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. 2020 Jan 24;44(2):349–354. doi: 10.1007/s12639-020-01195-8

Reliability of heterophyid antigens in heterologous protection against human schistosomiasis

Alaa H A Hegazy 1, Lamia A Galal 1, Tasneem M Hassan 1, Refaat M A Khalifa 1,✉
PMCID: PMC7244675  PMID: 32508409

Abstract

Schistosomiasis is a neglected tropical parasitic disease which has been controlled by praziquantel for many decades; however, reemergence of praziquantel resistant strains has been a continuous threat. Therefore, the development of reliable antischistosomal vaccine is significantly demanded for optimal control. In the present study, comparison among Schistosoma haematobium, Schsitosoma mansoni and Pygidiopsis genata crude antigens was carried out by Sodium dodecyl sulphate polyacrylamide gel electrophoresis. Hyperimmunization of rabbits with tested parasites’ crude antigens was done to obtain hyperimmune sera. Western blotting was applied to show cross reactivity between parasites’ crude antigens and either homologous or heterologous sera. Although there was no cross reaction between P. genata crude antigens and sera of both Schistosoma species and vice versa; it is supposed that the immunogenic band at 79 kDa might develop cross reactivity with Schistosma spp. SEA fractionation if used in future studies.

Keywords: Heterologous immunity, Pygidiopsis genata, Schistosomiasis, SDS-PAGE, Western blotting

Introduction

One of the neglected tropical diseases (NTDs), schistosomiasis is the second most prevalent parasitic disease regarding the number of both infected and prone to infection people. Recent estimates have shown that about 206.5 million people had received anti-schistosomal treatment in 2016 (WHO 2016). At least 255 million people, in 78 countries and territories, were at risk of infection (Barry et al. 2013). A high annual mortality rate, 200,000 deaths, has been attributed to schistosomiasis (WHO 2012). In Egypt, MENA (Middle East North Africa) country, at least 7.2 million individuals are infected with schistosomiasis (Hotez et al. 2012).

Integrated strategies must be applied to control the highly morbid schsitosmiasis. Although significantly effective against human –infecting Schistosoma species, Praziquantel (PZQ) is not reliable in schistosomiasis control due to recent arise of resistant schistosome strains (Caffrey and Secor 2011). Extensive efforts to construct antischistosomal vaccine is a global demand. Sh28GST and Sm-TSP-2 are considered to be promising candidates for vaccine (Cheng et al. 2013). Unfortunately, there is a minor progress in research area concerning vaccine identification (Chen et al. 2016).

Concerning heterologous immunity, Fasciola gigantica worm crude antigens induced a decrease in total male and female counts of S. mansoni worms (Maghraby et al. 2017). Also, H. heterophyes adult worm crude antigens have been noted to reduce not only worm and egg burdens but also granuloma size and number in S. mansoni challenge infection (Youssef et al. 1989). Experimental mice, given heterophyid encysted metacercariae (E.M.C), had shown a notable heterologous immunity in subsequent S. mansoni challenge infections (Al Azzouni 1988). Hence the current study aimed to identify and compare protein components of Schistosoma haematobium and S. mansoni on one hand and those of Pygidiopsis genata, a tear drop–shaped intestinal heterophyid, on the other hand by the use of the Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and to identify immunoreactive proteins, responsible for specific binding and cross reacting activities by using Western Blotting which may be a corner stone in the construction of a heterologous vaccine against intestinal and urinary schistosomiasis.

Materials and methods

Pygidiopsis genata crude antigens preparation

A total of 133 small-sized Tilapia zillii fish, weighing less than 50 grams each were collected randomly from commercial markets in Alexandria governorate and then transported in ice boxes to Medical Parasitology Laboratory, Faculty of Medicine, Assuit University, Egypt. From each fish, snips were obtained and examined to detect E.M.C according to Morishita et al. (1965) and Sohn et al. (2009). Chopped infected fish fillet was digested to obtain free metacercariae as described by Yokogawa and Sano (1968).

Subsequently, 54 albino mice, 5-week old, were utilized to obtain adult worms, after receiving 0.3 ml of solution containing about 300 metacercariae (infective dose). Mice were sacrificed by cervical decapitation (10 days post infection). For each one, small intestine was obtained, cut, scraped and incubated as described by El Assal (1974). Viable heterophyiid worms were collected by Pasteur pipettes with subsequent clarification by 2 wire sieves, with pore size 200 and 50 µ, respectively. After identification; the worms entrapped on the latter were gathered in aliquots and kept at − 4 °C. All of them were found to be those of P. genata whose antigens were prepared according to Mansour et al. (1983).

Schistosoma soluble worm antigen preparation (SWAP)

Schistosoma haematobium and S. mansoni (SWAP) were purchased from the Schistosome Biological Supply Program Unit at Theodore Bilharz Research Institute (SBSP, TBRI) El-Dokki, Giza, Egypt.

Hyperimmunization of laboratory—bred rabbits

According to Langley and Hillyer (1989), one rabbit was immunized with 40,400 ug protein of each parasite crude antigens (S. haematobium, S. mansoni and p. genata, respectively), mixed with 400 ug of Freund’s adjuvant for three consecutive doses. Each two doses were separated by 2 weeks. Two weeks following the last dose, capillary tube was introduced into retro-orbital vein of each rabbit to get blood. Drained blood was collected in red-topped tubes and centrifuged for 5 min at 3000 rpm. The supernatant for each one was kept in aliquots at − 20 °C.

Sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE)

According to Laemmli (1970). Electrophoresis was carried out on a vertical gel slab (Bio-Rad mini gel unit®, Bio-Rad, USA) in 12% polyacrylamide gel and 5% stacking gel at 150 V for approximately 1.5 h. One part of antigens was diluted with two parts (v/v) of sample buffer [2% SDS, 5% (v/v) b-mercaptoethanol, 0.1% bromophenol blue and 0.25% (v/v) glycerol in 62.5 mM Tris–HCl, pH 6.8]. The wells were filled with 30 µL antigen extracts. Non-prestained Molecular Weight (MW) standards (wide range, Bio Basic Inc Canada) were incorporated into the control gel lanes to determine the relative MW of the resolved proteins. The recovered gel was either stained using Coomassie® blue R-250 to visualize protein bands or transferred to Immobilon-p transfer membrane to perform immunobloting.

Immunoblotting of tested parasite crude antigens

Following SDS PAGE, separated protein fractions were transferred to PVDF membrane (Millipore Corporation, Bedford, USA) for 90 min. at 90 V in a transfer buffer (0.025 M Tris–glycine; pH 8.3, 10% v/v methanol) using a Mini Trans-Blot® Transfer Cell (Bio-Rad) as described by Towbin et al. (1979). The membrane was stripped into three portions that were blocked with 3% Skimmed milk in NET for 3 h. Each strip was incubated separately with either of the three tested sera diluted 1:25 in 2 ml blocking solution (3% skimmed milk in 0.1 M PBS, pH 7.4, containing 0.02% Tween 20 (EL Gomhoreya pharmaceutical CO.) for three hours. They were washed three times, 5 min. each, with blocking solution, and then incubated for 2 h at room temperature with peroxidase conjugated anti-rabbit IgG antibody (Sigma) at concentration 1:1000 in blocking buffer. They were then washed with distilled water to stop the reaction. Finally, MW values were determined as in electrophoresis (BLUelf Prestained Ladder, Gene Direx, USA).

Results

133 T. zilli fish were examined; 105 had been infected with heterophyid E.M.C with overall prevalence (78.94%).

The double-walled spherical E.M.C were aggregated in groups between striated muscle fibers of T. zillii, especially caudally.

Experimental infection of mice revealed high infectivity of the used E.M.C with detection of eggs in stool of mice 6–10 days post infection. Pyriform adult worms were obtained from intestines and all of them were identified as those of P. genata.

The electrophoresis of S. haematobium (SWAP) revealed nine polypeptide bands, whose MW were corresponding to 226, 86, 70, 57, 54, 45, 37, 32, and 26 kDa (Fig. 1-lane: 1). Regarding S. mansoni (SWAP) fractionation, nine bands with MW of 209, 73, 69, 57, 54, 45, 32, 28, and 26 kDa were detected (Fig. 1-lane: 2). On the other hand, electrophoresis of P. genata proteins revealed seven bands at 58, 56, 49, 33, 29, 27, and 20 kDa (Fig. 1-lane: 3).

Fig. 1.

Fig. 1

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Polypeptide composition of the tested antigens by SDS PAGE. M: Molecular weight marker, Lane (1): SWAP of S. haematobium, Lane (2): SWAP of S. mansoni, Lane (3): Crude antigens of P. genata. M: protein marker

Concerning S. haematobium hyperimmune serum, it reacted with S. haematobium (SWAP) at eight bands of MW: 102, 87, 70, 64, 57, 54, 31 and 27 kDa. Bands at 102, 87, 70, and 64 kDa had shown stronger reactions. Also, it reacted with S. mansoni (SWAP) at eight polypeptide bands of MW: 102, 82, 70, 64, 57, 50, 31, and 28 kDa. Among all of them, stronger reactions were observed at 70, 64, 55, and 31. However, no significant reactions developed between it and P. genata crude antigens (Fig. 2-lane 1, 2, 3).

Fig. 2.

Fig. 2

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Showing immunoblotting against S. haematobium anti serum (lane 1, 2, 3) M: Protein marker. Lane (1): S. haematobium (SWAP), lane (2): S. mansoni (SWAP), lane (3): P. genata crude antigens

Regarding P. genata hyper immune serum, it was apparently reactive with P. genata antigens alone, with no cross reactivity with either Schistosoma antigens. Reactive bands had MW of five reactions at 145, 111, 85, 79 and 67 kDa. Among them the strongest reaction was detected at 67 kDa (Fig. 3-lane 4, 5, 6).

Fig. 3.

Fig. 3

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Showing immunobloting against P. genata antiserum (Lane 4, 5, 6): lane (4) S. haematobium (SWAP). Lane (5): P. genata crude antigens, lane (6): S. mansoni (SWAP)

Regarding S. mansoni hyper immune serum, it reacted with S. haematobium (SWAP) at six polypeptide bands: 102, 70, 64, 54, 31 and 27 kDa.

It reacted with S. mansoni (SWAP) at 102, 87, 72, 70, 64, 57, 54, 31 and 28 kDa. However, no reactions were observed with P. genata crude antigens (Fig. 4-lane 7, 8, 9).

Fig. 4.

Fig. 4

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Showing immunobloting against S. mansoni antiserum (lane 7, 8, 9): lane (7): S. haematobium (SWAP), lane (8): S. mansoni (SWAP), lane (9): P. genata crude antigens

Discussion

All the present E.M.C. were found to be those of Pygidiopsis genata and their abundance on the expense of other heterophyids (particularly Heterophyes heterophyes) was discussed by the same authors Khalifa et al. (2019).

The current study aimed to interpret the protein components of S. haematobium, S. mansoni and P. genata crude antigens by SDS-PAGE and detect their immunoreactivity by WB technique, if found.

As far as we know, neither P. genata proteins nor their homologous hyperimmune sera have been involved in electrophoresis or blotting yet. Also, S. haematobium hyperimmune serum has never been used in blotting of neither homologous nor heterologous proteins. The present hyperimmune serum of P. genata failed to react with proteins of both S. haematobium and S. mansoni. Also, the heterologous sera of both S. haematobium and S. mansoni failed to be reactive with P. genata proteins.

Obviously, some reactions were stronger than others. It may be suggested that polyclonal antibodies differ regarding not only the epitopes they recognize on the immunizing antigen, but also their affinity for the same determinants (Kuhlmann 2008).

Electrophoresis of S. haematobium (SWAP) revealed a protein band at 26 kDa; it was also reported by Sharaf (2004), while Hillyer and Pacheco (1986) and El-Ossily et al. (2016) reported it at 25 kDa. The present band had been found reactive to both S. haematobium and S. mansoni hyper immune sera at 27 kDa.

Concerning S. mansoni (SWAP) fractionation, a protein band had been revealed at 28 kDa. El-Sagheir et al. (2015) and Chacon et al. (2002) detected protein bands at 25 and 26 kDa, respectively. The present band at 28 kDa had been found reactive with both S. haematobium and S. mansoni hyperimmune sera.

On the other hand, P. genata crude antigens fractionation detected a protein band at 27 kDa. Han et al. (2014) obtained one at 28 kDa from Metagonimus yokogawi crude antigens fractionation. The present band at 27 kDa had been found non-reactive to either homologous or heterologous hyperimmune serum.

The present immunogenic band at 26–28 kDa is suggested to be glutathione S-transferase (r28GST). Riveau et al. (1998) reported the safety and efficacy of the vaccine candidate recombinant 28-kDa (r28GST) designated as Bilhvax, in a phase3 trial conducted in Senegal.

The SDS fractionation of S. haematobium (SWAP) revealed a 32 kDa protein, also shared by El-Sagheir et al. (2015) and present S. mansoni (SWAP) fractionation. Chacon et al. (2002) revealed a protein band at 31 kDa, whereas Hayunga et al. (1981); Sharaf (2004); Basyoni and El-Wahab (2013) recorded it at 30 kDa.

The present protein bands, at 31 kDa in both S. haematobium and S. mansoni (SWAP), reacted to both Schistosoma spp. hyperimmune sera. Vendrame et al. (2001) also reported them at 31 kDa in both Shistosoma sp. (SWAP) by blotting with S. mansoni hyperimmune serum.

The present 32 kDa protein may be suggested to be the Legumain with asparaginyl endopeptidase activity detected by Dalton et al. (1996). A nearby protein 31 kDa (Cathepsin B) has sensitivity up to 86% in diagnosis of schistosmiasis as reported by (Skelly and Shoemaker 2001).

The present immunogenic 70 kDa protein in S. haematobium (SWAP) fractionation was reported by El-Ossily et al. (2016) at 73 kDa, whereas Hillyer and Pacheco (1986) and Sharaf (2004) detected it at 74 kDa. On the other hand, S. mansoni (SWAP) fractionation had shown a protein band at 69 kDa. El-Sagheir et al. (2015) and Basyoni and El-Wahab (2013) reported proteins at 70 kDa, whereas EL-Ossily et al. (2016) detected it at 68 kDa.

The 70 kDa protein was shared by both S. haematobium and S. mansoni (SWAP) and reacted with both S. haematobium and S. mansoni hyperimmune sera. It is suggested to be Heat Shock Protein 70 (HSP 70), a highly immunogenic, evolutionarily conserved protein (Mayer and Bukau 2005).

Finding a reaction between P. genata serum with P. genata crude antigen at 79 kDa is noteworthy, since similar reaction at 79 kDa was found by El-Ossily et al. (2016) occurring between S. mansoni SEA and S. mansoni hyperimmune seum. This reaction wasn’t detected in Schistosma haeamtobium nor S. mansoni (SWAP) when blotted with any serum at this study.

It is believed that this band may be specific Schsitosoma spp. or P. genata egg antigen, since no similar band was found in adult worm fractionation nor immunoblotting with either of the tested hyperimmune serum.

Although adult worms used were of both sexes, they are sexually immature with lack of fertilization. Sexual immaturity is attributed to their presence in portal vein and their absence from final destination where sexual maturity occur (Warren 1976). However, in the case of P. genata adult, they were obtained from their habitat with fertilization ensured. In addition, the largest organ in trematodes is the uterus and so generous number of P. genata eggs was involved in crude antigen preparation. Subsequently, the 79 kDa antigen was obviously reactive in WB. It is suggested that this 79 kDa band in P. genata hyperimmune serum may produce a cross reaction with the corresponding band in Schistsosma SEA, if allowed.

The observed variability in molecular weight of different proteins or antigens may be attributed to the different strains utilized in different researches may be due to procedure differences regarding buffer concentration, gel-running time, and voltage. In addition, manual measurement must be taken into account. Besides afore mentioned causes, result diversity in heterophyid fractionation and blotting is justified by their belonging to totally disparate genera.

Reactive P. genata antigens have different MW than those of Schistsoma spp. (SWAP). This suggests similarity in molecular weight with dissimilarity in epitope structure.

Conclusion

Although there was no cross reaction between P. genata crude antigens and sera of both Schistosoma species and vice versa; it is supposed that the immunogenic band at 79 kDa might develop cross reactivity with Schistosma spp. SEA fractionation if used in future studies.

Acknowledgements

Authors would like to express their deepest gratitude and thanks to Prof Ahmed Kamal Dyab, Head of Medical Parasitology Department, Assiut University, Egypt for his assistance in sing all the department’s equipment and reagents and Prof Mervat Al-Azzoni, (former head of Medical Parasitology Department) Faculty of Medicine Alexandria University, Egypt for her assistance in getting the needed infected fishes and supplying us with her fantastic Ph.D. thesis.

Author’s contribution

AHAH: Collection of materials, conducting all practical experiments and manuscript writing. TMH: Supervision of the experimental studies. LAG: Planning the study design and leading the research team. RMAK: Proposal of the study protocol and final manuscript revision.

Funding

This study was financed by the Grant office of Faculty of Medicine, Assiut University, Egypt (Reference code: 1168). The authors, therefore, acknowledge with thanks the Grant Office for technical and financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

The study protocol and all steps of practical experiments were approved by the Research and Ethics Committee of Faculty of Medicine, Assuit University.

Footnotes

Publisher's Note

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