The nanoparticle containing TpD induced robust anti-nicotine antibody titers, whereas nanoparticles lacking TpD showed no detectable antibody response (Fig. 4A). Antibody levels increased with each boost, particularly after the third boost on day 169, 141 days after the previous immunization, suggesting helper T cell memory was long lived. To further assess long-lived T cell memory, we immunized mice on days 0, 14 and 28 with nicotine nanoparticles containing R848 and either TpD or ovalbumin 323–339 (Ova) peptide (Fig. 4B). Spleens were harvested 122–152 days after final inoculation BYL719 in vitro and either not stimulated, or stimulated ex vivo with TpD or Ova peptide. Supernatants
were harvested after 18 h and evaluated for IFN-γ levels. In TpD immunized mice, IFN-γ secretion was not detectable when splenocytes were non-stimulated or challenged with the Ova peptide. In contrast IFN-γ was detected at significant levels when splenocytes were stimulated with TpD. Conversely, in Ova immunized mice only the Ova peptide was able to induce a response. The data suggests that TpD, when delivered in a nanoparticle, is able to provide long term CD4T cell memory and can function on re-challenge to provide a boost in a vaccine response. In order to
evaluate the dose-dependent effect of helper buy PD-0332991 peptide on anti-nicotine antibody titers in vivo, we designed an experiment using limiting levels of TpD. Mice were immunized on days 0, 14 and 28, and on day 46 serum analyzed for antibody titers (Fig. 4C). Increasing the amount of TpD during immunization resulted in elevated anti-nicotine antibody titers, suggesting that the magnitude of antibody response is helper peptide dependent. We further investigated TpD activity in non-human primate pre-clinical models. Data from rhesus monkeys immunized on days 0, 28, and 56 with escalating doses of nicotine old nanoparticles are shown in Fig. 5. As expected no anti-nicotine antibody titers were seen two weeks prior to immunization or at the time of the first immunization (Fig. 5A). Antibodies were detectable after the first immunization, and increased significantly
after the second and third immunization. Titers were variable at the lowest dose (0.3 mg) and plateaued at the 0.9 mg dose. Analysis of CD4 T cell recall responses showed detectable levels of TpD responding cells at the lowest dose, (Fig. 4B) but not prior to immunization. All 4 monkeys tested showed helper T cell responses. There was not a clear dose response, as expected given the small number of animals studied (N = 1 per group). T cell recall responses were detectable 63 days after the last immunization, suggesting memory T cells were being generated. We next studied TpD activity in a larger cohort of cynomolgus monkeys (N = 50) immunized with nicotine nanoparticles and evaluated them for both anti-nicotine antibody titers and T cell recall responses ( Fig. 6).
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