Limits...
Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice.

Mishra S, Lavelle BJ, Desrosiers J, Ardito MT, Terry F, Martin WD, De Groot AS, Gregory SH - PLoS ONE (2014)

Bottom Line: To date, therapeutic vaccines have demonstrated only limited success.Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array.A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.

ABSTRACT
Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

Show MeSH

Related in: MedlinePlus

IFN-γ ELISpot assays.The spleens were dissected and pooled on day 35 from groups of 4 mice immunized with vaccine construct-transfected, and IFN-γ ELISpot assays were performed in triplicate. The data, expressed as the means ± SD ELISpots/106 splenocytes minus the average negative control, 0.1% DMSO+2 SD (3,346 ELISpots/106 splenocytes), were obtained in a single experiment representative of duplicate experiments. The average number of ELISpots/106 splenocytes derived from mice immunized with non-transfected DCs was not significantly different from the DMSO control (not shown). *Splenocytes derived from mice immunized with transfected DCs and incubated with the HLA-A2- and -DRB1-restricted peptides indicated did not yield values that were significantly different from the DMSO control (ANOVA).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4128787&req=5

pone-0104606-g004: IFN-γ ELISpot assays.The spleens were dissected and pooled on day 35 from groups of 4 mice immunized with vaccine construct-transfected, and IFN-γ ELISpot assays were performed in triplicate. The data, expressed as the means ± SD ELISpots/106 splenocytes minus the average negative control, 0.1% DMSO+2 SD (3,346 ELISpots/106 splenocytes), were obtained in a single experiment representative of duplicate experiments. The average number of ELISpots/106 splenocytes derived from mice immunized with non-transfected DCs was not significantly different from the DMSO control (not shown). *Splenocytes derived from mice immunized with transfected DCs and incubated with the HLA-A2- and -DRB1-restricted peptides indicated did not yield values that were significantly different from the DMSO control (ANOVA).

Mentions: The gene sequences that encode highly conserved HLA-A*0201-restricted HCV epitopes and HLA-DRB1-restricted viral ICS (validated previously by their ability to elicit specific naïve human T cell responses invitro) were incorporated into two separate multi-epitope genes, and each gene was cloned into an expression vector (NTC8685-eRNA41H-EGFP). HLA-A2/DRB1 transgenic mice were immunized with these vaccine constructs using DCs (characterized in the preceding section) as a vector. DCs transfected with either of the two constructs were mixed in equal proportion and the mice were immunized s.c. Mice administered non-transfected DCs served as the control. Splenocytes derived from a group of four mice on day 35 following immunization and a single boost 2-weeks later demonstrated a negligible response to most peptides encoded by the vaccine constructs in IFN-γ ELISpot assays (data not shown). Splenocytes derived from four mice vaccinated and boosted twice, however, recognized and produced IFN-γ in response to incubation with ∼90% of the HLA-A2-restricted epitopes and all the HLA-DRB1-restricted ICS (Figure 4). In both cases, the responses to individual peptides quantified in ELISpot assays varied in terms of the number of spots.


Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice.

Mishra S, Lavelle BJ, Desrosiers J, Ardito MT, Terry F, Martin WD, De Groot AS, Gregory SH - PLoS ONE (2014)

IFN-γ ELISpot assays.The spleens were dissected and pooled on day 35 from groups of 4 mice immunized with vaccine construct-transfected, and IFN-γ ELISpot assays were performed in triplicate. The data, expressed as the means ± SD ELISpots/106 splenocytes minus the average negative control, 0.1% DMSO+2 SD (3,346 ELISpots/106 splenocytes), were obtained in a single experiment representative of duplicate experiments. The average number of ELISpots/106 splenocytes derived from mice immunized with non-transfected DCs was not significantly different from the DMSO control (not shown). *Splenocytes derived from mice immunized with transfected DCs and incubated with the HLA-A2- and -DRB1-restricted peptides indicated did not yield values that were significantly different from the DMSO control (ANOVA).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128787&req=5

pone-0104606-g004: IFN-γ ELISpot assays.The spleens were dissected and pooled on day 35 from groups of 4 mice immunized with vaccine construct-transfected, and IFN-γ ELISpot assays were performed in triplicate. The data, expressed as the means ± SD ELISpots/106 splenocytes minus the average negative control, 0.1% DMSO+2 SD (3,346 ELISpots/106 splenocytes), were obtained in a single experiment representative of duplicate experiments. The average number of ELISpots/106 splenocytes derived from mice immunized with non-transfected DCs was not significantly different from the DMSO control (not shown). *Splenocytes derived from mice immunized with transfected DCs and incubated with the HLA-A2- and -DRB1-restricted peptides indicated did not yield values that were significantly different from the DMSO control (ANOVA).
Mentions: The gene sequences that encode highly conserved HLA-A*0201-restricted HCV epitopes and HLA-DRB1-restricted viral ICS (validated previously by their ability to elicit specific naïve human T cell responses invitro) were incorporated into two separate multi-epitope genes, and each gene was cloned into an expression vector (NTC8685-eRNA41H-EGFP). HLA-A2/DRB1 transgenic mice were immunized with these vaccine constructs using DCs (characterized in the preceding section) as a vector. DCs transfected with either of the two constructs were mixed in equal proportion and the mice were immunized s.c. Mice administered non-transfected DCs served as the control. Splenocytes derived from a group of four mice on day 35 following immunization and a single boost 2-weeks later demonstrated a negligible response to most peptides encoded by the vaccine constructs in IFN-γ ELISpot assays (data not shown). Splenocytes derived from four mice vaccinated and boosted twice, however, recognized and produced IFN-γ in response to incubation with ∼90% of the HLA-A2-restricted epitopes and all the HLA-DRB1-restricted ICS (Figure 4). In both cases, the responses to individual peptides quantified in ELISpot assays varied in terms of the number of spots.

Bottom Line: To date, therapeutic vaccines have demonstrated only limited success.Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array.A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.

ABSTRACT
Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

Show MeSH
Related in: MedlinePlus