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Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice.

Yazdanian M, Memarnejadian A, Mahdavi M, Motevalli F, Sadat SM, Vahabpour R, Khanahmad H, Soleimanjahi H, Budkowska A, Roohvand F - Adv Biomed Res (2015)

Bottom Line: The cellular immune responses (proliferation, In vivo CTL assay and IFN-γ/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8(+)-epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals.Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting.Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.

ABSTRACT

Background: Fusion of Hepatitis B virus surface antigen (HBsAg) to a DNA construct might be considered as a strategy to enhance cellular and cytotoxic T-lymphocytes (CTL) responses of a Hepatitis C Virus core protein (HCVcp)-based DNA vaccine comparable to that of adjuvanted protein (subunit) immunization.

Materials and methods: pCHCORE vector harboring coding sequence of HBsAg and HCVcp (amino acids 2-120) in tandem within the pCDNA3.1 backbone was constructed. The corresponding recombinant HCVcp was also expressed and purified in Escherichia coli. Mice were immunized either by adjuvanted HCVcp (pluronic acid + protein) or by pCHCORE vector primed/protein boosted immunization regimen. The cellular immune responses (proliferation, In vivo CTL assay and IFN-γ/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8(+)-epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals.

Result: Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting. The immunization results indicated that the pCHCORE shifted the immune responses pathway to Th1 by enhancing the IFN-γ cytokine level much higher than protein immunization while the proliferative and CTL responses were comparable (or slightly in favor of DNA immunization).

Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization.

No MeSH data available.


Related in: MedlinePlus

In vitro expression analysis of pCHCORE plasmid by western blotting. Assessment of the expression for HBsAg-HCVcp fusion protein in cell lysates of pCHCORE transfected HEK 293T cells was carried out by using anti-HBsAg polyclonal antibody. In lane 1 of the blotted membrane presentation of the doublet bands in expected sizes (≈50 kDa) (indicated arrows) are due to the presence of both glycosylated and nonglycosylated forms of HBsAg-HCVcp fusion protein. Lane 2: Protein MW marker
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Figure 2: In vitro expression analysis of pCHCORE plasmid by western blotting. Assessment of the expression for HBsAg-HCVcp fusion protein in cell lysates of pCHCORE transfected HEK 293T cells was carried out by using anti-HBsAg polyclonal antibody. In lane 1 of the blotted membrane presentation of the doublet bands in expected sizes (≈50 kDa) (indicated arrows) are due to the presence of both glycosylated and nonglycosylated forms of HBsAg-HCVcp fusion protein. Lane 2: Protein MW marker

Mentions: Construction of pCHCORE vector by insertion of HCVcp (2-120) DNA sequence in pCHBS (HBsAg harboring pCDNA3.1 plasmid;[11] Figure 1a) is schematically presented in Figure 1b. Analysis by restriction enzymes followed by sequencing reactions approved the accuracy of the chimeric expression vector (results not shown). Analysis of the lysates of pCHCORE transfected HEK293T cells by western blotting indicated the in vitro expression of the HBsAg-HCVcp fusion protein with the expected size of around 50 kDa in the doublet shaped bands, which demonstrated the occurrence of both glycosysated and nonglycolsylated forms of the fused protein [Figure 2]. Accordingly and in agreement with our previous results,[202324] coomassie blue-stained gel of SDS-PAGE and western blot analyses of arabinose-induced lysates of BL21-AI strain of E. coli cells harboring pIVEX2.4a-core vector, indicated a 21-kDa protein band corresponding to HCVcp (residues 2-122). SDS-PAGE and densitometry analysis of the Ni-NTA chromatography-based purified protein demonstrated over 85% purification and less than 25 endotoxin units per 50 μg of the protein as previously described[20] (results are not shown due to their presence in our previous publications, which are accordingly referred here).


Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice.

Yazdanian M, Memarnejadian A, Mahdavi M, Motevalli F, Sadat SM, Vahabpour R, Khanahmad H, Soleimanjahi H, Budkowska A, Roohvand F - Adv Biomed Res (2015)

In vitro expression analysis of pCHCORE plasmid by western blotting. Assessment of the expression for HBsAg-HCVcp fusion protein in cell lysates of pCHCORE transfected HEK 293T cells was carried out by using anti-HBsAg polyclonal antibody. In lane 1 of the blotted membrane presentation of the doublet bands in expected sizes (≈50 kDa) (indicated arrows) are due to the presence of both glycosylated and nonglycosylated forms of HBsAg-HCVcp fusion protein. Lane 2: Protein MW marker
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300588&req=5

Figure 2: In vitro expression analysis of pCHCORE plasmid by western blotting. Assessment of the expression for HBsAg-HCVcp fusion protein in cell lysates of pCHCORE transfected HEK 293T cells was carried out by using anti-HBsAg polyclonal antibody. In lane 1 of the blotted membrane presentation of the doublet bands in expected sizes (≈50 kDa) (indicated arrows) are due to the presence of both glycosylated and nonglycosylated forms of HBsAg-HCVcp fusion protein. Lane 2: Protein MW marker
Mentions: Construction of pCHCORE vector by insertion of HCVcp (2-120) DNA sequence in pCHBS (HBsAg harboring pCDNA3.1 plasmid;[11] Figure 1a) is schematically presented in Figure 1b. Analysis by restriction enzymes followed by sequencing reactions approved the accuracy of the chimeric expression vector (results not shown). Analysis of the lysates of pCHCORE transfected HEK293T cells by western blotting indicated the in vitro expression of the HBsAg-HCVcp fusion protein with the expected size of around 50 kDa in the doublet shaped bands, which demonstrated the occurrence of both glycosysated and nonglycolsylated forms of the fused protein [Figure 2]. Accordingly and in agreement with our previous results,[202324] coomassie blue-stained gel of SDS-PAGE and western blot analyses of arabinose-induced lysates of BL21-AI strain of E. coli cells harboring pIVEX2.4a-core vector, indicated a 21-kDa protein band corresponding to HCVcp (residues 2-122). SDS-PAGE and densitometry analysis of the Ni-NTA chromatography-based purified protein demonstrated over 85% purification and less than 25 endotoxin units per 50 μg of the protein as previously described[20] (results are not shown due to their presence in our previous publications, which are accordingly referred here).

Bottom Line: The cellular immune responses (proliferation, In vivo CTL assay and IFN-γ/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8(+)-epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals.Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting.Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.

ABSTRACT

Background: Fusion of Hepatitis B virus surface antigen (HBsAg) to a DNA construct might be considered as a strategy to enhance cellular and cytotoxic T-lymphocytes (CTL) responses of a Hepatitis C Virus core protein (HCVcp)-based DNA vaccine comparable to that of adjuvanted protein (subunit) immunization.

Materials and methods: pCHCORE vector harboring coding sequence of HBsAg and HCVcp (amino acids 2-120) in tandem within the pCDNA3.1 backbone was constructed. The corresponding recombinant HCVcp was also expressed and purified in Escherichia coli. Mice were immunized either by adjuvanted HCVcp (pluronic acid + protein) or by pCHCORE vector primed/protein boosted immunization regimen. The cellular immune responses (proliferation, In vivo CTL assay and IFN-γ/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8(+)-epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals.

Result: Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting. The immunization results indicated that the pCHCORE shifted the immune responses pathway to Th1 by enhancing the IFN-γ cytokine level much higher than protein immunization while the proliferative and CTL responses were comparable (or slightly in favor of DNA immunization).

Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization.

No MeSH data available.


Related in: MedlinePlus