Vaccine 35 (2017) 6977–6983

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Vaccine

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Protection against Schistosoma haematobium infection in hamstersby immunization with Schistosoma mansoni gut-derived cysteinepeptidases, SmCB1 and SmCL3

https://doi.org/10.1016/j.vaccine.2017.10.0690264-410X/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors.E-mail addresses: j.dalton@qub.ac.uk (J.P. Dalton), rashikaelridi@hotmail.com

(R. El Ridi).

Hatem Tallima a,b, Marwa Abou El Dahab c, Sahira Kareem a, John Pius Dalton d,⇑, Rashika El Ridi a,⇑a Zoology Department, Faculty of Science, Cairo University, Giza 12613, EgyptbDepartment of Chemistry, School of Science and Engineering, American University in Cairo, New Cairo 11835, Cairo, Egyptc Zoology Department, Faculty of Science, Ein Shams University, Cairo 11566, Egyptd School of Biological Sciences, Medical Biology Centre, Queen’s University Belfast, BT9 7BL Northern Ireland, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 June 2017Received in revised form 15 September2017Accepted 20 October 2017Available online 6 November 2017

Keywords:SchistosomiasisSchistosoma haematobiumHamstersCysteine peptidasesVaccineType 2 immune responsesOogram

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 independentexperiments, eight Syrian hamsters were immunized twice with a three week-interval with 0 (controls),20 mg SmCB1, 20 mg SmCL3, or 10 mg SmCB1 plus 10 mg SmCL3, and then percutaneously exposed eightweeks later to 100 S. haematobium cercariae. Hamsters from each group were assessed for humoraland whole blood culture cytokine responses on day 10 post challenge infection, and examined for para-sitological parameters 12 weeks post infection. At day 10 post-infection we found that SmCB1 and SmCL3elicited low antibody titres and weak but polarized cytokine type 2 responses. Nevertheless, both cys-teine peptidases, alone or in combination, evoked reproducible and highly significant reduction in chal-lenge 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 SmCL3are efficacious adjuvant-free vaccines that induce protection in mice and hamsters against bothS. mansoni and S. haematobium.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Schistosomiasis is a severe parasitic disease caused by trema-tode blood flukes of the genus Schistosoma, predominantly Schisto-soma mansoni and Schistosoma haematobium. The disease iswidespread in many countries in the Middle East, Africa, and SouthAmerica [1,2]. Cercariae released by compatible freshwater snailsinvade the host through the skin and transform into schistosomulathat move into the dermal blood capillaries en route to the lung.Maturation begins in the liver sinusoids and is completed in thelower mesenteric vein (S. mansoni) or the peri-vesical venousplexus (S. haematobium). Migrating and mature parasites induceweak immune responses and inflict limited harm to the host, buteggs produced by fecund female worms cause the major clinicalmanifestations of schistosomiasis. Eggs exit the blood capillariesto the exterior with the stool or urine by impairing the integrity

of the tissues of the wall of the distal colon or rectum (S. mansoni)or lower urinary tract (S. haematobium). However, many eggs driftwith the blood circulation and become trapped in the host tissues,especially the liver, where they provoke vigorous granulomatousinflammatory reactions with an aim to destroy the egg and containthe spread of the egg-derived antigens. These egg-induced immunereactions result into local edema, congestion, nodules, tuberclesand polyps formation, neo-angiogenesis, fibrosis, scarring, and cal-cification in the liver and other tissues [1–3].

Praziquantel is the only drug available to treat schistosomiasisand despite its low cost, limited side-effects, and efficacy in curinglight and moderate infection in humans, the number of peopletreated with praziquantel in 2013 and 2014 represented only20.7% of the targeted population requiring chemotherapy [3,4]. Avaccine that is effective against intestinal/hepatic and urinary dis-ease is needed to reduce, or even eliminate the disease burden,especially in Africa [5,6].

We recently demonstrated that functionally active recombinantforms of two major gut-derived cysteine peptidases of S. mansoniadministered subcutaneously without adjuvant effectively protect

6978 H. Tallima et al. / Vaccine 35 (2017) 6977–6983

mice against infection with S. mansoni and S. haematobium [7–10].While second-order (primary adaptive) spleen cell and humoralimmune responses to experimental infection in mice are consis-tently weak and skewed towards the type 1 axis prior egg deposi-tion in tissues [11–18], immunization with cathepsin B1 (SmCB1)and cathepsin L3 (SmCL3) cysteine peptidases modulate innateimmune responses by inducing the secretion of cytokines that ini-tiated type-2 anti-schistosome immune responses. These vaccine-induced responses resulted in highly significant reduction of wormburden (>60%, P < 0.005), egg production and the viability of ovatrapped in the liver and intestine [7–11].

Because mice are poorly permissive to S. haematobium infection,we felt it was important to investigate the effectiveness of theSmCB1/SmCL3 cysteine peptidase vaccine in a more permissivemodel (hamster) as an important step towards advancing our vac-cine towards trials in non-human primates and pre-clinical stud-ies. To reduce the possibility of our vaccine inducing responsesthat may protect in an immunologically non-specific manner, asWilson and colleagues [19] suggest take place in murine models,our challenge infection were administered eight weeks followingthe second immunization.

2. Materials and methods

2.1. Ethics statement

All animal experiments were performed following the recom-mendations of the current edition of the Guide for the Care andUse of Laboratory Animals, Institute of Laboratory AnimalResources, National Research Council, USA, and were approvedby the Institutional Animal Care and Use Committee (IACUC) ofthe Faculty of Science, Cairo University, permit numbers CUFS FPHY 21 14 and CUFS-F-Imm-5-15.

2.2. Parasites and animals

Female Syrian hamsters (Mesocricetus auratus) were raised atthe Schistosome Biological Materials Supply Program, TheodoreBilharz Research Institute (SBSP/TBRI), Giza, Egypt, and when 6week-old, were maintained throughout experimentation at theanimal facility of the Zoology Department, Faculty of Science, CairoUniversity. Cercariae of an Egyptian strain of S. haematobium wereobtained from SBSP/TBRI and used immediately after sheddingfrom Bulinus truncatus snails. For challenge infections, hamsterswere anesthetized, the abdomen shaved and wetted with steriledeionized water, and then exposed to 100 cercariae in 100 mldeionized water which was protected from spreading by a sterilesteel ring.

2.3. Cysteine peptidases

Functionally active S. mansoni cathepsin B1 (SmCB1) andcathepsin L3 (SmCL3) were produced in the methyltrophic yeastPichia pastoris GS115 (Invitrogen) and PichiaPinkTM (ThermoFisher) strain, respectively, as described previously by our labora-tory [7–10].

2.4. Experimental design

For each of two consecutive experiments, three female Syrianhamsters were left unimmunized and uninfected and considerednaïve animals. A total of 32 hamsters were divided into four groupsof eight hamsters. Hamsters were subcutaneously injected twice atthe tail base region, with a three-week interval, with 200 ml Dul-becco’s phosphate-buffered saline, pH 7.1 (D-PBS) containing 0

(control group), 20 mg SmCB1, 20 mg SmCL3, or 10 mg SmCB1 plus10 mg SmCL3. Eight weeks after the second immunization, the con-trol and immunized hamsters were exposed to 100 cercariae of S.haematobium. On day 10, when a large proportion of migrating lar-vae are expected to be in the lung capillaries, serum and wholeblood cultures supernatants from three hamsters/group wereassessed for immunological responses to the immunogen. Serawere separated on an individual hamster basis, and stored at�20 �C until use. Parasitological parameters were evaluated for fivehamsters per group 12 weeks after the challenge infection.

2.5. Serum antibody assays

Hamster serum antibody titers were assessed by indirectenzyme-linked immunosorbent assay (ELISA) for binding to 250ng/well SmCB1 or SmCL3 in duplicate wells as described [9–11].Alkaline phosphatase (AKP)-labeled anti-hamster IgG (H + L) con-jugate was diluted 1:1000 (Kirkegaard and Perry Laboratories,Gaithersburg, MD, USA).

2.6. Whole blood cultures

Heparinized whole blood (150 ml) was diluted 1:4 with RPMI-1640 medium supplemented with 200 U/ml penicillin, 200 mg/mlstreptomycin, 25 mM HEPES, and 20 mg/ml polymyxin B (SigmaChemical Company, St. Louis, Missouri) as an inhibitor oflipopolysaccharide. Diluted blood samples were incubated induplicate in 48-well culture plates (Corning Costar, Corning, NY,USA) in the presence of 0 or 10 mg SmCB1 or SmCL3 for 72 h at37 �C/3% CO2. Plates were then centrifuged at 400g for 10 minand the cell-free supernatants transferred into wells of a sterileplate and stored at �76 �C until assayed for cytokine release.

2.7. Cytokine assays

Serum and culture supernatants were assayed by capture ELISAin duplicate for levels of hamster IL-4, IL-5, IL-13, IL-17, and IFN-cfollowing the manufacturer’s instructions (MyBioSource, Inc., SanDiego, CA, USA).

2.8. Parasitological parameters

Worm burden and total egg load in liver and intestine in indi-vidual hamsters were evaluated using the formula: % change = [mean number in infected controls -mean number in infected, trea-ted hamsters/mean number in infected controls] � 100. Percent-ages of egg developmental stages were evaluated using 3 to 5fragments of the ileum and the large intestine. After washing insaline solution and slight drying on absorbent paper, each intesti-nal fragment was placed between two slides and analyzed by lightmicroscopy to count and classify the eggs. For each fragment, up to100 eggs were counted and classified according to their develop-mental stage as immature, viable eggs/mature, viable eggs/non-viable calcified eggs as previously described [20]. Liver sectionsfrom each control and test hamster were stained with haema-toxylin and eosin and examined for the number and diameter ofgranulomas surrounding eggs. Of note, all ova counts, viability esti-mates and granuloma counts/diameters were recorded for eachhamster, based on a minimum of three replicates. The mean valuesfor the hamsters for every group was then calculated and differ-ences from unimmunized infected hamsters statistically analyzedby 2-tailed Student t and/or Mann-Whitney tests.

Fig. 2. Total serum IgG antibody responses of hamsters to SmCB1. Each columnrepresents mean ± SD absorbance of sera (1: 200-diluted) from three hamsters pergroup. Asterisks indicate significant (P < .005) differences between unimmunizedinfected control animals and animals vaccinated with SmCB1, SmCL3 and SmCB1 +SmCL3.

H. Tallima et al. / Vaccine 35 (2017) 6977–6983 6979

3. Results

3.1. Cytokine and humoral responses to the vaccines

Immune responses of hamsters were examined 10 days afterthe challenge infection with S. haematobium. Whole blood culturesfrom all groups of hamsters released negligible amounts of IL-4 fol-lowing incubation in antigen-free or antigen-supplemented med-ium (data not shown). Whole blood cells from animalsimmunized with SmCB1, SmCL3 or SmCB1 plus SmCL3, cultured10 days after infection, secreted lower IL-5, IL-13 and IL-17 levelsthan unimmunized controls, with a dramatic decrease in IFN-clevels (Fig. 1). Significantly heightened memory responses toSmCB1 and/or SmCL3 were predominantly recorded for IL-5 andIL-13, notably in hamsters immunized with SmCB1 plus SmCL3(Fig. 1).

Antibodies to SmCB1 were detected in the serum of hamsters(titer � 1:1600) immunized with this antigen either alone or incombination with SmCL3 using (Fig. 2). By contrast, significantantibody titers were not observed against SmCL3 in the serum ofhamsters immunized with this antigen alone or combined withSmCB1.

3.2. Parasitological findings

SmCB1 and SmCL3 alone elicited highly significant (P < .008)reduction in total worm burden, with the highest decreaseobserved in hamsters immunized with SmCB1. The highest reduc-tion (P < .005) in parasite egg load in the large intestine wasrecorded in hamsters immunized with the cysteine peptidases in

Fig. 1. Cytokine response of hamster whole blood cultures. Whole blood cultures from uhamsters were obtained 10 day after infection with S. haematobium and stimulated ex vimean ± SE of duplicate cultures of three hamsters per group minus mean values for two ndifference between non-stimulated and antigen-stimulated cultures. Data are derived fr

combination (Table 1). Immunization with the cysteine peptidases,alone or in combination, did not alter the number of eggs recov-ered from the whole liver tissue (Table 1) but was associated withhighly significant decrease (�50%, P < .02) in the diameter of

nimmunized, infected controls, SmCB1-, SmCL3-, and SmCB1 + SmCL3-immunizedvo with 0 (none), SmCB1 or SmCL3 at 10 mg/ml. Columns represent cytokine pg/mLaïve, unimmunized and uninfected hamsters. Asterisks indicate significant (P < .05)om two independent experiments.

Table 1Effect of immunization with SmCB1 or SmCL3 alone or in combination on parasitological parameters of challenge S. haematobium infection in hamsters.a

Parameter Infected controls Vaccine

SmCB1 SmCL3 SmCB1 + SmCL3

Experiment 1Total worm burdenMean ± SD 26.4 ± 3.2 9.2 ± 2.2 14.5 ± 2.8 6.6 ± 2.7P value <0.001 0.0006 <0.001Reduction % 65.1 45.0 73.0

Liver egg countsMean ± SD 18,200 ± 9203 20,600 ± 8414 11,525 ± 5588 7865 ± 2462P value NS NS NSReduction %

Large int egg countsMean ± SD 12,917 ± 2072 6975 ± 665 4366 ± 404 2400 ± 1058P value 0.0016 0.0013 0.0005Reduction % 46.0 66.2 81.4

Experiment 2Total worm burdenMean ± SD 25.4 ± 3.2 6.2 ± 1.9 9.8 ± 3.1 7.0 ± 3.7P value 0.0079 0.0079 0.0159Reduction % 75.6 61.4 72.4

Liver egg countsMean ± SD 18,500 ± 5220 20,000 ± 9000 32,900 ± 14,232 17,125 ± 5618P value NS NS NSReduction %

Large int egg countsMean ± SD 9900 ± 2631 4600 ± 2434 6500 ± 2397 450 ± 129P value 0.0108 0.0350 0.0002Reduction % 53.5 34.3 95.4

a Hamsters (5 per group) were vaccinated with SmCB1 or SmCL3 alone or in combination, challenged 8 weeks after second immunization with 100 cercariae of S.haematobium, and assessed for parasitological parameters twelve weeks post infection. Int. = intestine; NS = not significant, as assessed by the Mann-Whitney test (two-tailedP value). Liver and intestine egg counts are total, per hamster. Reduction % = mean number in unimmunized hamsters – mean number in cysteine peptidase-immunizedhamsters/mean number in unimmunized hamsters � 100.

Table 2Effect of immunization with SmCB1 or SmCL3 alone or in combination on egg development of challenge S. haematobium infection in hamsters.a

Mean % egg developmental stage ± SE Infected controls Vaccine

SmCB1 SmCL3 SmCB1 + SmCL3

Small IntestineImmature ova 15.7 ± 6.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0P value NA NA NAMature ova 73.5 ± 42.5 17.7 ± 48.2 10.4 ± 12.0 10.0 ± 0.1P value NS 0.021 0.024Dead ova 7.5 ± 1.7 83.8 ± 4.5 90.6 ± 9.2 90.0 ± 5.7P value <0.0001 <0.0001 <0.0001

Large IntestineImmature ova 10.5 ± 10.5 19.0 ± 32.0 1.0 ± 2.2 0.0 ± 0.0P value NS NS NAMature ova 76.5 ± 4.9 8.8 ± 12.0 9.0 ± 10.2 9.2 ± 14.2P value <0.0001 <0.0001 0.0003Dead ova 14.7 ± 6.9 72.2 ± 31.4 90.0 ± 11.7 88.2 ± 12.2P value 0.009 <0.0001 <0.0001

a Representative of 2 independent experiments. Hamsters (5 per group) were vaccinated with SmCB1 or SmCL3 alone or in combination, challenged 8 weeks after secondimmunization with 100 cercariae of S. haematobium, and assessed for parasitological parameters twelve weeks post infection whereby 10 fragments of small and largeintestine were examined. A minimum of 100 eggs were counted for each animal. Significance of differences of mean percentages of different developmental stages in smallintestine and large intestine of vaccinated versus infection control groups was assessed by Student’s t-test. NS = not significant. NA = not applicable.

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circumoval granulomas (Table 2, Fig. 3) as well as death of themajority of ova (up to 90.0%, P < 0.009 - <0.001) that reached thesmall and large intestine (Table 3).

4. Discussion

In the present study, we have demonstrated that immunizationof hamsters, a permissive laboratory model for S. haematobiuminfection, with SmCB1 and SmCL3, alone or in combination and

without formulation in adjuvant, induces high-level (60–75%) pro-tection against challenge infection. This data supports our previousresults using murine models [7–10] for both S. haematobium and S.mansoni; thus, we have shown that these proteases are capable ofeliciting cross-species protection. Whereas in our previous reportschallenge infections were administered four or six weeks after thesecond immunization, here the period was extended to eightweeks in consideration of the study of Wilson and colleague [19]that suggested spurious protection may result from non-specificimmune responses that hamper the passage of schistosomules

Fig. 3. Examples of transverse sections of hamster liver 12 weeks after challenge infection with S. haematobium. The sections illustrate the typical granuloma diameter inunimmunized hamsters (A) and hamsters immunized with SmCB1 (B), SmCL3 (C) and SmCB1 + SmCL3 (D). Magnification 200�.

H. Tallima et al. / Vaccine 35 (2017) 6977–6983 6981

through the lungs of their hosts. Extending the time-framebetween vaccination and challenge was suggested as a means ofavoiding these non-specific effects [19], lending more strength tothe idea that a vaccine is acting via immune-dependent factors.

The immunological mechanism by which the high level of pro-tection is achieved in our vaccine system is still unclear and needsfurther exploration. Cytokine responses examined in whole bloodcultures supernatants at 10 days post challenge infection revealedlow but significant responses indicative of activation of type-2

Table 3Effect of immunization with SmCB1 or SmCL3 alone or in combination on liver granuloma

Granuloma parameters Infected controls Va

Sm

Experiment 1NumberMean ± SD 15.3 ± 2.3 11P value NSReduction %

DiameterMean ± SD 306.5 ± 67.6 21P value 0.0Reduction % 30

Experiment 2NumberMean ± SD 22.5 ± 4.3 12P value 0.0Reduction % 45

DiameterMean ± SD 395.0 ± 45.6 19P value 0.0Reduction % 50

a Hamsters (5 per group) were vaccinated with SmCB1 or SmCL3 alone or in combhaematobium, and assessed for number and diameter of granulomas in liver. Granuloma csections. The mean of values for the hamsters for every group was then calculated andanalyzed by Student’s t-test (two-tailed P value). Reduction % = mean number in unimmnumber in unimmunized hamsters � 100.

responses. These findings confirm our previous data, whichshowed that pre-treatment of hosts with cysteine peptidase mod-ulates schistosome infection-induced first- and second-order cyto-kine signals. Indeed, vaccination of mice with the cysteinepeptidase papain, diminished early induction of type 1, type 17,and type 2 cytokines following Schistosoma mansoni infection,and skewed the immune system towards a polarized type 2immune milieu [11]. The in vivo administration of cysteine pepti-dases has been shown to cause epithelial and endothelial barrier

s induced by challenge S. haematobium in hamsters.a

ccine

CB1 SmCL3 SmCB1 + SmCL3

.3 ± 3.0 13.5 ± 2.1 9.0 ± 4.2NS NS

3.6 ± 19.9 217.5 ± 35.2 157.3 ± 22.11 NS 0.001.1 48.6

.2 ± 2.0 16.4 ± 1.8 16.0 ± 4.7021 NS NS.7

6.7 ± 46.3 199.7 ± 51.7 187.5 ± 29.7029 0.0059 0.0003.2 49.4 52.5

ination, challenged 8 weeks after second immunization with 100 cercariae of S.ounts and diameters were recorded for each hamster, based on a minimum of threedifferences from unimmunized infected hamsters (infected controls) statistically

unized hamsters – mean number in cysteine peptidase-immunized hamsters/mean

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disruption leading to release of alarmins [21] that bind to specificreceptors on innate lymphoid cells type 2 (ILC2). Activation of ILC2leads to release of type 2 cytokines, namely IL-5 and IL-13 [22,23].Additionally, cysteine peptidases may interact with protease-activated receptors (PAR) on naïve T cells, driving their differenti-ation towards the type 2 axis [24]. Our observed skewing of theimmune responses towards the type 2 immunity axis is in accor-dance with our previous results obtained in the murine S. mansoni[7,8,10,11] and S. haematobium [9] models. However, in this ham-ster model of S. haematobium infection vaccine-induced immuneresponses exhibited a greater impact on the host protectiveresponses to the parasite eggs and eggs-derived antigens whichresulted in significantly (P < .02) smaller granuloma diameterscompared to unimmunized S. haematobium-infected hamsters.

The role of immunogen-specific antibodies in protection is stillunclear. For example, despite the high level of protection achievedby immunization with SmCL3 there was no detectable levels ofspecific antibodies at 10 days post challenge even a serum dilu-tions of 1:50 (although we cannot rule out the role of class-specific SmCL3 antibodies such as IgA or IgE [7,8,10,11]). Signifi-cant but low levels of anti-SmCB1 were observed at this time-point which may have played an important role in host protectionas the highest reduction in challenge worm burdens and parasiteegg burdens in large the intestine were recorded in hamstersimmunized with SmCB1 or SmCB1 in combination with SmCL3.

Type 2 cytokines and antibodies induce recruitment and activa-tion of innate lymphoid cells type (ILC2), eosinophils, basophils,and connective tissue and mucosal mast cells, and expansion ofalternatively activated macrophages [17,25–33]. Parasite-inducedtype 2 immune responses are effective at eliminating worms inchronic infection with gastrointestinal nematodes, Heligmoso-moides polygyrus, Nippostrongylus brasiliensis and Trichuris muris[17,25–37]. In the case of the trematodes S. mansoni and S. haema-tobium potent type 2 immune responses are not elicited against themigrating parasites and these succeed in colonizing the blood-stream of the hosts for decades. However, following the onset ofegg deposition in the host tissues, powerful egg antigens-inducedpro-inflammatory type 1 immune responses are soon replaced bymodulatory type 2 immune responses, which are partially effectivein protecting against subsequent infections [1–3,13,38].

We have recently proposed that the cysteine peptidase induc-tion of type 2 cytokines from epithelia, endothelial, and innatelymphoid cells may be propelled and amplified via induction ofuric acids from injured and dead cells [39]. Uric acid was recentlyidentified a key player that regulates the development and expan-sion of type 2 immune responses to cysteine peptidases [40]. Moreimportantly, elevation in uric acid levels is associated withincreased synthesis of fatty acids, including arachidonic acid, inliver, vascular endothelial and muscle cells. Arachidonic acid wasshown to be an efficient schistosomicide of larval and adult S. man-soni and S. haematobium in vitro and in vivo [41], and appears toplay a natural schistosomicide role in water-rats [42]. Experimentsare in progress to elucidate the putative mechanisms by which par-asite cysteine peptidase initiate anti-schistosome protective type 2immune responses. Protective efficacy assays in independent labo-ratories followed by extensive safety tests are now required beforevaccine experiments in baboons and pre-clinical trials in humanscan be considered.

Acknowledgments

The research was funded by the Science and TechnologyDevelopment Fund (STDF) – Egypt, grant ID. 13874 to R. El Ridiand H. Tallima. Development and production of the cysteinepeptidases was funded by Queen’s University Belfast, NI, UK, andthe Royal Society, UK.

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