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Fusion protein comprised of the two schistosomal antigens, Sm14 and Sm29, provides significant protection against Schistosoma mansoni in murine infection model.

Mossallam SF, Amer EI, Ewaisha RE, Khalil AM, Aboushleib HM, Bahey-El-Din M - BMC Infect. Dis. (2015)

Bottom Line: In the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29.In addition, significant reduction of tissue egg burdens was observed in mice immunized with the fusion protein when compared with the infected saline/adjuvant negative control groups and groups immunized with the individual Sm14 and Sm29 antigens.The findings presented in this study highlight the importance of the fusion protein FSm14/29 as a potential vaccine candidate that is worthy of further investigation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Alexandria, Egypt. mossallamsh@hotmail.com.

ABSTRACT

Background: Schistosoma mansoni infection represents a major cause of morbidity and mortality in many areas of the developing world. Effective vaccines against schistosomiasis are not available and disease management relies mainly on treatment with the anthelmintic drug praziquantel. Several promising schistosomal antigens have been evaluated for vaccine efficacy such as Sm14, Sm29 and tetraspanins. However, most investigators examine these promising antigens in animal models individually rather than in properly adjuvanted antigen combinations.

Methods: In the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29. The fusion protein, FSm14/29, was administered to Swiss albino mice either unadjuvanted or adjuvanted with polyinosinic-polycytidylic acid adjuvant, poly(I:C). Mice were challenged with S. mansoni cercariae and different parasitological/immunological parameters were assessed seven weeks post-challenge. Data were analyzed using the ANOVA test with post-hoc Tukey-Kramer test.

Results: Mice pre-immunized with unadjuvanted or poly(I:C)-adjuvanted fusion protein showed reduction of adult worm burden of 44.7 and 48.4%, respectively. In addition, significant reduction of tissue egg burdens was observed in mice immunized with the fusion protein when compared with the infected saline/adjuvant negative control groups and groups immunized with the individual Sm14 and Sm29 antigens. Light microscope and scanning electron microscope (SEM) investigation of adult worms recovered from FSm14/29-immunized mice revealed appreciable morphological damage and tegumental deformities. Histopathological examination of liver sections of immunized mice demonstrated reduced granulomatous and inflammatory reactions when compared with infected unvaccinated mice or mice immunized with the individual Sm14 and Sm29 antigens.

Conclusion: The findings presented in this study highlight the importance of the fusion protein FSm14/29 as a potential vaccine candidate that is worthy of further investigation.

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Related in: MedlinePlus

Illustration of the created plasmids and purified antigens described in the study. (A) Schematic representation of the construction of pQE31-FSm14/29, pQE31-Sm14 and pQE31-Sm29. Amp: β-lactamase coding sequence; ColE1: ColE1 origin of replication; PT5: T5 promoter; lac O: lac operator element; RBS: Ribosomal Binding Site; H: Hexahistidine tag; MCS: Multiple Cloning Site; T: Terminator; B: BamHI recognition site; K: KpnI recognition site; P: PstI recognition site. (B) SDS-PAGE of antigens purified from corresponding induced E. coli M15 (pREP4) strains. Lane 1, protein ladder; lanes 2 and 3, two successive elutions of the fusion antigen FSm14/29; lanes 4 and 5, two successive elutions of Sm14 antigen; lanes 6 and 7, two successive elutions of Sm29 antigen. Buffer E (pH 4.5) was used for elutions under denaturing conditions following induction with 0.3 mM IPTG at 25°C for 16 h.
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Fig1: Illustration of the created plasmids and purified antigens described in the study. (A) Schematic representation of the construction of pQE31-FSm14/29, pQE31-Sm14 and pQE31-Sm29. Amp: β-lactamase coding sequence; ColE1: ColE1 origin of replication; PT5: T5 promoter; lac O: lac operator element; RBS: Ribosomal Binding Site; H: Hexahistidine tag; MCS: Multiple Cloning Site; T: Terminator; B: BamHI recognition site; K: KpnI recognition site; P: PstI recognition site. (B) SDS-PAGE of antigens purified from corresponding induced E. coli M15 (pREP4) strains. Lane 1, protein ladder; lanes 2 and 3, two successive elutions of the fusion antigen FSm14/29; lanes 4 and 5, two successive elutions of Sm14 antigen; lanes 6 and 7, two successive elutions of Sm29 antigen. Buffer E (pH 4.5) was used for elutions under denaturing conditions following induction with 0.3 mM IPTG at 25°C for 16 h.

Mentions: To create a fusion protein comprised of Sm14 and Sm29, herein designated FSm14/29, the following procedure was carried out. The above obtained Sm14 and Sm29 amplicons were used as templates for the following two PCR reactions respectively. First, Sm14 was amplified again using primers 5’- TGAAGGATCCGATGTCTAGTTTCTTGGGAAAGTGGAA-3’ and 5’- ACGCGGTACCGGATAGTCGTTTATAATTGC-3’ with BamHI and KpnI flanking restriction sites, respectively. Second, Sm29 was amplified using primers 5’- TATAGGTACCGTGCGTTGCTACGTCTGTGATTATT-3’ and 5’-ACGCCTGCAGTTATTTTGTCATTCCGTTACATAGAT-3’ with flanking sites of KpnI and PstI, respectively. Both Sm14 and Sm29 amplicons were cut with KpnI then ligated together with T4 DNA ligase and the ligation reaction was used as a template to amplify the fusion gene using Sm14 forward primer and Sm29 reverse primer with BamHI and PstI recognition sites, respectively. The resulting fusion gene amplicon was cut with BamHI and PstI and was ligated to a similarly digested pQE31 plasmid. The resulting pQE31-(FSm14/29) was transformed into E. coli TOP10 as an intermediate cloning host. Plasmid pQE31-(FSm14/29) was subsequently extracted from E. coli TOP10 and transformed into the expression host E. coli M15 (pREP4) by electroporation where cells were plated onto Amp/Km-containing LB agar plates. Positive colonies were confirmed by colony PCR to ensure correct creation of the construct. Plasmids pQE31-Sm14 and pQE31-Sm29 containing the single antigen genes were also created and transformed into E. coli M15 (pREP4) using similar cloning procedures. Figure 1A illustrates the three plasmids created in this study.Figure 1


Fusion protein comprised of the two schistosomal antigens, Sm14 and Sm29, provides significant protection against Schistosoma mansoni in murine infection model.

Mossallam SF, Amer EI, Ewaisha RE, Khalil AM, Aboushleib HM, Bahey-El-Din M - BMC Infect. Dis. (2015)

Illustration of the created plasmids and purified antigens described in the study. (A) Schematic representation of the construction of pQE31-FSm14/29, pQE31-Sm14 and pQE31-Sm29. Amp: β-lactamase coding sequence; ColE1: ColE1 origin of replication; PT5: T5 promoter; lac O: lac operator element; RBS: Ribosomal Binding Site; H: Hexahistidine tag; MCS: Multiple Cloning Site; T: Terminator; B: BamHI recognition site; K: KpnI recognition site; P: PstI recognition site. (B) SDS-PAGE of antigens purified from corresponding induced E. coli M15 (pREP4) strains. Lane 1, protein ladder; lanes 2 and 3, two successive elutions of the fusion antigen FSm14/29; lanes 4 and 5, two successive elutions of Sm14 antigen; lanes 6 and 7, two successive elutions of Sm29 antigen. Buffer E (pH 4.5) was used for elutions under denaturing conditions following induction with 0.3 mM IPTG at 25°C for 16 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389862&req=5

Fig1: Illustration of the created plasmids and purified antigens described in the study. (A) Schematic representation of the construction of pQE31-FSm14/29, pQE31-Sm14 and pQE31-Sm29. Amp: β-lactamase coding sequence; ColE1: ColE1 origin of replication; PT5: T5 promoter; lac O: lac operator element; RBS: Ribosomal Binding Site; H: Hexahistidine tag; MCS: Multiple Cloning Site; T: Terminator; B: BamHI recognition site; K: KpnI recognition site; P: PstI recognition site. (B) SDS-PAGE of antigens purified from corresponding induced E. coli M15 (pREP4) strains. Lane 1, protein ladder; lanes 2 and 3, two successive elutions of the fusion antigen FSm14/29; lanes 4 and 5, two successive elutions of Sm14 antigen; lanes 6 and 7, two successive elutions of Sm29 antigen. Buffer E (pH 4.5) was used for elutions under denaturing conditions following induction with 0.3 mM IPTG at 25°C for 16 h.
Mentions: To create a fusion protein comprised of Sm14 and Sm29, herein designated FSm14/29, the following procedure was carried out. The above obtained Sm14 and Sm29 amplicons were used as templates for the following two PCR reactions respectively. First, Sm14 was amplified again using primers 5’- TGAAGGATCCGATGTCTAGTTTCTTGGGAAAGTGGAA-3’ and 5’- ACGCGGTACCGGATAGTCGTTTATAATTGC-3’ with BamHI and KpnI flanking restriction sites, respectively. Second, Sm29 was amplified using primers 5’- TATAGGTACCGTGCGTTGCTACGTCTGTGATTATT-3’ and 5’-ACGCCTGCAGTTATTTTGTCATTCCGTTACATAGAT-3’ with flanking sites of KpnI and PstI, respectively. Both Sm14 and Sm29 amplicons were cut with KpnI then ligated together with T4 DNA ligase and the ligation reaction was used as a template to amplify the fusion gene using Sm14 forward primer and Sm29 reverse primer with BamHI and PstI recognition sites, respectively. The resulting fusion gene amplicon was cut with BamHI and PstI and was ligated to a similarly digested pQE31 plasmid. The resulting pQE31-(FSm14/29) was transformed into E. coli TOP10 as an intermediate cloning host. Plasmid pQE31-(FSm14/29) was subsequently extracted from E. coli TOP10 and transformed into the expression host E. coli M15 (pREP4) by electroporation where cells were plated onto Amp/Km-containing LB agar plates. Positive colonies were confirmed by colony PCR to ensure correct creation of the construct. Plasmids pQE31-Sm14 and pQE31-Sm29 containing the single antigen genes were also created and transformed into E. coli M15 (pREP4) using similar cloning procedures. Figure 1A illustrates the three plasmids created in this study.Figure 1

Bottom Line: In the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29.In addition, significant reduction of tissue egg burdens was observed in mice immunized with the fusion protein when compared with the infected saline/adjuvant negative control groups and groups immunized with the individual Sm14 and Sm29 antigens.The findings presented in this study highlight the importance of the fusion protein FSm14/29 as a potential vaccine candidate that is worthy of further investigation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Alexandria, Egypt. mossallamsh@hotmail.com.

ABSTRACT

Background: Schistosoma mansoni infection represents a major cause of morbidity and mortality in many areas of the developing world. Effective vaccines against schistosomiasis are not available and disease management relies mainly on treatment with the anthelmintic drug praziquantel. Several promising schistosomal antigens have been evaluated for vaccine efficacy such as Sm14, Sm29 and tetraspanins. However, most investigators examine these promising antigens in animal models individually rather than in properly adjuvanted antigen combinations.

Methods: In the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29. The fusion protein, FSm14/29, was administered to Swiss albino mice either unadjuvanted or adjuvanted with polyinosinic-polycytidylic acid adjuvant, poly(I:C). Mice were challenged with S. mansoni cercariae and different parasitological/immunological parameters were assessed seven weeks post-challenge. Data were analyzed using the ANOVA test with post-hoc Tukey-Kramer test.

Results: Mice pre-immunized with unadjuvanted or poly(I:C)-adjuvanted fusion protein showed reduction of adult worm burden of 44.7 and 48.4%, respectively. In addition, significant reduction of tissue egg burdens was observed in mice immunized with the fusion protein when compared with the infected saline/adjuvant negative control groups and groups immunized with the individual Sm14 and Sm29 antigens. Light microscope and scanning electron microscope (SEM) investigation of adult worms recovered from FSm14/29-immunized mice revealed appreciable morphological damage and tegumental deformities. Histopathological examination of liver sections of immunized mice demonstrated reduced granulomatous and inflammatory reactions when compared with infected unvaccinated mice or mice immunized with the individual Sm14 and Sm29 antigens.

Conclusion: The findings presented in this study highlight the importance of the fusion protein FSm14/29 as a potential vaccine candidate that is worthy of further investigation.

Show MeSH
Related in: MedlinePlus