone and in combination, in hamsters challenged with S. haematobium. For each of two independent experiments, eight Syrian hamsters were immunized twice with a three week-interval with 0 (controls), 20 µg SmCB1, 20 µg SmCL3, or 10 µg SmCB1 plus 10 µg SmCL3, and then percutaneously exposed eight weeks later to 100 S. haematobium cercariae. Hamsters from each group were assessed for humoral and whole blood culture cytokine responses on day 10 post challenge infection, and examined for parasitological parameters 12 weeks post infection. At day 10 post-infection we found that SmCB1 and SmCL3 elicited low antibody titres and weak but polarized cytokine type 2 responses. Nevertheless, both cysteine peptidases, alone or in combination, evoked reproducible and highly significant reduction in challenge worm burden (>70%, P < 0.02) as well as a significant reduction in worm egg counts and viability. The data support our previous findings and show that S. mansoni cysteine peptidases SmCB1 and SmCL3 are efficacious adjuvant-free vaccines that induce protection in mice and hamsters against both S. mansoni and S. haematobium.

Vaccine potential of HLA-A2 epitopes from Leishmania Cysteine Protease Type III (CPC).

Although the precise host-defence mechanisms are not completely understood, T-cell-mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA-A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA-A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8+ T-cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN-γ production, which indicates its pivotal role in protective immune response. The IFN-γ production was mainly in a CD8+ T-cells-dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I-dependent manner. Moreover, no role of the CD3+ T cell was observed in the IL-10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis.

Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores.

Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.