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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 9  |  Issue : 1  |  Page : 43-54

Trials of vaccination by lung schistosomula and Biomphalaria alexandrina vaccines against experimental Schistosoma mansoni


Department of Medical Parasitology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Date of Submission31-Dec-2015
Date of Acceptance27-Apr-2016
Date of Web Publication25-Oct-2016

Correspondence Address:
Naglaa F Abd El-Aal
Department of Medical Parasitology, Faculty of Medicine, Zagazig University, Zagazig
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-7942.192996

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  Abstract 

Background
Schistosomulum stage is believed to be the target of protective immunity. Over the past two decades, several investigators have demonstrated the antigenic communion between Schistosoma mansoni and Biomphalaria alexandrina.
Objective
The present study aimed to evaluate the effect of combining S. mansoni schistosomal lung antigen preparations and B. alexandrina antigen preparations for use as antischistosomal vaccination in murine models, and to compare their efficacy with and without the use of complete Freund’s adjuvant (CFA).
Materials and methods
Seventy laboratory-bred Swiss albino male mice were used in this study. They were classified into seven groups (10 mice each). Each mouse was sensitized with an initial subcutaneous injection of the extracted antigens. After 2 weeks, a second subcutaneous injection of the same antigen dose was given. Two weeks after the last dose of vaccination, all mice groups were infected with 100 S. mansoni cercariae subcutaneously. Mice were sacrificed by rapid decapitation 7 weeks post-infection for assessment of the inoculated antigens by parasitological (stool egg count, worm burden, tissue egg load, and oogram pattern) and histopathological (hepatic sections stained with hematoxylin and eosin for detection of granuloma number and diameter) studies.
Results
The data showed that vaccination with combined antigens (S. mansoni schistosomal lung antigen prepations + B. alexandrina antigen preparations with CFA) had the best protective effect.
Conclusion
The single antigen vaccination did not protect against infection by antigenically complexed S. mansoni. The cocktail vaccine apparently induced an agreeable immune response against many of the antigenic components. This new cocktail represents a promising approach toward the future development of vaccine strategy.

Keywords: Biomphalaria alexandrina, histopathological assessment, parasitological parameters, Schistosoma mansoni, schistosomula, vaccinations


How to cite this article:
Selim MA, Ahmed SM, El Settawy MA, Abo El-Maaty DA, Abd El-Aal NF, Abd El Hameed EF. Trials of vaccination by lung schistosomula and Biomphalaria alexandrina vaccines against experimental Schistosoma mansoni. Parasitol United J 2016;9:43-54

How to cite this URL:
Selim MA, Ahmed SM, El Settawy MA, Abo El-Maaty DA, Abd El-Aal NF, Abd El Hameed EF. Trials of vaccination by lung schistosomula and Biomphalaria alexandrina vaccines against experimental Schistosoma mansoni. Parasitol United J [serial online] 2016 [cited 2021 May 12];9:43-54. Available from: http://www.new.puj.eg.net/text.asp?2016/9/1/43/192996


  Introduction Top


In a recent report by WHO, it was estimated that at least 258 million schistosomiasis patients required preventive treatment in 2014. Schistosomiasis transmission was recorded from 78 countries. The report indicated that preventive chemotherapy for schistosomiasis is required in 52 endemic countries with moderate to high transmission, and advised that repeated preventive treatment over a number of years will reduce and prevent morbidity [1]. Despite the existence of the highly effective antischistosomal drug, praziquantel, schistosomiasis is spreading into new areas where climate change, especially temperatures, may become suitable for an increased S. mansoni transmission, such as over much of the eastern Africa, particularly Rwanda, Burundi, south-west Kenya, and eastern Zambia [2]. This may reduce the impact of control and elimination programs. S. mansoni may also spread to new areas outside existing control programs [2]. In Egypt, a comprehensive integrated schistosome control program combining chemotherapy, snail control, provision of potable water, and improved sanitation resulted in less morbidity, prevalence, and severity of infection [3]. A 3-year implementation of a combination of mass drug administration, an information and education campaign, provision of a safe water supply, and construction of latrines in Burkina Faso, Niger, and Mali led to a decreased prevalence [4]. Elimination and control of schistosomiasis was also successful in a number of countries including Japan, Tunisia, Puerto Rico, Iran, Mauritius, Venezuela, Morocco, and most of the Caribbean [5].

However, schistosomiasis is still an important public health problem in endemic countries with increasing concern due to the development of parasite resistance to praziquantel, which was recorded in several studies [6],[7]. This led to the supposition that antigens or cocktails of antigens are needed to induce higher and more consistent and durable levels of protection [8]. Such vaccines would contribute to the reduction of schistosomiasis morbidity through induction of specific immune responses leading to decrease in parasite load and reduced egg production [9]. According to one study, the efficacy of a vaccine depends on the choice of parasite stage and the use of an effective adjuvant [10]. McManus [11] mentioned that vaccination against schistosomes can be targeted toward the prevention of infection and/or the reduction of parasite fecundity. Because a reduction in worm numbers is regarded as the ‘gold standard’ for the antischistosomal vaccine development, the migrating schistosomulum stage was considered likely to be the major vaccine target for the induction of protective immune responses [12]. Consequently, a naturally or vaccine-acquired immunity could significantly decrease human pathology and disease transmission [13]. In an early report, Mountford and Harrop [14] indicated that lung-stage schistosomula antigens are capable of stimulating cellular immunity. They added that aggregation with improved antigen delivery systems can produce effective vaccines against schistosomiasis. They proved that the priority of the immune response is to target mainly the lung-stage and skin-stage followed by liver stage larvae. In addition, it is documented that the lung-stage schistosomula antigenic preparations elicit predominant T helper 1 (Th1) and Th17 immune responses, dominated by interferon-γ (IFN-γ), interleukin 17 (IL-17), IgG2a, and IgG2b antibodies [15].

Shalaby et al. [16] obtained specific reactivity using B. alexandrina snail-foot, 54 and 45 kDa polypeptide fractionated antigens for immunodiagnosis of Paramphistomum trematode infections, whereas no specific reactivity was obtained by the antigenically active polypeptides of B. alexandrina hepatopancreases antigen toward anti-Paramphistomum spp. antibodies. Nagy et al. [17] concluded that vaccination with susceptible and resistant vaccines of B. alexandrina snail ameliorated the oxidative stress due to S. mansoni infection with more regard for the vaccine of susceptible snail. It was revealed that the concept of shared common antigens between S. mansoni and its intermediate host B. alexandrina provides a basis for the preparation of a vaccine [18]. Other recorded schistosome vaccine antigens were discovered using attenuated schistosome larvae, protective monoclonal antibodies, or by analysis of human antibody and cytokine responses to recombinantly derived proteins [19]. High throughput antigens discovered are the recently published complete genomes of S. japonicum and S. mansoni [20], and the related postgenomic research on the schistosome proteome, transcriptome, glycome, and immunome [21].

In an attempt to introduce an effective vaccine, the present study aimed to evaluate the effect of the combination of S. mansoni schistosomal lung antigen preparations (SLAP) and B. alexandrina antigen preparations (BAAP) for use in antischistosomal vaccination of murine models. The efficacy of vaccination with and without the use of an adjuvant was also compared.


  Materials and methods Top


This nonrandomized, control-trial study was performed at the laboratories of the Parasitology Department, Faculty of Medicine, Zagazig University and Theodor Bilharz Research Institute (TBRI), Giza, Egypt, from May 2013 to September 2014.

Experimental animals

Seventy laboratory-bred Swiss albino male mice were used in this study. Mice were fed on standard diet with free accessibility to water at the Schistosome Biological Supply Program, TBRI, Giza, Egypt. All procedures related to experimental animals in the present study met the International Guiding Principles for Biomedical Research Involving Animals, as issued by the International Organizations of Medical Sciences [22] and approved by Ethics Committee of the Faculty of Medicine, Zagazig University.

Experimental design

Seven groups of Swiss albino mice (10 mice each group) were classified as follows: group A was nonimmunized and served as a control group, group B was immunized with 50 μg of CFA and served as a control, group C was immunized with 50 μg of BAAP, group D was immunized with 50 μg of SLAP, group E was immunized with 50 μg of BAAP and 50 μg of CFA, group F was immunized with 50 μg of SLAP and 50 μg of CFA, and group G was immunized with 25 μg of SLAP, 25 μg of BAAP, and 50 μg of CFA. Booster doses of vaccine were given 2 weeks later.

Schistosomula extraction

Laboratory-bred B. alexandrina snails were purchased from the Schistosome Biological Supply Unit, TBRI, Giza, Egypt, and infected in the laboratory according to the guidelines proposed by Becker and Lamprecht [23]. After exposure to light for at least 4 h, S. mansoni cercariae from the snails were used to infect the experimental mice through the subcutaneous route. Schistosomula were extracted from the lungs 18 days post-infection by a slight modification in the perfusion technique [24].

Preparation of antigens

SLAP was prepared [25] and its protein content was determined [26]. BAAP was prepared according to a study by Nabih [27]. CFA was obtained from Sigma Chemical Co. (St Louis, Missouri, USA), and emulsified in phosphate-buffered saline at a ratio of 2:1 (v/v).

Antigen administration

The protein content of each extraction was determined by the Bradford method [28]. The antigen administration protocol was performed according to Maghraby et al. [29]. Each mouse was sensitized with a single subcutaneous injection of the selected antigen in a dose of 50 μg. After 15 days, a second inoculation with the same antigen concentration was administered; thus, each mouse received a total dose of 100 μg protein.

Infection procedure

Two weeks after the last dose of vaccination, all mice were administered 100 S. mansoni cercariae subcutaneously.

Antigens assessment

Mice were sacrificed by rapid decapitation 7 weeks post-infection for the assessment of the inoculated antigens by parasitological and histopathological studies.

Parasitological studies

  1. Egg count per gram stool was determined by using the Kato thick smear method [30], which was carried out with modifications proposed by Martin and Beaver [31].
  2. Worm burden was calculated by perfusion of adult worms from the liver and the portomesenteric system [32].
  3. Tissue egg load was studied by calculating the number of eggs per gram tissue (liver and intestine) [33].
  4. For the oogram pattern, the percentage of eggs at various developmental stages in the small intestine was determined and classified according to the categories defined by Pellegrino et al. [34].


Histopathological study

The liver was removed from each one of the sacrificed mice, rinsed with phosphate-buffered saline, and fixed in Bouin fluid for 4 h, and then transferred to 70% alcohol for several days. After dehydration in absolute alcohol and clearing in xylol, it was embedded in paraffin wax, and sectioned at a thickness of 5 μm. Sections were stained with hematoxylin and eosin, and examined for periocular granulomas. The diameter of granulomas was measured using an ocular micrometer. This was done only for granulomas containing ova in their centers and not for confluent ones. The mean diameter for each group was calculated. Granuloma count was determined in five successive fields of magnification, from serial tissue sections more than 25 μm apart.

Statistical analysis

Data were entered, checked, and analyzed using the statistical computer program Statistical Package for the Social Sciences (SPSS Inc., Chicago, Illinois, USA, version 19 Windows). Data were expressed as mean±SD. Comparison between the mean values of different parameters in the studied groups was performed using the Student t-test. A P-value of less than 0.05 was considered statistically significant.


  Results Top


Parasitological results

There was an insignificant reduction (P>0.05) in the mean egg count per gram stool in the immunized groups C (without adjuvant BAAP) and D (SLAP) (17.5 and 19.5%, respectively) compared with the nonimmunized infected control group A (0%); and the percent reduction was higher in group D (19.5%) compared with group C (17.5%). Whereas the reduction was significant (P<0.01) in groups E (BAAP+CFA) and F (SLAP+CFA) (24.1 and 34.1%, respectively), and significant (P<0.001) in group G (SLAP+BAAP+CFA) (54.5%) compared with the nonimmunized infected control group A (0%). The reduction was highest in group G ([Table 1] and [Figure 1]).
Table 1 Mean egg count per gram stool in different studied groups of mice

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Figure 1 S. mansoni mean egg count per gram stool in different studied groups of mice.

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As regards the worm burden, there was an insignificant reduction (P>0.05) in the mean number of worms in the immunized group C, and significant reduction (P<0.01) in group D (15.3 and 24.7%, respectively) compared with the nonimmunized infected control A. On the other hand, the highest percent reduction (91%) in the mean number of worm burden was found in group G immunized with the combination of both vaccines and adjuvant ([Table 2]).
Table 2 Worm burden in different studied groups of mice

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Concerning the mean egg count per gram liver, there was a significant reduction in the immunized groups without adjuvant (groups C and D) (61.4 and 70.2%, respectively) compared with the nonimmunized infected control group A (0%). This reduction was significant in groups C (P<0.01; 61.4%) and D (P<0.001; 70.2%). The reduction was higher in group D than in group C, and in groups E, F, and G (P<0.001; 72.2, 75, and 94.4%, respectively) compared with the nonimmunized infected control group. Moreover, the difference between all groups was significant (P<0.01). Regarding these results, it was observed that the highest reduction percentages in the number of eggs per gram of liver (94.4%) was in group G ([Table 3] and [Figure 2]).
Table 3 Mean egg count per gram liver in different studied groups of mice

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Figure 2 S. mansoni mean egg count per gram liver in different studied groups of mice.

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The mean number of eggs per gram intestine was decreased in the immunized groups without adjuvant compared with the nonimmunized infected control group. The reduction was significant in groups C (P<0.01; 48%) and D (P<0.001; 61.3%). The reduction was higher in group D than in group C (61.3 and 48%, respectively), and was significant in groups E, F, and G (P<0.001; 69.4, 87.7, and 89.1%, respectively) compared with the nonimmunized infected control group. Moreover, the difference between all groups was significant (P<0.001) except between groups F and G (P<0.01). These results revealed that the highest reduction percentage (89.1%) was observed in group G ([Table 4] and [Figure 3]).
Table 4 Mean egg count per gram intestine in different studied groups of mice

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Figure 3 S. mansoni mean egg count /g intestine in different studied groups of mice. A, control infected; B, control+CFA (complete Freund’s adjuvant); C, BAAP (B. alexandrina antigen preparations); D, SLAP (S. mansoni schistosomal lung antigen preparations); E, BAAP+CFA; F, SLAP+CFA; G, SLAP+BAAP+CFA.

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Moreover, there was a decrease in the percentage of immature stages in the immunized groups compared with the nonimmunized infected control group. The decrease was significant in groups C and D (P<0.05), and in groups E and F (P<0.01), and in group G (P<0.001). There was a decrease in the percentage of immature stages in combined immunized groups with adjuvant compared with the CFA control group. The decrease was significant in group E (P<0.05), in group F (P<0.01), and in group G (P<0.001). The percentage of mature ova was decreased in all immunized groups compared with the nonimmunized infected control group. The decrease was significant in groups C and D (P<0.05), in groups E and F (P<0.01), and in group G (P<0.001). The reduction was significant in group E (P<0.01), and in groups F and G (P<0.001) when compared with the CFA control group. As regards dead ova, there was an increase in the percentage of dead ova in all the immunized groups compared with the nonimmunized infected control group. The increase was significant in groups C and D (P<0.01), in groups E, F, and G (P<0.001). The increase was significant in group E (P<0.01), in groups F and G (P<0.001) when compared with the CFA control group ([Table 5]).
Table 5 Oogram in intestines of mice in different studied groups

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Regarding the mean granuloma number, the reduction was insignificant in groups B and C (P>0.05), significant in groups D, E, and F (P<0.05), and in group G (P<0.001) with highest reduction (78.8%) ([Table 6] and [Figure 4] and [Figure 5]). Whereas the reduction in granuloma size was insignificant in group B (P>0.05); it was significant in groups C, D, E (P<0.05), and F, and in group G (P<0.001) with highest reduction (76.3%) ([Table 7] and [Figure 6] and [Figure 7]).
Table 6 Mean granuloma number in different studied groups of mice

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Figure 4 Section in liver of nonimmunized S. mansoni-infected control group showing multiple granulomatous reaction (hematoxylin and eosin, ×60).

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Figure 5 Section in liver of mice group immunized with S. mansoni schistosomal lung antigen preparations+B. alexandrina antigen preparations+complete Freund’s adjuvant showing the least number of S. mansoni granulomatous reactions (hematoxylin and eosin, ×60).

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Table 7 Mean granuloma diameter in different studied groups of mice

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Figure 6 Section in liver of nonimmunized S. mansoni-infected control group showing extensive granulomatous reaction (arrow) (hematoxylin and eosin, ×300).

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Figure 7 Section in liver of S. mansoni-infected mice group and immunized with S. mansoni schistosomal lung antigen preparations+B. alexandrina antigen preparations+CFA showing mild granulomatous reaction (arrow) (hematoxylin and eosin, ×300). Arrow shows decreased granulomatous reaction.

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  Discussion Top


Schistosomiasis continues to be one of the most prevalent parasitic diseases in the world. Despite the existence of a highly effective antischistosome drug, the disease is spreading into new areas, and national control programs do not usually achieve desired results, particularly in low endemic areas. The availability of a vaccine could represent an additional component to chemotherapy [35]. Consequently, vaccine strategies, as a complement to chemotherapy, represent an essential step for the future control of schistosomiasis. An improved understanding of the immune response to schistosome infection, both in animal models and in humans, suggests that the development of a vaccine may be possible [36]. It was suggested that control based on chemotherapy followed by vaccination would result in a short-term effect with long-term protection [37].

In their study, El Ridi et al. [15] documented that a lung-stage schistosomula antigenic preparation elicited predominant Th1 and Th17 immune responses, dominated by IFN-γ, IL-17, and IgG2a and IgG2b antibodies. Later, Basyoni et al. [38] provided support to the assumption of antigenic communion between S. mansoni and B. alexandrina, through the exhibition of common antigenic epitopes. They concluded that B. alexandrina antigens were the best antigens among the tested ones that can replace S. mansoni adult worm crude antigen in diagnosis and thus for vaccination.

As the levels of protection elicited by a single antigen proved to be low, it was suggested that the development of novel cocktail vaccine formulations was necessary to enhance protection [39]. Therefore, the present study concerned itself with evaluation of the combination of SLAP and BAAP preparations as a vaccine against S. mansoni infection in Swiss albino mice. For assessing the effect of both SLAP and BAAP components on S. mansoni-infected mice, we used fecal egg count per gram stool, the egg count per gram of liver, the egg count per gram intestine, the intestinal tissue egg number, worm burden, in addition to granuloma number and size as parameters. Subcutaneous administration, being the best and rarely painful route of immunization, was used [40], leading to accumulation of antigens in the draining lymph nodes and stimulation of cellular immunity [41].

Conventional approaches asserted that CFA was the best enhancer for the assessment of potential candidate vaccines in the mouse model [36]. Furthermore, Geldhof et al. [42] indicated that CFA is the most common adjuvant used in the preclinical evaluation of schistosome vaccine candidates. Preclinical research on CFA with Schistosoma vaccines showed the induction of a mixed Th1/Th2 immune response, with minimal evidence of toxicity or allergic reactions [43]. CFA has been reported to promote both cell-mediated and humoral immunity, primarily via toll-like receptor activation [44]. In our study, we used CFA combined with antigens prepared from S. mansoni schistosomula and B. alexandrina snails.

In the present study, reduction in the fecal egg count was obtained in all vaccinated groups. It was observed that the greater decrease in percentages of fecal egg counts was obtained in groups immunized by antigens combined with the adjuvant than in groups immunized by antigen preparations alone. Interpretation of reduction in the percentage in fecal egg count showed that it was highest with SLAP, BAAP, and CFA immunization (54%), indicating that this combination gave better results than did each vaccine alone with or without the adjuvant. This reduction can be attributed to the reduction in worm burden and also in the tissue egg count. These results asserted those of a former report from Brazil [45], in which mice immunization with schistosomula tegument (Sm-teg) induced significant reduction in fecal egg counts (59–60%), and were in agreement with the results obtained by Mountford and Harrop [14], who indicated that the delivery of two doses of SLAP and IL-12 induced significant levels (37.9–53.4%) of protective immunity to challenge infection. In an earlier study, Mountford et al. [46] reported that vaccination with SLAP alone failed to induce any protection. Moreover, our results were in agreement with those obtained by Etewa et al. [47], who stated that antischistosomal vaccination by mixed crude antigens preparations of cercariae, soluble worm antigens, soluble egg antigens (SEAs), and CFA induced a reduction of 70.87% in eggs per gram stools. Any difference between their results and ours may be attributed to our use of three combinations of antigens and/or their use of larger dosage of 200 μg of the extracted antigen. The infection of mice with 60 cercariae may be another factor. In this context, it must be considered that, as Ribeiro de Jesus et al. [48] had reported, different specific antigens induce different types of protective immune responses that inversely correlate with infection levels.

In their study, Capron et al. [49] had declared that the reduction in worm burden is the gold standard of anti-Schistosoma spp. vaccine development, and in our study the highest reduction in the mean percentage number of worm burden was obtained with the combination of SLAP, BAAP, and CFA (97%). That may be attributed to different proteins obtained from S. mansoni schistosomula and from B. alexandrina, resulting in greater ability for stimulation of protective Th1 cell-mediated immune responses. It is evident that the use of different antigen preparations by different researchers resulted in variations in reported percentages of reduction in worm burdens. In their report, Teixeira de Melo et al. [45] stated that mice immunization with Sm-teg induced significant reduction in worm burden of 43–48%. Using the nucleoprotein of susceptible snails as antigen, Hamed et al. [18] reported about 71% reduction in worm burden, and El Ridi and Tallima [50] indicated that mice immunization with glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and other larval excretory–secretory products caused a percentage reduction of 62–78% in worm burden, with a highly significant difference. Similarly, in their study, Etewa et al. [51] stated that cercarial, soluble worm, and SEAs preparations combined with CFA was the most protective, with significant reduction in the percentage of worm burden (90.28%). El-Ahwany et al. [52] also considered their 52.4% reduction in the mean number of S. mansoni adult worms in a group of infected mice immunized with purified schistosomula antigen as highly significant. These results are still lower than our record, possibly because we extracted 18-day-old schistosomula, whereas they used 14-day-old schistosomula. In another report, immunization with lung-stage schistosomula resulted in a 48% reduction in worm burden after using mechanically transformed schistosomula [53], whereas we used schistosomula extracted by lung perfusion. On the other hand, after three subcutaneous vaccinations in the absence of adjuvant, primary juvenile worm cells induced remarkable average reductions in worm burden of 54.3% [54]. A protective effect of 58.4% reduction in worms load was also achieved for cultured juvenile worm cells from S. japonicum 12-day-old schistosomula, which were extracted from rabbits and used without adjuvant. All these factors explain the variability between our recorded responses to vaccination and those of other researchers.

Concerning the mean egg count per gram liver in the present study, the mean number of eggs was decreased in the immunized groups without adjuvant compared with the nonimmunized infected control group. It was observed that the highest percentage reduction (94.4%) in the number of eggs per gram of liver was in the group immunized with SLAP, BAAP, and CFA, indicating that the cocktail vaccine gave the best result. The results obtained in our study exceeded those obtained in a study conducted by Teixeira de Melo et al. [45], who reported that mice immunization with Sm-teg induced significant reduction in liver-trapped eggs (65%). Liver eggs per gram reduction (76.5%) was reported in mice immunized only with live S. japonicum larval cells [55]. Again, the use of a different species or single antigen must be considered. In contrast to this, El-Ahwany et al. [52] stated that 14 days of immunization with schistosomula antigen resulted in 41.4% reduction in liver egg load, which may be attributed to the different mode of infection and timing of larval extraction. Moreover, Hamed et al. [18] reported 51.31% reduction in ova count by using the nucleoprotein of susceptible snails as antigen.

As regards the percentages of eggs per gram intestine in the present work, the mean number of eggs per gram intestine was decreased in the immunized groups without adjuvant compared with the nonimmunized infected control group. Our results also asserted that the highest reduction percentage (89.1%) occurred in the group immunized with SLAP, BAAP, and CFA. This reduction in tissue egg load of intestine may be attributed to the fact that some ova were entrapped intravascularly while others did not induce an inflammatory reaction. Etewa et al. [51] confirmed that combined antigens composed of cercarial antigen, soluble worm antigen, and soluble egg antigen and Freund’s adjuvant are the most protective, with significant reduction in the tissue egg load in intestines (93.98%). On the other hand, the effect of 14 days of immunization with schistosomula antigen resulted in only 49.2% reduction in intestinal egg load [52], whereas using mechanically transformed schistosomula for immunization recorded 75% reduction in tissue egg load [53].

Moreover, egg viability is an important parameter for the evaluation of efficacy of vaccines. In our study, there were changes in the oogram pattern, including a significant reduction in the percentage of immature and mature ova, and significant increase in the percentage of dead ova in the immunized groups compared with the nonimmunized infected control group. These changes were marked in the group immunized with SLAP, BAAP, and CFA, indicating that the cocktail vaccine gave the best result. Our results are in agreement with results obtained by Ismail [56] and those by Etewa et al. [51], who reported marked oogram changes in mice vaccinated with cocktail antigens, cercarial antigen, soluble worm and egg antigens, and Freund’s adjuvant. The reasonable explanation for these results may be the presence of predictable antibodies IgM, IgG, and IgA classes in response to worm antigens as reported by Dunne et al. [57]. Our results are also in agreement with those obtained by El-Ahwany et al. [52], who confirmed that immunization with schistosomula antigen causes a significant increase in specific conventional antibodies, IgG and IgM, together with a highly significant increase in IgG1, which cause reduction in ova count and worm burden.

Our overall results of tissue egg load are in agreement with those of Afifi et al. [53], who stated that the addition of IFN-γ to SLAP in the immunization program raised the protection level of mice tissue egg load (in liver and intestine) from 22 to 75%. In our study, the percentage tissue egg count reduction was raised from 61.4% in group C to 94.4% in group G in liver and from 48% in group C to 89.1% in group G in intestine.

The histopathological examination of livers from vaccinated groups revealed that the highest reduction percentages in number and size of granulomas were obtained in the cocktail vaccinated group; these observations can be explained by the statement of Chitsulo et al. [58], who pointed out that a vaccine that induces even a partial reduction in worm burdens could reduce pathology and limit parasite transmission. The obtained data were in agreement with those of El-Ahwany et al. [52], who reported that experimentally induced liver granulomas were significantly reduced with immunization of 14-day-old schistosomula antigenic preparation compared with infected control and attributed that to the reduction in the CD4+ cells and increase in the CD8+ cells in the immunized groups with purified schistosomula antigen. In another study, the tegument protein from S. mansoni-immunized mice produced significant reductions in liver granuloma size and pathology induced by parasite infection [59]. The resulting amelioration of hepatic pathological changes in our group of mice immunized with SLAP, BAAP, and CFA may be attributed to the neutralization of most of the toxic components released from eggs by high serum levels of total immunoglobulins and the blocking antibody IgG1, as reported by Mountford and Harrop [14].

The obtained data indicate that SLAP antigen is a very promising antigen because the immunity produced by SLAP is represented more by Th1-type rather than by Th2, giving the highest ratio in case of IFN-γ:IL-4 secretion, as indicated by Mountford and Harrop [14], leading to the preferable production of Th1 rather than Th2 cytokines during infection [60]. It has been suggested that the lung-stage schistosomulum protectively confines antigenic molecules in specific lipid-rich sites of the outer surface membrane as a physical barrier, which in addition to biochemical and immunological barriers must exist to protect the parasite from the host immune response [61],[62]. This may reflect the presence of lipid-rafts on the schistosome surface membrane that effectively allow compartmentalization of regions, as an immunological camouflage [63]. In addition, antigens of SLAP and BAAP are involved in multiple functions by reducing the worm and ova counts and the number and size of liver granulomas. These types of antigens play a significant role in cell activation and the modulation of granulomatous hypersensitivity.

Taken as a whole, immunization with cocktail vaccine of SLAP, BAAP, and CFA induced marked protection in our study. These results can perhaps be attributed to the fact that the schistosome parasite is such an antigenically complex organism that the immune responses induced by single antigen vaccination may not be strong enough to combat or challenge the infection. Moreover, CFA emulsions restrain the antigen at the site of injection and cause a granulomatous response that activates macrophages, and it strongly enhances immunoglobulin-forming cells for IgM, IgG1, IgG2a, and IgA [64],[65], and can be considered as a potent stimulant of Th1 and Th2 responses [66].

In conclusion, SLAP and BAAP could be considered as partially protective vaccines. The protection was better when combining the two antigens with CFA. Apparently, a single antigen vaccination could not combat the challenge of antigenically complex S. mansoni, whereas a cocktail vaccine can induce an immune response against many antigenic components. This new cocktail represents a promising approach toward the future development of vaccine strategy.

Authors Contributions

MA Selim, SM Ahmed, MA El Settawy conceived and designed the experiments; SM Ahmed, MA El Settawy, DA Abo El-Maaty, NF Abd El-Aal, EF Abd El-Hameed analyzed the data, contributed agents/materials/analysis tools and wrote the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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