Immunogenicity and Antigenicity of Opisthorchis felineus Proteins

| Three antigenic preparations of the human liver fluke Opisthorchis felineus were analyzed and characterized by immunochemical and serological reactions. The antigenic profiles of the excretory-secretory antigen (ES-Ag) contained 21 predominant protein bands in the range of 25-283 kDa, 20 protein bands in the range of 24-302 kDa of the somatic antigen (S-Ag), and 33 protein bands in the range of 23-313 kDa of the egg antigen. Stimulation of the immune system by these antigen preparations resulted in the production of specific mouse and hamster antibodies, which confirmed their immunogenicity. The antigenic characteristic of O. felineus antigens were measured by indirectELISA with specific sera. The average antibody titers when interacting with ES-Ag, S-Ag and E-Ag were in the range of 1:300, 1:3, 680 and 1:1,600, respectively. The 105-, 105and 250-kDa protein bands of the ES-Ag, S-Ag and E-Ag preparations, respectively, were specific to positive serum from dogs infected with O. felineus. The activity and specificity of the antigen preparations in ELISA were positively correlated with dot immunoblot and Western blot assays.

The comparative antigenicity and immunogenicity of O. felineus ES-Ag, S-Ag and E-Ag remain poorly understood, and available data in the literature are ambiguous and at times contradictory.
The aim of our study was to evaluate the serodiagnostic potential of these three groups of antigens to determine common immunoreactive proteins that can be used as antigens in ELISA for the diagnosis of opisthorchiasis at all stages of the disease.

experimental animals
Fifty Syrian golden male hamsters (Mesocricetus auratus) (100-120 g body weight) at 8-10 weeks and twenty outbred laboratory male mice (Mus musculus L.) at 8-10 weeks (20-25 g body weight) were kept under good hygienic conditions at a vivarium of Saken Seifullin Kazakh Agrotechnical University (KATU). Their use and care were approved by the Animal Ethics Committee of Veterinary Medicine Faculty of KATU (Ethical approval letter, No.: 1, 09.11.2017) All activities involving animals were carried out according to Guidance for Accommodation and Care of Animals: Species-specific provisions for laboratory rodents and rabbits (Interstate Standard, GOST 33216-2014). The animals were provided with food and water ad libitum.

obtaining O. felineus adult worms
Thirty hamsters were infected orally with 50 freshly isolated metacercariae. The infections were verified by coproovoscopy (Pavlyukov et al., 1991). On the 40 th day post infection (p.i.), hamsters were euthanized with CO 2 , and adult trematodes were isolated from the livers to obtain antigens.

preparation of excretorysecretory antigen (es-ag)
Adult trematodes were washed 4-5 times in PBS, pH 7.2-7.4, supplemented with penicillin (Simbirskaya Veterinary Company, Ulyanovsk, Russia) and streptomycin (Himfarm, Shymkent, Kazakhstan) (500 mg/ml), and immediately cultured. Viability was assessed by motility and general contractile movements. Obtained liver flukes were used to accumulate ES-Ag by cultivating in RPMI-1640 nutrient medium supplemented with 1 M HEPES, 200 mM L-glutamine, 100 mM sodium pyruvate (all from Sigma-Aldrich, St. Louis, USA), and 5,000 units of penicillin/streptomycin. Adult worms were cultivated as previously described (Bulashev et al., , 2011 with some modifications. Briefly, 100 specimens of isolated O. felineus were cultivated per 15 ml during 5-6 days to accumulate maximum ES-Ag concentrations in nutrient medium. Cultivations were conducted at 37°C and 5% carbon dioxide. ES-Ag preparation included collection of culture supernatant, centrifugation at 15,000×g for 10 min, membrane filtration (cellulose acetate; 0,20 μm) and dialysis against PBS. Dialysis was performed using dialysis tubing cellulose membrane (Sigma-Aldrich, USA) with a pore diameter of 51 μm for 12 hours at 4 C. The protein concentration was determined by Bradford method (Bradford, 1976).

preparation of somatic antigens (s-ag)
S-Ag of O. felineus was prepared as previously described (Borovikov et al., 2010), with some modifications. Adult trematodes were washed several times in PBS (pH 7.2, containing 0.45% NaCl) and kept at -20°C for 18-24 hours (200 pieces per 1 ml of PBS solution). Adult worms were triturated after freezing by homogenizing (mechanically) and centrifuged at 3,000×g for 30 min. Supernatant was collected and proteins were precipitated with 30% ammonium sulfate at 6-8°C for 12 hours. The precipitate (S-Ag fraction 1) was separated by centrifugation (3,000×g for 30 min) and dissolved in 2 ml of 0.05 M Tris-HCl buffer (pH 8.2) containing 0.5 M NaCl. The protein concentration was determined by Bradford method (Bradford, 1976).

preparation of egg antigen (e-ag)
To prepare the egg antigen, marites were cultured in a nutrient medium as described above. After 24 hours, the culture medium was analyzed under a microscope for the

Academic Publishers
Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Issue 9 | Page 935 presence of egg release in sexually mature marites. The culture medium with eggs was centrifuged at 1,000×g for 5 min, and the supernatant was saved for ES-Ag purification. The egg pellet was collected, washed twice in PBS and used for antigen preparation. Briefly, 0.5 ml of lysis buffer was added to the pellet, then sonicated (amplitude 40, at 4°C for 30 seconds, 2 times) and centrifuged at 21,000×g for 10 min. The resulting supernatant was used as an egg antigen.

the immuniZation of laboratory animals
The immunogenicity of O. felineus antigens was studied by immunizing hamsters and outbred mice. Blood was centrifuged at 1,000×g for 10 min, and serum was transferred into a clean tube. Fifteen hamsters were immunized with these antigens according to the same scheme, but the concentration of injected antigens was 0.2 mg.

canine/dog serum samples
The serum samples of 6 experimentally infected dogs were collected during our previously study and were stored at −70°C until use . Fife dogs were infected by feeding skinned fish fillets containing metacercariae of O. felineus every 5-7 days over the course of two months and one dog served as control. The infection of dogs was carried out without taking into account the number of larvae. From three weeks post infection onward, stool samples from each dog were collected weekly for microscopic examination by conventional Fülleborn's method (Fülleborn, 1920). Serum samples were taken every seven days after infection.

indirect elisa for the detection of antibodies against O. felineus antigens (es-ag, s-ag and e-ag)
A microtiter plate was filled with antigen (5.0 μg/ml) in PBS (pH 7.2) and incubated at 4°C overnight. After washing 3 times with PBS-T, the active centers of the wells were neutralized with 1% BSA, serum samples were added and the plate was incubated for 1 h at 37°C. Nonspecific components were removed by washing with PBS-T. The presence of specific antibodies was detected using peroxidase-conjugated goat anti-mouse IgG (H+L) ( Jackson Immuno Research, West Grove, USA) or peroxidase-conjugated goat anti-hamster IgG (H+L) (Sigma-Aldrich) and its substrate. As a control negative serum, a sample taken from mice and/or hamsters on day 0 before first immunization was used. ELISA results were considered positive if the OD value of the well with test serum was higher by at least twice the OD value of the control well. Analysis was performed in triplicate.
Statistical analysis was performed to determine the significance of differences between antibody titres (Sayduldin, 1992).

sds-page and western blot
A 4-12% SDS-PAGE gradient gel (BioRad, California, USA) was performed as described by (Laemmli, 1970). Proteins of O. felineus antigens were visualized by Coomassie blue staining or Silver Quest Staining Kit (Invitrogen, Waltham, Massachusetts, USA). The molecular masses of the protein bands were determined using Photo-Capt, Version 12.4 (Vilber Lourmat, France). After electrophoresis, proteins were transferred onto a PVDF membrane (Millipore, Darmstadt, Germany), and immunoblotting was carried out as described by (Towbin et al., 1979). After blocking in a solution of 5% bovine serum albumin (BSA) at room temperature for 1 h, the PVDF membrane was incubated with specific antibodies at room temperature for 2 h. HRP-conjugated goat antidog IgG-heavy and light chains (Bethyl Laboratories, Inc., Montgomery, TX, USA) were used at 1:10,000 dilution in phosphate buffered saline with Tween-20 (PBS-T) at room temperature for 1 h for detection of antigenantibody complexes. Complexes were detected by enhanced chemoluminescence (ECL) using an Immobilon Western kit (Millipore, Darmstadt, Germany).

immunodot blot analysis
Immunodot blot analysis was conducted using a nitrocellulose membrane (Bio-Rad, Hercules, California, USA) on which various antigen dilutions in the form of dots were fixed (1:2-1:1,024). The nitrocellulose membranes were left at room temperature for 30 min to allow antigen immobilization. The dots were subsequently blocked for an hour with 5% skim milk in PBS containing 0.05% Tween-20 and then washed three times with PBS-T. Thereafter, the membranes were incubated for 2 h with dog's sera diluted 1:100 in 5% skim milk-PBS and washed three times with PBS-T. This was followed by addition of HRP-goat anti-dog IgG-heavy and light chains diluted 1:30,000 in 5% skim milk-PBS. The subsequent washing steps and detection procedures were performed according to the ECL Plus manual. All analyses of serum samples were performed in triplicate.

immunogenicity of O. felineus antigens
On day 16, the average antibody titers in sera of mice and hamsters immunized with ES-Ag were 1:260 and 1:1,060 (t=1.84, P<0.2), respectively (Table 1).  The stimulation of the immune system by S-Ag resulted in production titer yields of specific antibodies of mice and hamsters of 1:530 and 1:1,390 (t=1.12, P<0.15). The mean titers of mice and hamsters immunized with E-Ag during this period increased significantly and were 1:700 and 1:1,600 (t=2.21, P<0.1), respectively. Thus, the studied antigens were able to induce immune responses in the form of antibody formation, which confirmed their immunogenicity.
The ES-Ag dilutions interacting with positive sera were 1:8 to 1:32 ( Figure 3A). The use in the dot immunoblot assays of S-Ag dilutions and same positive sera demonstrated

Academic Publishers
Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Issue 9 | Page 937 better results and were 1:128 to 1:512 ( Figure 3B). The E-Ag dilutions under the same reaction conditions showed good results and were 1:64 to 1:256 ( Figure 3C).

dIScuSSIon
In Kazakhstan, the diagnosis of opisthorchiasis is made on the basis of the detection of Opisthorchis eggs in feces and duodenal contents. The results of serological methods of research (ELISA) are not the basis for diagnosis because there are additional diagnostic studies and indirect signs of opisthorchiasis invasion (Order No. 283, 2015). This is because the available immunological tests for detecting specific Opisthorchis antibodies are not satisfactory. Applicable serological diagnoses of O. felineus are an unmet need Waikagul et al., 2002;Starkova et al., 2011;Kotelkin et al., 1997;Glupov et al., 1997;Borovikov et al., 2010).
The results presented in this study show that the three type preparations obtained from O. felineus can be used as antigens for immunodiagnosis. Recent proteomic analyses of excretory-secretory products of O. felineus showed complex protein compositions. Thirty-seven proteins in ES-Ag O. felineus have been identified by high-resolution proteomics; such an approach could be useful for identifying proteins that cannot be detected by immunoblotting because of their low content in comparison to major parasite protein components (L'vova et al., 2014). However, no data exist in the literature for proteomic studies of somatic and egg antigens. In this regard, it is important to isolate immunogenic proteins specific for O. felineus from antigenic preparations as potential antigens for vaccine and immunodiagnostic development.
Our results showed that we identified more proteins in antigens than other studies. First, this was because we used a 4-12% SDS-PAGE gradient gel to separate the antigens, which is permeable to high m.w. protein components ( Figure 1). Second, the Silver Quest Staining Kit used to color the SDS-PAGE gel has a higher resolution than the Coomassie blue method.
All three types of antigens elicited strong immune responses in laboratory animals. E-Ag has the highest immunogenicity; the antibody titers of laboratory animals immunized with E-Ag were higher than the ES-Ag and S-Ag antigens. This was observed in two types of laboratory animals. Hamster immune status was more sensitive to O. felineus antigens than the mice; specific antibody titers in the blood serum after immunization were much higher in all experimental groups (Table 1).
Antibodies of the infected dogs in the indirect-ELISA were specific to all prepared antigenic preparations of Opisthorchis felineus. Moreover, according to the titers of specific antibodies, S-Ag was more active than ES-Ag and E-Ag by its antigenicity (Figure 2). The present study also showed that the three types of antigens interacted in different ways with the sera of the infected dogs because of substantial differences between titers of specific antibodies. Thus, when we used ES-Ag, the highest titers showed 3 and 4 sera (1:1,600), S-Ag interacted better with 4 and 5 sera (1:12,800), and the highest antibody titers showed serum 3 in the interaction with E-Ag (1:6400). Similar data were observed from the study of antigen activity by dot immunoblot assay. S-Ag was also more active than ES-Ag and E-Ag by its antigenicity as shown in Figure 3.
E-Ag was the most immunogenic in the laboratory animals, but less antigenic with the sera of the infected dogs. This was because after the dogs were infected, parasite

Academic Publishers
Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Issue 9 | Page 938 egg secretions only began after 30 days i.p. Thus, the dog immune response only began later to respond to the eggs and not to the parasite itself.
The results presented in Figure 4 show that the positive sera obtained from infected dogs with the ES-Ag antigen of O. felineus recognized the immunoreactive 105-kDa protein component of the parasite. This immunoreactive 105-kDa protein component is also present in the S-Ag and E-Ag antigens. Previous studies have also reported on the immunogenicity of this 105-kDa protein and the possibility of its use in diagnoses (Kotelkin et al., 1997;Glupov et al., 1997). For the first time, we described an immunoreactive 250-kDa protein component in the S-Ag and E-Ag antigens. It should be noted that 250-kDa protein component is more antigenic than the 105-kDa protein component. This is clearly illustrated in Figure 4. The presence of two dominant immunoreactive proteins in the S-Ag and E-Ag antigens explains their high immunogenic (Table 1) and antigenic activity ( Figures  2 and 3). Proteins with the lowest molecular weights in the antigens have relatively large proportions of the total composition of proteins but do not possess pronounced immunogenicity.

concluSIon
Opisthorchis felineus ES-Ag, S-Ag and E-Ag contain a common immunoreactive protein with a m.w. of 105 kDa that can be used for the serological diagnosis of opisthorchiasis at all stages. However, further studies are necessary to determine the sequence of this immunoreactive protein and its production by recombinant DNA technology. The use of a recombinant antigen for the diagnosis of opisthorchiasis is advantageous because the antigen can then be produced at preparative scales to develop more convenient and inexpensive serological assays.

AcKnowlEdgEMEntS
The authors are grateful to Orken S. Akibekov, Sadibek S. Tokpan and Zhasulan K. Baibolin (Saken Seifullin Kazakh Agrotechnical University, Nur-Sultan, Kazakhstan) for their technical support during the research activity. This investigation was financially supported by the Ministry of Education and Science of the Republic of Kazakhstan in the frame of the projects No. AP05131132 for 2018-2020.