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Protection against Schistosoma mansoni infection using a Fasciola hepatica-derived fatty acid binding protein from different delivery systems.

Vicente B, López-Abán J, Rojas-Caraballo J, del Olmo E, Fernández-Soto P, Muro A - Parasit Vectors (2016)

Bottom Line: In contrast, mice vaccinated with rFh15b showed only reductions in eggs trapped in the liver and intestine (53 and 60%, respectively), and hepatic lesions (45%).Moreover, mice immunised with rFh15b showed an increase in IFNγ and a decrease in B220 cells compared to untreated mice, and less production of IgG1 and IgM than in mice immunised by rFh15.Native FABP is more effective than both recombinant systems.

View Article: PubMed Central - PubMed

Affiliation: Parasite and Molecular Immunology Laboratory, Tropical Disease Research Centre, Universidad de Salamanca (IBSAL-CIETUS), Avda. Licenciado Méndez Nieto s/n, 37007, Salamanca, Spain.

ABSTRACT

Background: Schistosomiasis is a water-borne disease afflicting over 261 million people in many areas of the developing countries with high morbidity and mortality. The control relies mainly on treatment with praziquantel. Fatty acid binding proteins (FABP) have demonstrated high levels of immune-protection against trematode infections. This study reports the immunoprotection induced by cross-reacting Fasciola hepatica FABP, native (nFh12) and recombinantly expressed using two different expression systems Escherichia coli (rFh15) and baculovirus (rFh15b) against Schistosoma mansoni infection.

Methods: BALB/c mice were vaccinated with native nFh12 or recombinant rFh15 and rFh15 FABP from F. hepatica formulated in adjuvant adaptation (ADAD) system with natural or chemical synthesised immunomodulators (PAL and AA0029) and then challenged with 150 cercariae of S. mansoni. Parasite burden, hepatic lesions and antibody response were studied in vaccination trials. Furthermore differences between rFh15 and rFh15b immunological responses (cytokine production, splenocyte population and antibody levels) were studied.

Results: Vaccination with nFh12 induced significant reductions in worm burden (83%), eggs in tissues (82-92%) and hepatic lesions (85%) compared to infected controls using PAL. Vaccination with rFh15 showed lower total worm burden (56-64%), eggs in the liver (21-61%), eggs in the gut (30-77%) and hepatic damage (67-69%) using PAL and AA0029 as immunomodulators. In contrast, mice vaccinated with rFh15b showed only reductions in eggs trapped in the liver and intestine (53 and 60%, respectively), and hepatic lesions (45%). We observed a significant rise in TNFα, IL-6, IL-2, IL-4 and high antibody response (IgG, IgG1, IgG2a, IgM and IgE) in mice immunised with either rFh15 or rFh15b. Moreover, mice immunised with rFh15b showed an increase in IFNγ and a decrease in B220 cells compared to untreated mice, and less production of IgG1 and IgM than in mice immunised by rFh15.

Conclusions: Higher level of protection is obtained by using Fasciola hepatica-derived FABP protein against Schistosoma mansoni infection. Native FABP is more effective than both recombinant systems. It could be due to post-translational modifications or FABP isoform or changes in the recombinant proteins.

No MeSH data available.


Related in: MedlinePlus

The expression and purification of rFh15b using the baculovirus system. a The generated vector pFBFh15His; b The nucleotide sequence from Fh15, including the Kozak sequence, the C-terminus 6-His tag and the restriction sequences for BamHI and Xbal; c The expression of rFh15b detected with Coomassie blue staining (Lane 1, molecular weight marker; Lane 2: non-induced baculovirus; Lane 3, induced baculovirus) and Western blot using anti-6His monoclonal antibody (Lane 1, molecular weight marker; Lane 2, induced baculovirus; Lane 3: non-induced baculovirus); d Purification of rFh15b by affinity chromatography detected by Coomassie blue staining and Western blot using anti-6His monoclonal antibody
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Fig1: The expression and purification of rFh15b using the baculovirus system. a The generated vector pFBFh15His; b The nucleotide sequence from Fh15, including the Kozak sequence, the C-terminus 6-His tag and the restriction sequences for BamHI and Xbal; c The expression of rFh15b detected with Coomassie blue staining (Lane 1, molecular weight marker; Lane 2: non-induced baculovirus; Lane 3, induced baculovirus) and Western blot using anti-6His monoclonal antibody (Lane 1, molecular weight marker; Lane 2, induced baculovirus; Lane 3: non-induced baculovirus); d Purification of rFh15b by affinity chromatography detected by Coomassie blue staining and Western blot using anti-6His monoclonal antibody

Mentions: The second method to obtain the recombinant rFh15 protein was based on the use of a baculovirus expression vector system, using standardised protocols of ALGENEX (Madrid, Spain). Briefly, to clone into pFasBacHis vector a nucleotide sequence from 15 kDa FABP protein (GenBank M95291.1) was synthesised and a Kozak sequence was inserted into the N-terminus extreme, along with BamHI and XbaI restriction sites at the N- and C-terminus respectively. The plasmid pMA (ampR) with the cloned Fh15 gene between KpnI/Sacl sites was used to amplify DNA by transformation of E. coli (DH5alpha) cells and isolation of ampicillin-resistant colonies. The resulting amplified DNA together with the cloning vector (pFasBacHis) were cut with restriction enzymes BamHI and Xbal and the corresponding band (412 bp) from Fh15 insert was isolated and purified. pFasBacHis vector was dephosphorylated with alkaline phosphatase treatment and the Fh15 insert was subsequently ligated. The resulting product was then used to transform E. coli (DH5alpha) cells and ampicillin- and gentamicin-resistant colonies were then isolated. The DNA from these isolated colonies was isolated and characterised using the restriction enzymes for BamHI and Xbal sites, respectively, and automated sequence was performed to verify the sequence of the insert. The resulting vector and the sequence of the Fh15 insert is depicted in Fig. 1a. To obtain the recombinant baculovirus, E. coli special competent (DH10B) cells were transformed starting from a previously generated vector (pFBFh15His). These cells carry the receptor bMON14272 that contains a beta-galactosidase codifying gene. Upon incorporation in the same cell vector and receptor, the recombinant baculovirus presents resistance to kanamycin, tetracyclin and geneticin and loses its beta-galactosidase activity. One colony resistant to the three antibiotics was selected and the DNA was isolated and used to transfect insect cells sf21 using the cellfectin reagent (Invitrogen, Waltham, MA USA). Seventy-two hours after the transfection, the so-called progeny 1 from the recombinant baculovirus was collected and stored until further use. Finally, thirty Trichoplusia ni larvae were inoculated with the previously obtained recombinant virus. Larvae were harvested during the next 48–96 h and the expression of the recombinant protein was assessed using both Coomassie blue staining and Western blot with monoclonal anti-6His antibodies.Fig. 1


Protection against Schistosoma mansoni infection using a Fasciola hepatica-derived fatty acid binding protein from different delivery systems.

Vicente B, López-Abán J, Rojas-Caraballo J, del Olmo E, Fernández-Soto P, Muro A - Parasit Vectors (2016)

The expression and purification of rFh15b using the baculovirus system. a The generated vector pFBFh15His; b The nucleotide sequence from Fh15, including the Kozak sequence, the C-terminus 6-His tag and the restriction sequences for BamHI and Xbal; c The expression of rFh15b detected with Coomassie blue staining (Lane 1, molecular weight marker; Lane 2: non-induced baculovirus; Lane 3, induced baculovirus) and Western blot using anti-6His monoclonal antibody (Lane 1, molecular weight marker; Lane 2, induced baculovirus; Lane 3: non-induced baculovirus); d Purification of rFh15b by affinity chromatography detected by Coomassie blue staining and Western blot using anti-6His monoclonal antibody
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836169&req=5

Fig1: The expression and purification of rFh15b using the baculovirus system. a The generated vector pFBFh15His; b The nucleotide sequence from Fh15, including the Kozak sequence, the C-terminus 6-His tag and the restriction sequences for BamHI and Xbal; c The expression of rFh15b detected with Coomassie blue staining (Lane 1, molecular weight marker; Lane 2: non-induced baculovirus; Lane 3, induced baculovirus) and Western blot using anti-6His monoclonal antibody (Lane 1, molecular weight marker; Lane 2, induced baculovirus; Lane 3: non-induced baculovirus); d Purification of rFh15b by affinity chromatography detected by Coomassie blue staining and Western blot using anti-6His monoclonal antibody
Mentions: The second method to obtain the recombinant rFh15 protein was based on the use of a baculovirus expression vector system, using standardised protocols of ALGENEX (Madrid, Spain). Briefly, to clone into pFasBacHis vector a nucleotide sequence from 15 kDa FABP protein (GenBank M95291.1) was synthesised and a Kozak sequence was inserted into the N-terminus extreme, along with BamHI and XbaI restriction sites at the N- and C-terminus respectively. The plasmid pMA (ampR) with the cloned Fh15 gene between KpnI/Sacl sites was used to amplify DNA by transformation of E. coli (DH5alpha) cells and isolation of ampicillin-resistant colonies. The resulting amplified DNA together with the cloning vector (pFasBacHis) were cut with restriction enzymes BamHI and Xbal and the corresponding band (412 bp) from Fh15 insert was isolated and purified. pFasBacHis vector was dephosphorylated with alkaline phosphatase treatment and the Fh15 insert was subsequently ligated. The resulting product was then used to transform E. coli (DH5alpha) cells and ampicillin- and gentamicin-resistant colonies were then isolated. The DNA from these isolated colonies was isolated and characterised using the restriction enzymes for BamHI and Xbal sites, respectively, and automated sequence was performed to verify the sequence of the insert. The resulting vector and the sequence of the Fh15 insert is depicted in Fig. 1a. To obtain the recombinant baculovirus, E. coli special competent (DH10B) cells were transformed starting from a previously generated vector (pFBFh15His). These cells carry the receptor bMON14272 that contains a beta-galactosidase codifying gene. Upon incorporation in the same cell vector and receptor, the recombinant baculovirus presents resistance to kanamycin, tetracyclin and geneticin and loses its beta-galactosidase activity. One colony resistant to the three antibiotics was selected and the DNA was isolated and used to transfect insect cells sf21 using the cellfectin reagent (Invitrogen, Waltham, MA USA). Seventy-two hours after the transfection, the so-called progeny 1 from the recombinant baculovirus was collected and stored until further use. Finally, thirty Trichoplusia ni larvae were inoculated with the previously obtained recombinant virus. Larvae were harvested during the next 48–96 h and the expression of the recombinant protein was assessed using both Coomassie blue staining and Western blot with monoclonal anti-6His antibodies.Fig. 1

Bottom Line: In contrast, mice vaccinated with rFh15b showed only reductions in eggs trapped in the liver and intestine (53 and 60%, respectively), and hepatic lesions (45%).Moreover, mice immunised with rFh15b showed an increase in IFNγ and a decrease in B220 cells compared to untreated mice, and less production of IgG1 and IgM than in mice immunised by rFh15.Native FABP is more effective than both recombinant systems.

View Article: PubMed Central - PubMed

Affiliation: Parasite and Molecular Immunology Laboratory, Tropical Disease Research Centre, Universidad de Salamanca (IBSAL-CIETUS), Avda. Licenciado Méndez Nieto s/n, 37007, Salamanca, Spain.

ABSTRACT

Background: Schistosomiasis is a water-borne disease afflicting over 261 million people in many areas of the developing countries with high morbidity and mortality. The control relies mainly on treatment with praziquantel. Fatty acid binding proteins (FABP) have demonstrated high levels of immune-protection against trematode infections. This study reports the immunoprotection induced by cross-reacting Fasciola hepatica FABP, native (nFh12) and recombinantly expressed using two different expression systems Escherichia coli (rFh15) and baculovirus (rFh15b) against Schistosoma mansoni infection.

Methods: BALB/c mice were vaccinated with native nFh12 or recombinant rFh15 and rFh15 FABP from F. hepatica formulated in adjuvant adaptation (ADAD) system with natural or chemical synthesised immunomodulators (PAL and AA0029) and then challenged with 150 cercariae of S. mansoni. Parasite burden, hepatic lesions and antibody response were studied in vaccination trials. Furthermore differences between rFh15 and rFh15b immunological responses (cytokine production, splenocyte population and antibody levels) were studied.

Results: Vaccination with nFh12 induced significant reductions in worm burden (83%), eggs in tissues (82-92%) and hepatic lesions (85%) compared to infected controls using PAL. Vaccination with rFh15 showed lower total worm burden (56-64%), eggs in the liver (21-61%), eggs in the gut (30-77%) and hepatic damage (67-69%) using PAL and AA0029 as immunomodulators. In contrast, mice vaccinated with rFh15b showed only reductions in eggs trapped in the liver and intestine (53 and 60%, respectively), and hepatic lesions (45%). We observed a significant rise in TNFα, IL-6, IL-2, IL-4 and high antibody response (IgG, IgG1, IgG2a, IgM and IgE) in mice immunised with either rFh15 or rFh15b. Moreover, mice immunised with rFh15b showed an increase in IFNγ and a decrease in B220 cells compared to untreated mice, and less production of IgG1 and IgM than in mice immunised by rFh15.

Conclusions: Higher level of protection is obtained by using Fasciola hepatica-derived FABP protein against Schistosoma mansoni infection. Native FABP is more effective than both recombinant systems. It could be due to post-translational modifications or FABP isoform or changes in the recombinant proteins.

No MeSH data available.


Related in: MedlinePlus