الفهرس 
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Abstract
Schistosomiasis, is an endemic disease in many tropical and subtropical countries, mainly in poor communities. About 258 million people are globally infected, with up to 779 million at risk of acquiring the infection. chronic morbidity in S. mansoni infection is mainly caused by schistosomal eggs that lodge within the liver causing extensive tissue damage. Hosts mount a strong immunological response against the parasites՚ eggs forming collagen-rich granulomatous reaction around them. Treatment of schistosomiasis depends solely on praziquantel; which started to develop resistance both in field and laboratory. Also, reinfection is common due to repeated exposures. Therefore, vaccination is considered the most promising control measure against schistosomiasis.
Many vaccine trials have been conducted against Schistosomal infection, most of which did not show high levels of protection due to the complex life cycle of the parasite. To overcome this obstacle, multivalent proteins have been investigated as new anti-schistosomal vaccine strategies. Multivalent vaccines have the advantage of combining more than one antigenic moiety leading to higher degrees of protection.
Currently, among the popular applications in the medical field, EVs are being evaluated as immunizing agents against different parasitic infections; demonstrating promising degrees of protection, via their established roles in modulating the immune system. In addition to this, they carry many proteins that are considered promising vaccine candidates. In the current study, S. mansoni egg-derived EVs were evaluated as an immunizing agent against experimental schistosomiasis mansoni.
To achieve this aim, S. mansoni eggs were isolated from S. mansoni infected livers by artificial digestion and several centrifugations to ensure removal of dead eggs. Following isolation, viable eggs were cultured in RPMI media in 5% CO2 for 24 hours. After being cultured, EVs were isolated from the culture supernatant using cooling ultra-centrifugation technique. Isolated EVs were characterized ultrastructurally for assessing their sizes and integrities, while the Lowry’s technique was used to quantify their protein content.
For carrying out the experimental study, 120 Swiss male Albino mice were divided into two groups; group I (constituted 80 control mice) and group II (constituted 40 experimental mice). Control mice were subdivided into six subgroups: two infected controls and three non-infected controls.
Infected controls included: Subgroup Ia (20 infected non-vaccinated non-adjuvanted mice), subgroup Ib (20 adjuvanted infected mice), while thenon-infected controls included: Subgroup Ic (10 non-infected non-vaccinated non-adjuvanted mice), subgroup Id (10 adjuvanted non-infected mice), subgroup Ie (10 vaccinated non-infected, mice) and subgroup If (10 vaccinated adjuvanted non-infected).
Experimental mice were equally subdivided into two subgroups; each constituting 20 mice: subgroup IIa (vaccinated infected mice) and subgroup IIb (vaccinated-adjuvanted infected mice).
Vaccination process was done by receiving three subcutaneous injections of 20 µg EVs; two weeks apart (on day 1, 14, and 28). On the same respective vaccination schedule, adjuvanted mice received three doses of 100 µl alum, while vaccinated-adjuvanted mice received a combination of 20 µg EVs and 100 µl alum in three subcutaneous doses. Non-vaccinated non-adjuvanted control mice received three doses of PBS on the same schedule. All infected mice were challenged by 100 cercariae via the paddling technique two weeks after the last injection. All mice were scarified on the 84th day of the experiment.
Infected mice were evaluated by parasitological parameters including; adult worm load, tissue egg counting and assessment of the egg maturity using Pellegrino’s technique. Ultra-structure examination of retrieved adult worms by SEM and TEM was further performed. Histopathological examination of parts of infected livers was done using H&E stain and Masson’s trichrome stain for calculation of granulomata sizes and numbers, besides assessing of the inflammatory reaction and collagen deposition.
To assess the immunological response induced by immunization with EVs, IFN γ and IgG profiles were measured in sera of all studied mice two weeks after each injection and at the time of sacrifice using ELISA technique.
Western blot was performed to determine the molecular weight of EVs’ protein components targeted by the antibody produced using the sera of the experimental subgroups and sera of the non-vaccinated infected control mice.
Results of the current work proved the efficacy of S. mansoni egg-derived EVs as immunizing agent against experimental schistosomiasis mansoni. Parasitological measures showed that the EVs vaccination (subgroup IIa) caused significant reduction of the parasite burden compared to the infected control subgroup (Ia). Adult worm load was reduced by 46.58%, while egg burden showed 93.14% and 93.17% reduction in the hepatic and intestinal count respectively. Pellegrino’s method showed that 69% of the eggs died after reaching maturity. This was accompanied by reduction in the granulomata numbers and sizes by 76.19% and 65.63% respectively. Using alum as an adjuvant did not result in further enhancement of the vaccine efficacy, where vaccinated-adjuvanted infected mice (subgroup IIb) showed 68.7% reduction of the total worm load, 69.32% reduction of the hepatic egg count, while intestinal egg count showed 72.56% reduction. Granuloma numbers and sizes were reduced by 53.97% and 58.39%, respectively, 78% of the studied eggs died after reaching maturity.
Histopathological examination of H&E-stained liver sections showed reduced inflammatory infiltrates surrounding the egg in both experimental subgroups (IIa and IIb) compared to infected control subgroup (Ia). Neutrophils were the dominating cell type in the hepatic granuloma detected in subgroup IIa, while histocytes were dominating in these of subgroup IIb. Examining Masson’s trichrome stained liver sections obtained from experimental subgroups showed reduced fibrosis especially around the central veins in comparison to the infected control subgroup (Ia).
Ultra-structural examination of adults obtained from the experimental subgroups showed severe tegumental derangement of the adult male worm with complete loss of tone of the subtegumental muscle layer, leading to widening of the gynecophoric canal with decreased ability to maintain the female worm inside it, resulting in decoupling effect on the adult worms. Female worms showed swelling of the suckers with cauliflower appearance of the tegument.
Assessment of the immunological response in the experimental subgroups showed continuous rise in the IFN γ profile after each vaccination dose and at the time of sacrifice compared to the control subgroups. This rise in the IFN γ level was accompanied by increment in the titre of IgG profile as well.
Upon using sera from experimental subgroup IIa, western blot analysis showed that antibodies were produced against two protein moieties of molecular wights approximately 14 KDa and 21 KDa, while using sera from infected controls did not show any bands. Proteomic analysis is planned to identify the nature of the obtained proteins.
In conclusion, the current study highlighted the immune protective role of S. mansoni egg-derived EVs as a potential vaccine candidate against murine schistosomiasis mansoni. Increment of IFN γ and IgG profiles in the experimental subgroups proved that egg-derived EVs were able to modulate the immune system resulting in significant reduction of adult worm load and tissue egg count, with consequent reduction of the pathological changed associated with the infection. Whereas, adjuvanted EVs did not significantly influence the protection any further