Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis

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Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis

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Upninder Kaur a, Sumeeta Khurana a, Uma Nahar Saikia b, M.L. Dubey a,⇑ a b

Departments of Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India Departments of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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h i g h l i g h t s  Heparan Sulphate Binding Proteins (HSBPs) of E. histolytica were immunogenic.

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 HSBPs elicited both humoral and cellular immune response in guinea pig model.

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 Vaccination with HSBPs limits pathology after challenge infection.

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 Histopathological studies also supported the protective role.

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a r t i c l e 2 3 2 3 23 24 25 26 27 28 29 30 31 32

i n f o

Article history: Received 27 August 2012 Received in revised form 7 August 2013 Accepted 12 August 2013 Available online xxxx Keywords: Entamoeba histolytica Heparan Sulphate Binding Proteins Guinea pig model amoebiasis

a b s t r a c t Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30 lg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings. Ó 2013 Elsevier Inc. All rights reserved.

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1. Introduction

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Amoebiasis is one of the most common causes of death from protozoan parasitic diseases, second to malaria (Calderaro et al., 2006). Worldwide 50 million people suffer from amoebiasis, developing disabling colitis or extraintestinal complications leading to 50,000–100,000 deaths every year (Haque and Petri, 2006; WHO, 2007). Ingestion of Entamoeba histolytica cysts in faecally contaminated food or water initiates infection and parasite usually resides in the large intestine. Clinical symptoms range from asymptomatic colonization to amoebic dysentery and invasive extra intestinal amoebiasis.

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⇑ Corresponding author. Fax: +91 172 2744401. E-mail addresses: (M.L. Dubey).

[email protected],

[email protected]

Adherence of the parasite to intestinal epithelial cells is a prerequisite for the pathogenesis of a disease (Ravdin, 1986). A large array of glycoproteins, glycolipids and proteoglycans are present on the surface of eukaryotic cells and several pathogenic organisms use these surface proteoglycans as receptors for attachment, a process that ultimately facilitates tissue colonization and invasion. These proteoglycans include heparan sulphate, dermatan sulphate, chondroitin sulphate, keratin sulphate, heparin etc. A large number of microbial pathogens bind to heparan sulphate on eukaryotic cell surfaces, facilitating the microbial adherence and cellular invasion of the pathogen (Rostand and Esko, 1997). Heparan sulphate occurs as a proteoglycan in which two or three heparan sulphate chains are attached in close proximity to cell surface or extracellular matrix proteins (Gallagher and Lyon, 2000; Lozzo, 1998). Several protozoan parasites such as Leishmania amazonensis (Love et al., 1993); Plasmodium falciparum (Pancake et al., 1992) and Trypanosoma cruzi (Ortega-Barria and Pereira, 1991) have also been reported to bind to

0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.08.011

Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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heparan sulphate on eukaryotic cell surfaces which facilitate the microbial adherence and cellular invasion by the pathogen (Hirmo et al., 1997). However, this has not been demonstrated in E. histolytica previously. We recently identified heparan sulphate binding proteins in E. histolytica and non-pathogenic form Entamoeba dispar. In E. histolytica, two proteins (51.2 and 61.0 KDa) were identified which showed reactivity to heparan sulphate on immunoblotting. The aim of the present study was to evaluate immunogenicity and protective efficacy of heparan sulphate binding proteins of E. histolytica in a guinea pig model of intestinal amoebiasis. HSBPs were found to be immunogenic and capable of limiting the pathology of experimental infection in guinea pigs. Therefore HSBPs of E. histolytica has the potential as a vaccine candidate against amoebiasis.

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2. Materials and methods

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2.1. Isolation and purification of E. histolytica Heparan Sulphate Binding Proteins (HSBPs)

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The axenic strain of E. histolytica (HM1: IMSS) was maintained Q2 in TYI-S-33 medium (Diamond et al., 1978) supplemented with 10% heat inactivated horse serum and 100 U/ml of penicillin and 100 mg/ml of streptomycin. Trophozoites were harvested at 48–72 h (mid log phase), by chilling, and pelleted at 150 g for 5 min. Before lysis, the trophozoites were incubated in serum free medium for 48 h and then lysed in 10 ml of buffer containing 150 mM NaCl, 50 mM Tris, 0.5% (v/v) Nonidet P-40 (Sigma, USA) and 20 ll protease inhibitor cocktail (Serine & Cysteine proteases), pH 8.3 (Sigma, USA). The solubilized amoebic trophozoites were microcentrifuged at 10,000 g for 10 min. The supernatant was stored at 20 °C and was used for further protein isolation by ammonium sulphate. Proteins were precipitated by using different concentrations of ammonium sulphate (40%, 60%, 80% & 100%) from the culture lysates of axenically grown E. histolytica. The HSBPs were purified by affinity chromatography with Heparin Hi Trap column (Amersham Biosciences, UK). Further, the heparan

Fig. 1A. SDS PAGE analysis of E. histolytica protein preparations purified by affinity chromatography. Lane M: Protein molecular weight marker (Rainbow); Lane 1: 40% (NH4)2SO4 precipitate eluted with 0.25 M & 0.5 M NaCl; Lane 2: 60% (NH4)2SO4 precipitate eluted with 0.25 M & 0.5 M NaCl; Lane 3: 80% (NH4)2SO4 precipitate eluted with 0.25 M & 0.5 M NaCl; Lane 4: 100% (NH4)2SO4 precipitate eluted with 0.25 M & 0.5 M NaCl.

Fig. 1B. Determination of Heparan sulphate binding proteins of E. histolytica by immunoblotting with Heparan sulphate- HRP conjugate (Westeren Blot analysis). Lane M: Protein molecular weight marker, Rainbow Lane 1: 40% (NH4)2SO4 precipitate; Lane 2: 80% (NH4)2SO4 precipitate; Lane 3: 60% (NH4)2SO4 precipitate.

sulphate binding proteins were identified by SDS–PAGE followed by coomassie blue staining (Fig. 1A). The HSBPs were confirmed by immunoblotting with heparan sulphate (Sigma Aldrich, USA) conjugated with horse radish peroxidase which showed the presence of two proteins with heparan sulphate binding activity at 51.2 kDa and 61.0 kDa in E. histolytica in precipitation with 40% and 80% ammonium sulphate (Fig. 1B). As the yield of the protein was maximum with 80% ammonium sulphate, for further studies this concentration was used for protein precipitation.

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2.2. Preparation of antigen (HSBPs) for immunogenicity studies

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For immunogenicity and protection studies, large amount of HSBPs were needed. The bulk production of HSBPs was done by mass culturing of parasites. The parasite lysates were precipitated with 80% ammonium sulphate for the isolation of proteins. From these proteins, the HSBPs were purified by affinity chromatography with Heparin Hi Trap column (Amersham Biosciences, UK) and purified proteins were separated by SDS–PAGE on multiple gels. Proteins were eluted electrically from the gels using model 422 Elecro-eluter (Bio Rad apparatus, USA), reconfirmed by immunoblotting and stored at 20 °C for further use. Following electroelution, salts, SDS and dye were removed by dialysis (Lei et al., 2007).

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2.3. Vaccination of animals

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A total of 36 healthy, 2–4 weeks old, male guinea pigs (weighing 100–150 g) were used in the study. Animals were fed on standard pellet diet, 30 g/day which gave 100–110 calories/day with supplements of fresh green vegetables and spinach leaves. Twenty-one out of 36 animals were immunized with HSBP (Vaccinated group) and 15 were inoculated with PBS (Non-vaccinated control group). Immunization was done subcutaneously with 30 lg of HSBP of E. histolytica by three weekly inoculations, first with Freund’s complete adjuvant and subsequent inoculations with Freund’s incomplete adjuvant (in equal amounts). Due to non- availability of adequate number of animals, the control group inoculated with Freund’s complete/incomplete adjuvant was not included. However, a prior study using three guinea pigs inoculated with Freund’s complete adjuvant/incomplete

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Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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U. Kaur et al. / Experimental Parasitology xxx (2013) xxx–xxx Table 1 Vaccination and challenge infection schedule for immunogenicity and efficacy studies in Guinea pigs. Total number of guinea pigs = 36 Immunization Schedule

Group 1 (Experimental) (n = 21)

Group 2 (Control) (n = 15)

0 day (1st dose)

Heparan sulphate binding protein (HSBP) (30 lg) In FCA$ do In FIA do n=3 n = 12

PBS

do n=3 n=9

n=6 n=4

n=3 n=3

n=2

n=1

n=4

n=3

n=2

n=1

n=4

n=3

n=2

n=1

7th day (2nd dose) 14th day (3rd dose) 20th day

25th day (5th day – post challenge)

30th day (10th day – post challenge)

40th day (20th day – post challenge)

Immunogenicity tests* Challenge 2  105 E. h trophozoites intracaecally Unchallenged Immunogenicity tests* & Protection Studies #

Immunogenicity tests* & Protection Studies#

Immunogenicity tests* & Protection Studies#

Challenged Animals Unchallenged Animals Challenged Animals Unchallenged Animals Challenged Animals Unchallenged Animals

do

$

FCA: Freund’s Complete Adjuvant;  FIA: Freund’s Incomplete Adjuvant. For Immunological Studies: Serum IgG, IgM & IgA antibody, splenocyte proliferation & direct amoebicidal assays done. For this 5.0 ml blood and spleens were collected from sacrificed animals. # For Protection Studies: Caecum of animals was removed. Caecal scoring and histopathology was done as described in methods. *

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adjuvant showed that the results of immunological parameters were similar to that of PBS group. Therefore, only control group (PBS) was used for analysis.

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2.4. Challenge infection with E. histolytica

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Six days after the last booster dose, 12 guinea pigs from vaccinated group and nine animals from control group were challenged intracaecally with E. histolytica trophozoites infection for efficacy studies. Table 1 shows the immunization and challenge schedule and time points of immunogenicity assay and efficacy studies.

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2.4.1. Feeding of cholesterol As cholesterol is an important nutritional requirement for E. histolytica and is reported to increase the virulence of amoeba (Biagi et al., 1962), before challenge infection guinea pigs were fed with cholesterol. Guinea pigs of both the groups were fed on cholesterol purchased from Hi Media, INDIA (Mol. Wt. 386.66, melting point 145–149 °C) from three days prior to infection and it was continued till day of sacrifice or death of animals. All animals were kept fasting for 24 h before giving infection but allowed to have water ad lib. 2.4.2. Intracaecal challenge infection Trophozoites of virulent strains of E. histolytica (HM1: IMSS) purified from the log phase axenic culture were used for the challenge infection. Suspension of 2  105 trophozoites in physiological saline (0.5 ml) was used for intracaecal inoculation. Guinea pigs were anaesthetized using anesthetic ether. The abdomen was shaved and cleaned first with tincture iodine and then with methylated spirit and covered with sterile muslin cloth. A small (1–2 cm) incision was made on the abdomen by cutting through the skin up to peritoneum. The caecum was exposed, recognized by its large size. The requisite inoculum (0.5 ml) containing trophozoites was inoculated with a 23 gauge needle. The site of incision was cleaned with sterile spirit swab and the caecum inserted back. The incision was closed by stitching the peritoneum, muscle layer and the skin together, with a surgical needle and

black silk surgical thread. Tincture iodine was applied to the site and animals kept comfortably in cage and were under observation for recovery for two hours. They were given water after 4 h and food after 8 h.

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2.5. Immunogenicity assays

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The immunogenicity of vaccine was studied by determining specific antibody response, splenocyte proliferation and direct amoebicidal assays. All the assays were performed on vaccinated and nonvaccinated (control) animals just before challenge infection and on days 5, 10 and 20 days post challenge infection (Table 1). For antibody response, blood samples were taken by cardiac puncture. For splenocyte proliferation and direct amoebicidal assays, spleens were removed from the sacrificed animals. The splenocytes from the animals in each group were pooled for the assays.

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2.5.1. Antibody response IgG, IgM and IgA antibodies to heparan sulphate binding proteins of E. histolytica were detected by ELISA. Affinity purified heparan sulphate binding protein (1 lg/well) was coated on wells of a 96 well microtitre plates (Nunc, Germany). The dilution of antigen to be coated on the wells and test sera was predetermined by checker board titration. The conjugates (Anti guinea pig IgG, IgM and IgA) labeled with Horse Radish Peroxide (HRP) were purchased from Immunological Consultants Laboratory, Inc. Newberg, USA and the conjugates were used in dilutions as per the manufacturer’s instructions. The dilutions used in ELISA were: Goat anti-Guinea Pig IgG – 0.2 lg/ml; Goat anti-Guinea Pig IgM – 1:10,000; Sheep anti-Guinea Pig IgA – 0.1 lg/ml. Standard protocol for ELISA (Voller et al., 1978) was adopted with slight modifications. The results were read by ELISA reader at 492 nm.

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2.5.2. Splenocyte proliferation assay Splenocytes from vaccinated and control animals were isolated and cultured in RPMI medium (Sachdeva et al., 2009). The in vitro proliferation response to purified (by affinity chromatography)

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Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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HSBP antigen stimulation (5 lg/ml) was studied by incubating the cultures with 100 lM Bromodeoxyuridine (Brd Urd, Sigma, USA) at 37 °C in 5% CO2 for 48 h (Sharma et al., 2005). Analysis was done using CELL QUEST software on FACS CALIBUR flowcytometer (BD Biosciences, USA). Stimulation Index was calculated as the ratios of Brd Urd uptake in HSBP stimulated splenocytes versus the Brd Urd uptake in unstimulated splenocytes.

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2.5.3. Direct amoebicidal assay The splenocytes were separated into lymphocytes and monocytes by incubating cells in plastic petri dishes at 37 °C for 1 h in 5% CO2. For direct amoebicidal assay, lymphocytes (non-adherent cells) and monocytes (adherent Cells) were incubated with affinity purified HSBP in vitro at 37 °C for 2 h and then centrifuged at 150g for 5 min. These activated lymphocytes and monocytes (20X105) were incubated with 1X105 trophozoites of E. histolytica (ratio of 20:1) at 37 °C, 5% CO2 for 2 h (Vohra et al., 2003). E. histolytica trophozoites without immune effector cells (lymphocytes & monocytes) incubated in similar conditions were included as control. Spontaneously killed E. histolytica trophozoites were eliminated by gating in a flowcytometer. The cells were incubated for 30 min with 10 ll of propidium iodide (1 mg/ml, Sigma Chemical Co.), a fluorescent viable stain. Twenty thousand cells (both E. histolytica trophozoites with lymphocytes or monocytes) were acquired using FACS CALIBUR (BD Biosciences) and CELL QUEST software. Uptake of propidium iodide was measured in all the cells by ‘gating’ the particular cell type. Percentage of killed E. histolytica (percentage cytotoxicity) when incubated with lymphocytes and monocytes was calculated based on the total number of cells taken.

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2.6. Protection studies

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Protective efficacy of vaccination was evaluated by giving challenge infection to vaccinated and control animals as described above and comparing the severity of infection by caecal scoring (for lesions) and histopathological analysis of caecum of sacrificed animals of both the groups.

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2.6.1. Caecal scoring Specified number of challenged animals as shown in Table 1 from vaccinated and control groups were sacrificed on post challenge days 5, 10 and 20 for caecal scoring and histopathological studies. Unchallenged animals in each group were included as controls. After dissection, caeca of all the animals sacrificed on days 5, 10 and 20 were taken out. The caeca were cut open and the status

of mucosa and development of lesions, if any, were observed. The results were recorded as caecal scores (Neal, 1951). Grading of caecal lesions was done as Score I: Normal caecum; Score II: Hyperemic plaques or thickened caecal wall without any frank ulceration; Score III: Ulcers, each less than 3 mm in size; Score IV: More than two ulcers of more than 1 mm in diameter.

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2.6.2. Histopathological analysis For this, portion of caecum showing lesions, like ulcers and nodular masses were cut out into 5 cm strips from tissue already fixed in 10% formal saline. The tissue was processed for histopathology and 4–5 lm thick sections were cut and stained with Hematoxylin and Eosin (H & E). Periodic Acid Schiff’s (PAS) stain was used as and when required (Pittman and Hennigar, 1974). The stained sections were examined microscopically for ulceration, type of inflammation, extent of inflammation, presence or absence of perforation and identification and location of the parasite.

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2.7. Statistical analysis

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SPSS 17.0 software was used to analyze results. Data was analysed for statistical significance by using Mann Whitney’s U Test. For comparing caecal scoring of various groups, Chi-square test was applied. Results were expressed as mean ± S.D. p value 6 0.05 was considered statistically significant.

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2.8. Ethical approval

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The study was approved by the Institutional Ethics Committee & Animal Ethics Committee of the institute.

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3. Results

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3.1. Antibody response

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IgG, IgM and IgA antibodies to heparan sulphate binding proteins of E. histolytica were detected by ELISA and the results are shown in Tables 2 and 3 and 4.

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3.1.1. IgG antibody response On day 0 i.e., six days after the last booster dose of HSBP vaccine, all the three animals tested were positive for IgG and the mean O.D. in vaccinated animals was significantly higher (p = 0.050) than that of control animals (Table 2). On days 5, 10 and 20 P.I. also the mean O.D. values for IgG antibodies in unchallenged vaccinated animals

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Table 2 Anti - HSBP IgG antibody in vaccinated and control guinea pigs. Days

Unchallenged (n = 6)

Mean O.D.

No. tested

No. + ve

3 1 1 1

0 0 0 0

Challenged (n = 9) No. tested

No. + ve

0.052 0.018 0.017 0.07

3 3 3

3 3 3

Mean O.D.

Challenged (n = 12)

Mean O.D.

p Values

0.099e 0.078⁄ 1.032$

0.18 0.18 0.18

Mean O.D.

p values

0.337ð 0.912# 0.801¥

1 0.06 0.06

Control Group 0 5 10 20 Days

Vaccinated Group 0 5 10 20

Unchallenged (n = 9) No. tested

No. + ve

3 2 2 2

3 2 2 2

0.106^ 0.326 0.652 0.443

No. tested

No. + ve

4 4 4

4 4 4



vs. ^ p value = 0.05; e vs. ð; ⁄ vs. #; $ vs. ¥; p value < 0.05 (Day 5, 10 & 20). à p Values when the mean O.D. in unchallenged and challenged animals were compared in the same group.

Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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were higher than that in control unchallenged group though the difference was statistically not significant (Table 2). However, the mean IgG antibody levels were significantly higher in vaccinated challenged group than that in non-vaccinated (control) challenged group on all the P.I. days (p < 0.050) (Table 2). In the vaccinated animals, when the mean O.D. values of unchallenged and challenged group were compared, on day 5 P.I. it was not different in two groups. However, it was higher in vaccinated challenged group on day 10 and 20 P.I. In the vaccinated group, both in unchallenged and challenged animals the highest IgG values were seen on day 10 P.I. which declined slightly on day 20 P.I. in both groups. Though, the difference between two groups was not statistically significant. 3.1.2. IgM antibody response On day 0 i.e. just before challenge infection (6 days after the last booster dose), the mean O.D. in vaccinated group was significantly higher (p = 0.050) than that of control animals (Table 3). In vaccinated (unchallenged) animals the mean O.D. values for IgM antibodies on days 5, 10 & 20 P.I. were still higher as compared to non-vaccinated (unchallenged) group, though the difference was not significant statistically (Table 3). When mean O.D. values of control challenged and vaccinated challenged groups were compared, the mean O.D. values were higher in vaccinated animals on all the days of assay (day 5 and 10 P.I.) though the difference was statistically significant

(p = 0.034) on day 10 only, while on day 20, the mean O.D. values were not different.

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3.1.3. IgA antibody response On day 0, the mean O.D. values for IgA antibodies in vaccinated animals was significantly higher (p = 0.050) than that of control animals (Table 4). There was no significant difference in mean O.D. values for IgA antibodies on days 5, 10 and 20 P.I. between unchallenged control and unchallenged vaccinated group. Moreover there was no significant difference in IgA antibody production between non-vaccinated (control) challenged group and vaccinated challenged group on all the days after challenge infection. Amongst the vaccinated animals, the mean O.D. values for IgA were not different even after the challenge infection on all the days P.I. Amongst the non-vaccinated animals (both unchallenged and challenged), none of the animals in unchallenged group had IgA antibody and only 2 animals sacrificed on day 5 after challenge infection had IgA antibody (Table 4).

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3.2. Splenocyte proliferation assay

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Just before the challenge infection (day 0), the mean stimulation index (SI) in vaccinated animals was significantly higher (p = 0.050) (Fig. 2) than that in non-vaccinated animals. On 5, 10 and 20 P.I. among the vaccinated animals, the stimulation index was significantly higher (p < 0.050) in challenged group than that

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Table 3 Anti – HSBP IgM antibody in vaccinated and control guinea pigs. Days

Unchallenged (n = 6)

Mean O.D.

No. Tested

No. +ve

Control group 0 5 10 20

3 1 1 1

0 0 0 0

Days

Unchallenged (n = 9)

Vaccinated group 0 5 10 20

No. Tested

No. + ve

3 2 2 2

3 2 1 2

Challenged (n = 9) No. Tested

No. + ve

0.054$ 0.054 0.05 0.048

3 3 3

3 3 3

Mean O.D.

Challenged (n = 12)

0.09 0.09 0.053 0.09

No. Tested

No. + ve

4 4 4

4 4 4

Mean O.D.

p Values

0.16 0.124 0.13

0.24 0.06 0.06

⁄

Mean O.D.

p Values

0.197 0.171 0.125

0.18 0.18 0.18

#

$ vs.  p value = 0.05; ⁄ vs. # p value < 0.05. à p Values when the mean O.D. in unchallenged and challenged animals were compared in the same group.

Table 4 Anti – HSBP IgA antibody in vaccinated and control guinea pigs. Days

Control Group 0 5 10 20 Days

Vaccinated Group 0 5 10 20

Unchallenged (n = 6)

Mean O.D.

No. Tested

No. + ve

3 1 1 1

0 0 0 0

Unchallenged (n = 9) No. tested

No. + ve

3 2 2 2

3 0 2 0

Challenged (n = 9) No. Tested

No. + ve

0.009⁄ 0.023 0.023 0.02

3 3 3

2 0 0

Mean O.D.

Challenged (n = 12)

0.042$ 0.02 0.027 0.013

No. tested

No. + ve

4 4 4

2 2 0

Mean O.D.

p values

0.027 0.151 0.015

0.655 0.655 0.157

Mean O.D.

p Values

0.02 0.02 0.01

1 0.48 0.1

vs. $ p value = 0.05. à p Values when the mean O.D. in unchallenged and challenged animals were compared in the same group.

⁄

Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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animals as compared to unchallenged group on all P.I. days (Fig. 3), though the difference between the two groups was statistically not significant (p > 0.05). It was also observed that in vaccinated challenged group at day 20 after challenge infection percent cytotoxicity was maximum with lymphocytes and monocytes (36.7 ± 1.57 and 26.0 ± 1.40). Overall the lymphocytes showed higher cytotoxicity as compared to monocytes irrespective of groups and days after challenge infection (Fig. 3).

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of unchallenged group. However, the mean stimulation index was higher in non-vaccinated challenged group on day 5, 10 and 20 post challenge as compared to non-vaccinated unchallenged animals, but the difference was not statistically significant. The mean stimulation index in vaccinated challenged group was significantly higher (p < 0.050) when compared to non- vaccinated challenged group on all the days after challenge infection (Day 5, 10 & 20); (Fig. 2). The highest splenocyte proliferation response was seen in vaccinated challenged animals on day 20 P.I. (Fig. 2).

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3.3. Direct amoebicidal assay

3.4. Protective Efficacy

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Just before challenge infection (day 0) the mean percentage in vitro amoebicidal activity of lymphocytes and monocytes in vaccinated group was 3.7 ± 0.20 and 3.0 ± 0.20 respectively which was significantly higher (p = 0.050) than that in non vaccinated group (1.96 ± 0.40 and 1.96 ± 0.15 respectively). Following challenge infection also the mean amoebicidal activity of both lymphocytes and monocytes in vaccinated (challenged) group was significantly higher (p value < 0.05) than that of nonvaccinated challenged group on days 5, 10 and 20 after challenge infection (Fig. 3). Within both vaccinated and control animals, the mean cytotoxicity of both lymphocytes and monocytes was higher in challenged

Protective efficacy of the vaccine was studied by caecal scoring (Table 5) on gross examination of infected cacea on different days after challenge infection and histopathological analysis.

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3.4.1. Caecal scores All the nine animals in non-vaccinated challenged group had hyperemic plaques or thickened caecal wall (Score II) while one of these caeca showed frank ulcers (Score III) (Table 5). On the contrary, ten out of the 12 animals in vaccinated challenged group had normal caecum while only two had score II i.e., they showed hyperemic plaques (Table 5). The difference in caecal scores between non-vaccinated challenged and vaccinated challenged

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P

R

Fig. 2. Comparison of Splenocyte Proliferation Response in control and vaccinated groups. ⁄ vs. # p value (0.05); $ vs. 1; vs. €; vs. e p value (<0.05). Vac- just before challenge infection (Vaccinated); non-vac- just before challenge infection (Non-Vaccinated); CC- non-vaccinated challenged; CU-Non-vaccinated unchallenged; VC-vaccinated challenged; VU- vaccinated unchallenged.

¯

R

Fig. 3. Cytotoxicity of lymphocytes and monocytes against E. histolytica from control and vaccinated groups. ⁄ Vs € p value (0.05); Ï Vs £; O Vs H; ð Vs §; e Vs ; ¤ Vs ¥; $ Vs # p value 0.05). Vac- Just Before Challenge Infection (Vaccinated); Non-Vac- Just Before Challenge Infection (Non-Vaccinated); CC- Non-vaccinated Challenged; CU- Non-vaccinated Unchallenged; VC- Vaccinated Challenged; VU- Vaccinated Unchallenged.

Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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U. Kaur et al. / Experimental Parasitology xxx (2013) xxx–xxx Table 5 Gross pathological findings (Caecal Scores) in caeca of vaccinated and control challenged guinea pigs. Groups

Days After Challenge Infection

Animal Number

Non-vaccinated Unchallenged

Day 5 Day 10 Day 20 Day 5 Day 5 Day5 Day 10# Day 10# Day 10# Day 20 Day 20 Day 20 Day 5 Day 5 Day 10 Day 10 Day 20 Day 20 Day 5 Day 5 Day 5 Day 5 Day 10⁄ Day 10⁄ Day 10⁄ Day 10⁄ Day 20 Day 20 Day 20 Day 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Non-vaccinated Challenged

Vaccinated Unchallenged

Vaccinated Challenged

#

vs.

⁄

387

388

3.5. Histopathological analysis

389

All the unchallenged animals (Vaccinated & Non-Vaccinated) showed normal histological findings on all the days (5, 10 and 20) in the sections of caeca examined. The histopathological findings in challenged vaccinated and non-vaccinated animals on various days P.I. were as follows.

386

390 391 392 393

394 395 396 397 398 399 400 401 402 403 404 405 406 407

408 409 410 411 412 413

Score II

Score III

Score IV

p p p p p p p p p p p p p p p p p p p p p p p p p p p

(p value < 0.05) at day 10.

group was significant (p value < 0.05) at day 10 after challenge infection. All the animals in unchallenged groups had normal caecum (Table 5).

385

Score I p p p

3.5.1. Day 5 after challenge infection In non-vaccinated challenged group, 1 out of 3 animals showed ulcers with acute, active and heavy inflammation (Fig. 4A, Fig. 4B) and presence of amoebae in sections stained with PAS (Fig. 4C). Remaining 2 animals showed signs of mucosal/submucosal vascular congestion and hemorrhages with moderate degree of inflammation. In vaccinated challenged group, all the four animals had intact lining with mild to moderate degree of inflammation (Fig. 5) which was limited to mucosa in 3 out of 4 animals and only 1 animal showed mild submucosal inflammation. One animal in this group had moderate and one had focal cellular activity. Whereas two other animals did not show any cellular activity. Lymphoid follicles were seen in 3 out of 4 animals. 3.5.2. Day 10 after challenge infection On day 10 P.I., all (3/3) animals, in non-vaccinated challenged group had moderate degree of inflammation but the mucosal lining was intact. Extent of inflammation was up to mucosa in one animal and predominally mucosal and focally submucosal in rest two animals. Focal cellular activity was seen in one animal. The features of

reactive lymphoid follicles, oedema and submucosal congestion were also seen (Fig. 6). In vaccinated challenged group, all the four animals had intact lining and had mild inflammation which was limited to mucosa (Fig. 7). Two out of four animals in this group had moderate and focal cellular activity. Lymphoid follicles were seen in all the animals of this group.

414

3.5.3. Day 20 after challenge infection The animals in non-vaccinated challenged group had features of reactive lymphoid follicles, muscle necrosis, submucosal oedema submucosal haemorrahages and mild serositis (Fig. 8A, Fig. 8B, Fig. 8C, Fig. 8D).These animals also had moderate degree of active inflammation which extended predominally up to mucosa and fo-

421

Fig. 4a. Ulcer with neutrophilic infiltrate (;) and fibrin (;) in non-vaccinated challenged animal. (H & E stain 550X).

Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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Fig. 4b. Acute ulcer with fibrin rich neutrophil exudate and congested vessels in non-vaccinated challenged animal. (H & E stain 550X).

Fig. 4c. Ulcer with amoebae (arrow) in non-vaccinated challenged animal. (PAS stain 280X).

Fig. 5. Normal mucosa and intact lining in vaccinated challenged animal 5 day after challenge infection. (H & E stain 550X).

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cally up to submucosa. Cellular activity was focal in one animal of this group though the mucosal lining was intact in all the animals. All the four animals in vaccinated challenged group had intact lining. Degree of inflammation was mild to minimal with inflammation up to mucosa in one animal and up to submucosa in 3 animals. Cellular activity was focal and varied from mild to moderate in this group (Fig. 9). Lymphoid follicle was found only in one animal.

Fig. 6. Reactive lymphoid follicle (;) and congested vessels (;) in non- vaccinated challenged animal 10 day after challenge infection. (H & E stain 550X).

Fig. 7. Intact lining with focal activity and moderate inflammation in vaccinated challenged animal 10 day after challenge infection. (H & E stain 550X).

4. Discussion

435

Amoebiasis, especially the extraintestinal form is associated with significant morbidity and mortality. Therefore, prevention in the form of vaccination, could greatly decrease the incidence of the disease, and possibly help its eradication. Many vaccine candidates for amebiasis have been tested (Stanley, 2006), like the serine-rich E. histolytica protein, peroxiredoxin, the EhCP112 molecule, and the galactose/N-acetyl-D galactosamine

436

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Fig. 8a. Reactive lymphoid follicle (;) and submucosal oedema (;) in nonvaccinated challenged animal 20 day after challenge infection. (H & E stain 550X).

9

Fig. 8d. Ulcerated mucosa (;) submucosal haemorrahages, mild serositis (;) in nonvaccinated challenged animal 20 day after challenge infection. (H & E stain 550X).

Fig. 8b. Myonecrosis (arrow) in non-vaccinated challenged animal 20 day after challenge infection. (H & E stain 550X).

Fig. 9. Mild inflammation and focal activity in vaccinated challenged animal 20 day after challenge infection. (H & E stain 550X).

Fig. 8c. Submucosal oedema (arrow) in non-vaccinated challenged animal 20 day after challenge infection. (H & E stain 550X).

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– inhibitable lectin (Gal/GalNAc lectin). Much work in the literature has focused on the latter, i.e., vaccination with either parasite-purified Gal/GalNAc lectin (Cheng and Tachibana, 2001; Houpt et al., 2004; Ivory et al., 2006; Lotter et al., 1997; Petri and Ravdin, 1991) or recombinant lectin subunits has provided protection in rodent models against amebic liver abscess (Houpt et al., 2004; Lotter et al., 2000) and amebic colitis (Soong et al., 1995; Zhang and Stanley, 1994). But there are two limitations. Firstly, in most of these vaccine studies, the adjuvants and delivery routes are not compatible with eventual use in humans. Secondly, the

mechanisms involved in protection with most candidate antigens are not well understood. However, the most studied vaccine candidate is N-acetyl-d-galactosamine (GalNAc) inhibitable adherence lectin mediates attachment of trophozoites to colonic mucins or mammalian target cells (Parija, 2010). Amebic cytolysis of target cells requires Gal/GalNAc-lectin-mediated adherence, parasite phospholipase A activity, and maintenance of an acid pH in amebic intracellular vesicles. The native Gal-lectin, purified by immunoaffinity chromatography from detergent-solubilized amoebae, was first found to confer 67% protection against ALA in gerbils when immunized intraperitoneally or subcutaneously (Petri and Ravdin, 1991). Immunization of gerbils with purified recombinant LC3 (170 kDa subunit of lectin)-encoded protein elicited a high-titer serum anti-LC3 IgG antibody response and protective immunity against intrahepatic challenge with 0.5X106 virulent axenic trophozoites showing 71% vaccine efficacy (Soong et al., 1995). The cysteine-rich region of the lectin’s heavy subunit (Mann et al., 1991) especially portions closest to and including the newly identified carbohydrate-binding domain (Dodson et al., 1999) were found to be the most effective. Heparan sulphate binding proteins have been used as a vaccine against Helicobacter pylori. It has been demonstrated that oral immunization of BALB c mice with vaccine composed of

Please cite this article in press as: Kaur, U., et al. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Exp. Parasitol. (2013), http://dx.doi.org/10.1016/j.exppara.2013.08.011

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H. pylori HSBP resulted in a reduction of the adhesion of the bacteria to the gastrointestinal tract, from 100% in unvaccinated mice to 6.6% in the HSBP – immunized group (Ruiz-Bustos et al., 2000). Therefore, we, for the first time purified HSBP(s) from E. histolytica and demonstrated their immunogenicity and protective efficacy against a challenge infection with E. histolytica in guinea pig model. Cholesterol is known to be an important nutritional requirement for E. histolytica (Hansen and Anderson, 1948; Rees et al., 1994; Synder and Meleny, 1943). Virulence of E. histolytica has been reported to be enhanced when cholesterol is fed to the guinea pigs (Biagi et al., 1962; Bhatti et al., 1992) or when it is added to cultures of E. histolytica (Bos and Griend, 1977). Amoebae in the gut engulf cholesterol particles which perhaps increases their phagocytic capacity resulting in higher degree of caecal invasion .Thus, we used cholesterol in our study to increase the virulence of E. histolytica. Natural infection with E. histolytica is associated with significant IgG and IgM antibody responses (Abd-Alla et al., 1992, 1998). The experimental infection in the present study also resulted in good IgG and IgM response to HSBP antigen as shown by antibody detection in control (non-vaccinated) challenged animals. The vaccination with E. histolytica HSBP resulted in anti-HSBP IgG, IgM and IgA antibody response. The IgG and IgM responses were further boosted on challenge infection with E. histolytica in the vaccinated animals, indicating that vaccinated animals recognized the E. histolytica antigens and produced a heightened antibody production typical of a secondary response. The poor serum IgA response after challenge infection may be attributed to the fact that IgA is mainly secretary immunoglobulin i.e., can be detected mainly in secretions as it is predominantly involved in mucosal immunity. Prabir et al. (1998) also studied serum antibody response to intestinal amoebiasis in C3H/HeJ mice from 5 to 60 days post-inoculation with E. histolytica by enzyme-linked immunosorbent assay (ELISA). The serum antibody showed high IgG titre from 30 to 60 days post-inoculation as compared to IgM and IgA serum titres. The authors concluded that serum antibody responses to E. histolytica were induced in C3H/HeJ mice by intracaecal infection as in our study. Moreover, it is considered that suitable vaccines are those that elicit a mixture of antibodies response and can act at several levels against the pathogens. Further the experimental intracaecal infection in guinea pigs was associated with cell mediated immune response demonstrated by higher values of splenocyte proliferation response to HSBP in non-vaccinated challenged group as compared to unchallenged group. The HSBP vaccination also resulted in in vitro splenocyte proliferation in response to HSBP antigenic stimulation. This response was further enhanced on challenge infection in these vaccinated animals, indicating that in HSBP vaccinated animals the immune memory cells recognized the parasite infection and elicitated an immune response. This response was anti-amoebic as seen by direct amoebicidal assay. Indeed, there is increasing evidence for a role of cell-mediated immunity (CMI) in protection from intestinal amebiasis (Denis and Chadee, 1989; Xiaoti et al., 2007). CMI plays a critical role in lectin-elicited protective immunity to intestinal amebic infection was demonstrated by Xiaoti et al. (2009). It was shown that vaccination with purified E. histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In our study, most vaccinated animals, active lymphoid follicles were seen which is an indicator of protective response to challenge infection. The data on both of caecal scoring and histopathology showed that HSBP vaccination was capable of limiting the pathology of experimental infection in guinea pigs. These findings

suggest that HSBPs of E. histolytica are capable of stimulating mucosal immune responses and has the potential as a vaccine candidate against amoebiasis. Further studies of vaccination with this molecule in other models and exploration of other aspects of immunity are indicated.

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5. Conclusion

548

The results of the study showed that the HSBP was immunogenic in guinea pig model eliciting antibody response as well as direct amoebicidal activity in immune effector cells. Further the HSBP inoculation resulted in the immune responses which were capable of greatly limiting the pathology of challenge infection. Thus, the HSBP of E. histolytica has the potential as a vaccine candidate against amoebiasis. Further studies on vaccination with this molecule in other models and exploration of other aspects of immunity are indicated.

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Uncited references

558

Campbell and Chadee (1997), Ghadirian et al. (1980), Petri Q3 (2002) and Víctor and Shibayama (2005).

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Acknowledgments

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The study was supported by a grant received from Indian Council of Medical Research, New Delhi to Dr. Upninder Kaur.

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