Peanut allergy, a potentially life-threatening condition, has emerged as a major public health concern in developed countries. There are currently no approved treatment options, with dietary avoidance being the only recommended strategy for prevention of fatal anaphylactic reactions. We propose the use of a novel, proprietary Sementis Copenhagen Vector (SCV)-based peanut hypoallergenic vaccine (SCV-PHAV) to induce sustained unresponsiveness and immune tolerance to peanuts, an objective that eludes current immunotherapy modalities in allergy. We established an ex vivo human dendritic cells (DCs): CD4+ T cell culture system to investigate the capacity of SCV-PHAV instructed dendritic cells (DCs) to facilitate the production of a tolerant Th1-biased peanut-specific CD4+ T cell response. Briefly, monocyte-derived DCs and CD4+ T cells were isolated from the blood of peanut allergic patients. Peanut-pulsed and SCV-PHAV vaccinated DCs were incubated with CD4+ T cells for 2 weeks. At the end of the incubation period, cultures were labelled with a proliferation dye, re-stimulated with peanut extract and the cytokine profiles of proliferating antigen-specific T cells was examined by flow cytometry. A significantly higher Th1/Th2 ratio was detected in SCV-PHAV vaccinated cultures compared to peanut-pulsed cultures (n = 5). A consistent increase was observed in separate ex vivo DC:CD4 T cultures set up 6-months apart from the same patient, confirming both assay reproducibility and vaccine efficacy. In addition, we were able to demonstrate an antigen-specific Th1 bias following long-term cultures (5–6 weeks) in the presence of peanut antigens. The SCV-PHAV vaccine can modulate the cytokine signatures of peanut-reactive CD4+ T cells from an allergic, Th2-biased to a tolerant, Th1-biased profile.