Dendritic Cell-Mediated, DNA-Based Vaccination Against Hepatitis C Induces the Multi-Epitope-Specific Response of Humanized, HLA Transgenic Mice

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

Schematic: DC vector-mediated vaccination of HLA-A2/DR1 mice.

<p>Humanized HLA-A2/DRB1 transgenic mice were vaccinated s.c. with multi-HCV epitope-expressing plasmids using DCs derived from transgenic mice administered Flt3L-secreting B16 melanoma cells as a vaccine vector.</p

DCs derived from Flt3L-treated, HLA-A2/DRB1 mice exhibit an immature phenotype^a.

a<p>CD11c⁺PDCA-1⁺ DCs, purified from 2.55×10⁸ splenocytes/HLA-A2/DRB1 transgenic mouse inoculated s.c. with Flt3L-secreting B16 myeloma cells 12 days previously, were quantified and characterized by flow cytometry.</p>b<p>Not determined.</p

IFN-γ ELISpot assays.

<p>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).</p

Cytokine bead array.

<p>The spleens were dissected and pooled on day 35 from groups of four mice immunized with vaccine construct-transfected or non-transfected DCs. Single cell suspensions were transferred to quadruplicate wells and cultured 40 hours in the presence of the peptide indicated. The data, obtained in a single experiment representative of two experiments, are the means ± SD pg/ml cytokine produced by splenocytes obtained from mice immunized with transfected DCs minus that produced by splenocytes derived from control mice immunized with non-transfected DCs. Concentrations of IFN-γ, TNF-α, IL-4 and IL-10 (where indicated by *) are significantly greater than that produced by splenocytes derived from control animals (<i>P<</i>0.05; Student’s <i>t</i> test or Mann-Whitney Rank Sums test).</p

HLA-A2/DRB1 DCs transfected by electroporation.

<p>Purified HLA-A2/DRB1 DCs were transfection by electroporation with a GFP-expressing plasmid (NTC8685-eRNA41H-EGFP) and cultured overnight in the presence of GM-CSF to promote viability. The cells were examined visually on the following day: UV microscopy (left), DAPI-counter stain (center), merged images (right).</p