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Abstract
Schistosomiasis is a major public health problem in sub-Saharan Africa, South America, China, and Southeast Asia caused by the blood parasites belonging to genus Schistosoma that infect 200 million people, endanger 400 million others and the disease is associated with a chronic and debilitating morbidity.
The present work studied the kinetics of the changes in the immune responses developing in mice after schistosome infection starting from the very early stages post cercarial penetration till reaching the egg stage. We looked at the Immunoglobulin classes as well as subclasses levels by ELISA. Moreover we studied cellular phenotypic changes in different lymphoid organs.
We dissected the changes in both humoral and cellular immune responses after immunizing mice with crude antigens derived from different S. mansoni developmental stages.
We quantified immunoglobulins classes and subclasses levels in sera from immunized mice against the homologous antigens used in immunization. Moreover, we used sera from immunized mice with each of the used antigens to characterize the immunogenic peptides and the degree of cross reactivity among such antigens by Western blotting.
We investigated cellular phenotypic changes in different lymphoid organs in response to immunization. In addition, we compared the influence of infection or immunization on the nature of immunoglobuline classes and subclasses responses.
Results of ELISA:
1. Using CAP as an antigen in ELISA:
The obtained generalized high levels of both IgG and IgA against CAP at different time intervals post infection. At all intervals post infection except 30 days post infection IgM level in INMS was nearly the same as CMS against CAP
ِِِِAt 3 days post infection, sera showed significantly higher levels of IgG1, IgG2b and IgG3 than CMS. Although, level of IgG2a did not significantly vary, it was 1.5 fold higher among INMS than CMS.
Also, INMS showed significantly higher levels of IgG1 and IgG3 than CMS. Although levels of IgG2a did not significantly vary, was 1.7 fold higher among INMS than CMS, while the level of IgG2b did not show difference compared to CMS.
At 15 days post infection INMS showed significantly higher levels of IgG and IgA than CMS. IgM level of in INMS was nearly the same as CMS. Also, INMS showed significantly higher levels of all measured subclasses than CM.
At 30 days post infection INMS showed significantly higher levels of IgM, IgG and IgA than CMS. Also, INMS showed significantly higher levels of IgG1, IgG2a and IgG2b CMS. Although level IgG3 did not significantly vary, it was 15.5 fold higher among INMS than CMS.
2. Using SWAP as an antigen in ELISA:
At 3 days post infection INMS had significantly higher levels of IgM and IgG than CMS. Sera levels of IgA did not show any difference when compared to CMS. Also, INMS showed significantly higher levels of IgG1, IgG2a and IgG3 CMS. Level of IgG2b in INMS did not show any difference when compared to CMS.
At 9 days post infection INMS had significantly higher levels of IgM and IgG than CMS. IgA levels in INMS did not show any difference when compared to CMS. Also, INMS showed significantly higher levels of IgG1, IgG2a than CMS. Levels IgG2b and IgG3 nearly unchanged in comparison to CMS.
At 15 days post infection INMS had significantly higher levels of IgM and IgG. Level of IgA was nearly unchanged when compared to CMS. Levels of IgG1, IgG2a were significantly higher among INMS than in CMS. Levels of IgG2b in INMS nearly unchanged in comparison to CMS. Although, IgG3 level did significantly vary it was 1.5 fold higher among INMS than CMS.
At 30 days post infection INMS had significantly higher levels of IgM and IgG than CMS. Level of IgA did not show any difference when compared to CMS. Levels of IgG1, IgG2a were significantly higher among INMS than in CMS, while, level IgG2b did not show difference compared to CMS. Level of IgG3 was 1.5 fold higher among INMS than CMS but such increase was not significant.
3. Using SEA as an antigen in ELISA:
At 3 days post infection INMS had significantly higher levels of IgG than CMS. Although IgM and IgA did not significantly vary, both classes were were 2 fold higher among INMS than CMS. Also, when compared to CMS levels of IgG1, IgG2a, IgG2b and IgG3 were respectively 2, 3.4, 1.8 and 3 fold higher among INMS, yet, such increase was not significant.
At 9 days post infection Level of IgM among INMS was 1.5 fold higher than CMS. Both IgG and IgA levels were significantly higher among INMS than CMS. Also, levels of IgG1, IgG2a, IgG2b and IgG3 were significantly higher among INMS than in CMS.
At 15 days post infection INMS had significantly higher levels of IgM, IgG and IgA than CMS. Also, levels of IgG1, IgG2a and IgG2b were significantly higher among INMS compared to CMS. Although level of IgG3 was 2 fold higher among INMS than CMS, such increase was not significant.
At 30 days post infection INMS had significantly higher levels of IgM, IgG and IgA than CMS. Also, levels of IgG1, IgG2a, IgG2b and IgG3 were significantly higher among INMS compared to CMS.
Immunization with CAP and using CAP as an antigen in ELISA:
CAP-IMS at one week post 1st immunization showed significantly higher levels of IgM, IgG and IgA than CMS. CAP-IMS at one week post 1st immunization showed significantly higher levels of IgG2a and IgG2b than CMS. Although levels of IgG1 as well as IgG3 did not significantly vary, both subclasses were respectively 2 and 9.6 fold higher among IMS than CMS.
One week post 2nd immunization, levels of IgM, IgG and IgA were respectively 26.8, 3 and 6.7 fold higher among IMS than CMS, yet, such increase was not significant. IgG1 level was significantly higher in IMS than CMS. Levels of IgG2a, IgG2b and IgG3 were respectively 7.9, 4.8 and 3.2 fold increase among IMS than CMS, yet, such increase was not significant.
Immunization with SWAP and using SWAP as an antigen in ELISA:
One week post 1st immunization, level of IgG was significantly higher among IMS than CMS. Levels of IgM and IgA, although did not significantly differ, were respectively 1.44 and 1.37 fold higher among IMS than CMS. Sera levels of IgG1, IgG2a, IgG2b and IgG3 although did not significantly differ, were respectively 1.87, 1.36, 1.86 and1.58 fold higher among IMS than CMS.
One week post 2nd immunization, levels of IgA nearly did not change, while, IgG level remained significantly higher as post 1st immunization than CMS. Level of IgM was 1.29 fold higher among IMS than CMS, yet, such increase was not significant. While levels of IgG1, IgG2a, IgG2b and IgG3 were respectively 1.35, 1.75, 1.4, 1.58 and fold higher among IMS than CMS, yet, such increase was not significant.
Immunization with SEA and using SEA as an antigen in ELISA:
One week post 1st immunization, sera had significantly higher levels of IgM than CMS. Although levels of IgA and IgG did not significantly vary they were respectively 3 and 2.8 fold higher among IMS than CMS. Level of IgG2b was significantly higher among IMS than CMS. Although levels of IgG1, IgG2a, and IgG3 did not significantly vary, the values were almost 5 fold higher among IMS than CMS.
One week post 2nd immunization, levels of IgM, IgG and IgA were significantly higher in IMS than CMS. Although levels of IgG1, IgG2a, IgG2b and IgG3 did not significantly vary, the values were respectively 6.1, 3.5, 2.8 and 6.5 fold higher among IMS than CMS.
Characterization of the immunogenic peptides in different schistosomal antigens by Western blotting using individual sera from immunized mice
1. Characterization of the immunogenic CAP:
A. IgG reactivity in sera from mice immunized with CAP against SDS-PAGE fractionated CAP:
IgG reactivity of CAP-IMS were further tested against SDS- PAGE fractionated CAP on Western blots (Figure 2; strips 1-4). Results demonstrated that individual sera from mice that received single immunization (Figure 2; strips 1-4) recognized peptides at 65, 78, 85, 92 and 97 kDa. Only 2 individuals recognized peptides at 31 kDa (strips 2&3) and 40 kDa (strips 1&2). Booster CAP- IMS (strips 5-7) recognized peptides at 23, 48, 63, 71, 78, 85, 92 and 97 kDa. Only 2 individuals strongly reacted to a 40 kDa peptide (strips 6-7). Noteworthy, CAP-peptides at 65, 80, 85, 92 and 97 kDa were poorly specific as they cross reacted with CMS (Figure2; strip 8).
Cross reactivity of sera from immunized mice with SEA that were IgG reactive in ELISA was further tested against SDS-PAGE fractionated CAP antigen (Figure4) where strips 1-3 were treated with SIMS while strips 4&5 with BIMS and strip 6 with CMS.
SEA-SIMS (strips 1-3) or BIMS (strips 4&5) as well as CMS (strip 6) cross reacted with peptide bands at 65, 80, 85, 91 and 97 kDa reflecting poor specificity of such peptides.
B. Cross reactivity of sera from immunized mice with SWAP against SDS-PAGE fractionated CAP:
Cross reactivity of sera from immunized mice with SWAP that were IgG reactive in ELISA was further tested against SDS-PAGE fractionated CAP antigen (Figure 3) where strips 1-3 were treated with individual single IMS (SIMS) while strips 4-6 with booster IMS (BIMS) and strip 7 with CMS. SWAP-SIMS (strips 1-3) or BIMS (strips 4-6) and CMS (strip 7) recognized peptide bands at 65, 80, 85, 91 and 97 kDa, except for strip 5 which did not show any reactivity. Noteworthy, the previous CAP-peptides were poorly specific as they cross reacted as they recognized with CMS (Figure 3; strip 7).
C. Cross reactivity of sera from immunized mice with SEA against SDS-PAGE fractionated CAP:
Cross reactivity of sera from immunized mice with SWAP that were IgG reactive in ELISA was further tested against SDS-PAGE fractionated CAP antigen (Figure 3) where strips 1-3 were treated with individual single IMS (SIMS) while strips 4-6 with booster IMS (BIMS) and strip 7 with CMS. SWAP-SIMS (strips 1-3) or BIMS (strips 4-6) and CMS (strip 7) recognized peptide bands at 65, 80, 85, 91 and 97 kDa, except for strip 5 which did not show any reactivity. Noteworthy, the previous CAP-peptides were poorly specific as they cross reacted as they recognized with CMS (Figure 3; strip 7).
2. Characterization of the immunogenic SWAP peptides:
A. IgG reactivity in sera from mice immunized with SWAP antigen against SDS-PAGE fractionated SWAP:
Among SWAP-SIMS (Figure 5), two (strips 2&3) recognized SWAP peptides at 45, 50, 53, 59 and 75 kDa, while, the rest recognized peptide bands at 48 and 53 kDa (strip 1). Of the SWAP-BIMS, 2 recognized SWAP peptide band at 40 kDa (strips5&6), whereas the rest reacted with peptide bands at 50 and 57 kDa (strip 4). The CMS (strip 7) did not react to any of the above mentioned SWAP-peptides.
B. Cross reactivity of sera from immunized mice with CAP against
SDS-PAGE fractionated SWAP antigen:
IgG reactive CAP-IMS (Figure 6) in ELISA were tested for their cross reactivity against SWAP. Strips 1-3 were reacted with individual CAP-SIMS, while, strips 4-6 were treated with individual CAP-BIMS and strip 7 with CMS.
Among CAP-IMS, only one serum (Figure 6; strip 3) cross reacted with SWAP peptides at 30, 59 and 70 kDa. All CAP-BIMS recognized peptide band at 50 kDa (Figure 6). Both the 50 and 59 kDa were previously recognized by SWAP-IMS.
C. Cross reactivity of sera from immunized mice with SEA against SDS-PAGE fractionated SWAP:
IgG reactive SEA-IMS (Figure 7) in ELISA were tested for their cross reactivity against SWAP. Strips 1-3 were treated with individual SEA-SIMS, while, strips 4-6 were reacted with individual booster SEA-SIMS and strip 7 with CMS.
SEA-SIMS did not react with any of SWAP fractionated peptides, whereas two individual sera from SEA-BIMS (Figure 7; strips 4-6) recognized a band at 40 kDa, which was also previously recorded by SWAP-IMS. CMS did not show cross reactivity with any of the SWAP peptides (Figure 7; strip 7).
3. Characterization of the immunogenic SEA peptides:
A. IgG reactivity in sera from mice immunized with SEA against SDS-PAGE fractionated SEA:
SEA-SIMS (Figure 8; strips 1&2) or BIMS (strips 3&4) recognized SEA peptides at 33, 55, 96 & 101 kDa, while, only two IMS (strip 2&3) strongly reacted to additional peptides at 44, 61, 69, 71, 78 and 84 kDa. The CMS (Figure 8; strip 5) did not react to any of the above mentioned bands.
B. Cross reactivity of sera from immunized mice with CAP against SDS-PAGE fractionated SEA:
IgG reactive CAP-IMS (Figure 9) in ELISA were tested for their cross reactivity against SEA, where (strips 1&2) were treated with CAP-SIMS, while, (strips 3&4) with CAP-BIMS and strip 5 with CMS. CAP-IMS weakly cross reacted to 101, 107 & 111 kDa SEA peptides while only 2 individual CAP-IMS (Figure 9; strips 2&3) cross reacted with two extra bands at 63 and 66 kDa. Noteworthy, the 101 kDa SEA-peptide was previously recognized by SEA-IMS. The CMS (Figure 9; strip 5) did not react to any of the above mentioned bands.
C. Cross reactivity of sera from immunized mice with SWAP against SDS-PAGE fractionated SEA antigen:
IgG reactive SWAP-IMS (Figure 10) in ELISA were tested for their cross reactivity against SEA, where (strips 1&2) were treated with SWAP-SIMS, while, (strips 3&4) with SWAP-BIMS and strip 5 with CMS. SWAP-SIMS weakly cross reacted to 51, 55, 73, and 103 kDa SEA peptides (Figure 10; strip 1&2), while BIMS recognized two sharp bands at ~ 55 and 71 kDa (Figure 10; strip 3&4). The 71 kDa was previously recognized with SEA-IMS and the CMS did not react to any of the above mentioned SEA-peptides (Figure 10; strip 5).
VI. Cellular immune responses after infection or after immunization with S. mansoni antigens: percentage positive CD4+, CD8+-T and B-lymphocytes
A. At 30 days post infection:
At 30 days post infection, the mean percentages of MLNs CD4+, CD8+-T & B-lymphocytes as well as CD4+, CD8+-T & B-splenocytes were significantly higher among infected mice than CM. Noteworthy, CD4+ and CD8+-T thymocytes were nearly unchanged upon infection.
B. Immunized mice with CAP
A week post 1st immunization, the mean percentages CD4+, CD8+-T & B-lymphocytes from MLNs, CD8+-T thymocytes and CD4+ as well as B-splenocytes were significantly higher in IM than CM. Although mean percentages CD4+-T thymocytes and CD8+-T splenocytes were almost 1.5 fold higher in tissues from IM than CM, this difference was not significant.
One week post 2nd immunization, MLNs CD4+, CD8+-T & B-lymphocytes, CD4+-T and B-splenocytes remained significantly higher in IM than CM. Although mean percentages CD4+-T thymocytes & CD8+-T splenocytes were 1.47 and 1.69 fold higher in IM than CM this difference was not significant, while, mean percentage CD8+-T thymocytes was nearly unchanged in IM.
C. Immunized mice with SWAP:
A week post 1st immunization, the mean percentages MLNs CD8+_T lymphocytes, CD4+-T thymocytes and B-splenocytes were significantly higher in IM than CM. Although mean percentages CD4+-T, B-lymphocytes from MLNs, CD8+-T splenocytes and thymocytes in IM were 1.65, 1.45, 1.3 and 1.29 fold higher than in CM, these differences were not significant.
One week post 2nd immunization, the mean percentages CD8+-T, B-lymphocytes from MLNs, CD4+-, CD8+-T thymocytes, CD4+-T, and B- splenocytes from IM were significantly higher than CM. Although mean percentages MLNs CD4+-T lymphocytes, and CD8+-T splenocytes were 1.97 and 1.72 fold higher in IM than CM such differences were not significant.
D. Immunized mice with SEA post 1st immunization
A week post 1st immunization, the mean percentages CD4+-T cells from all tested lymphoid organs and B-splenocytes were significantly higher in IM than CM. Although the mean percentages MLNs CD8+-T, B-lymphocytes and CD8+-T splenocytes were respectively 1.4, 1.6 and 1.6 fold higher in IM than CM, such differences were not significant.
One week after 2nd immunization, the mean percentages MLNs CD4+-T thymocytes, CD8+-T and B-splenocytes were significantly higher among IM than CM. Although mean percentage CD4+-T thymocytes was 1.4 fold higher in IM than CM, this increase was not significant. CD8+-T thymocytes was nearly unchanged upon immunization.
In conclusion, varieties of both cellular and humoral immune responses are induced upon immunization with crude antigens prepared from different developmentary stages of the parasite S. mansoni. Although antigens from the, infective stage, cercariae were reported to be transiently present, CAP was the most immunogenic as it stimulated, CD4+, CD8+ T- and B-cells as well as Ig subclasses suggested to be protective in previous vaccination trials. In contrast, while SWAP was the poorest stimulator for antibody response, it mildly stimulated cellular response confirming the repeated disappointing protection results upon using worm stage antigens in immunization trials. The SEA- specific CD4+T response confirms the evidence that egg antigens have regulatory effect on modulating granuloma formation.
Since both cellular and humoral responses are required for protection against schistosomiasis.
Also our Western blotting results support the hypothesis of immunological cross reactivity existing between cercariae, adult worms and egg stages of the parasite S. mansoni.
Comparison of the influence of infection or immunization on the nature of immunoglobuline classes and subclasses responses against different developmental stages revealed that IgM, IgG, IgG1 and IgG2a were generally higher in IMS with different antigens than in INMS against CAP and SWAP antigens while the level of IgA in INMS was generally higher in infection than in IMS, in this respect immunization lead to recruitment of antibodies necessary for protection in vaccination trials or resistance to re-infection and such response could not be stimulated in higher levels post natural infection. We believe that there is a need for more studies to define and characterize the appropriate antigens’ combinations capable of stimulating immunoprotective responses regardless of the source stage.