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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

Lymphoproliferative and CTL responses to HCVcp and pCHCORE DNA vaccine immunizations. (a) Lymphocytes isolated from the spleens of vaccinated mice were cultured and pulsed with C39 peptide. The proliferative response was measured by cell proliferation ELISA, BrdU colorimetric kit (Roche Diagnostics, Germany) as per the manufacturer's protocol. The data represents mean SI of two determinations ± S.D. The figure is representative of two different experiments with similar observations (* indicates statistical significance, P value < 0.05). (b) In vivo CTL responses measurement of the relative proportion of CFSEhigh and CFSElow cells by fl ow cytometry via a FACS Calibur (BD Biosciences) indicated that Mice immunized with pCHCORE and HCVc P + F127 showed significantly higher specific lysis than control groups with no significant difference between both groups
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Figure 3: Lymphoproliferative and CTL responses to HCVcp and pCHCORE DNA vaccine immunizations. (a) Lymphocytes isolated from the spleens of vaccinated mice were cultured and pulsed with C39 peptide. The proliferative response was measured by cell proliferation ELISA, BrdU colorimetric kit (Roche Diagnostics, Germany) as per the manufacturer's protocol. The data represents mean SI of two determinations ± S.D. The figure is representative of two different experiments with similar observations (* indicates statistical significance, P value < 0.05). (b) In vivo CTL responses measurement of the relative proportion of CFSEhigh and CFSElow cells by fl ow cytometry via a FACS Calibur (BD Biosciences) indicated that Mice immunized with pCHCORE and HCVc P + F127 showed significantly higher specific lysis than control groups with no significant difference between both groups

Mentions: To evaluate the lymphocyte proliferation response, splenocytes of immunized mice were stimulated with C39 peptide and incorporation of 5-bromo-2 deoxyuridine (BrdU) into the stimulated splenocytes was detected by ELISA as described in the method section. Results indicated that the lymphocytes obtained from the animals immunized with HCVcp and DNA vaccine proliferated significantly higher compared with control groups (saline, F127 and pCHBS) following in vitro stimulation with peptide C39 [Figure 3a]. Additionally, the proliferative response of animals that were immunized with pCHCORE had a significantly higher SI mean compared with those immunized with HCVc P + F127 (P < 0.05).


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)

Lymphoproliferative and CTL responses to HCVcp and pCHCORE DNA vaccine immunizations. (a) Lymphocytes isolated from the spleens of vaccinated mice were cultured and pulsed with C39 peptide. The proliferative response was measured by cell proliferation ELISA, BrdU colorimetric kit (Roche Diagnostics, Germany) as per the manufacturer's protocol. The data represents mean SI of two determinations ± S.D. The figure is representative of two different experiments with similar observations (* indicates statistical significance, P value < 0.05). (b) In vivo CTL responses measurement of the relative proportion of CFSEhigh and CFSElow cells by fl ow cytometry via a FACS Calibur (BD Biosciences) indicated that Mice immunized with pCHCORE and HCVc P + F127 showed significantly higher specific lysis than control groups with no significant difference between both groups
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Lymphoproliferative and CTL responses to HCVcp and pCHCORE DNA vaccine immunizations. (a) Lymphocytes isolated from the spleens of vaccinated mice were cultured and pulsed with C39 peptide. The proliferative response was measured by cell proliferation ELISA, BrdU colorimetric kit (Roche Diagnostics, Germany) as per the manufacturer's protocol. The data represents mean SI of two determinations ± S.D. The figure is representative of two different experiments with similar observations (* indicates statistical significance, P value < 0.05). (b) In vivo CTL responses measurement of the relative proportion of CFSEhigh and CFSElow cells by fl ow cytometry via a FACS Calibur (BD Biosciences) indicated that Mice immunized with pCHCORE and HCVc P + F127 showed significantly higher specific lysis than control groups with no significant difference between both groups
Mentions: To evaluate the lymphocyte proliferation response, splenocytes of immunized mice were stimulated with C39 peptide and incorporation of 5-bromo-2 deoxyuridine (BrdU) into the stimulated splenocytes was detected by ELISA as described in the method section. Results indicated that the lymphocytes obtained from the animals immunized with HCVcp and DNA vaccine proliferated significantly higher compared with control groups (saline, F127 and pCHBS) following in vitro stimulation with peptide C39 [Figure 3a]. Additionally, the proliferative response of animals that were immunized with pCHCORE had a significantly higher SI mean compared with those immunized with HCVc P + F127 (P < 0.05).

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