P18-08. Characterization of CD34+ derived dendritic cells generated in vitro and transfected with HIV gene as potential therapeutic vaccine in macaque

International audiencen.

Role of dendritic cells in HIV-immunotherapy

HIV remains one of the most important deadly infections today, due to the lack of a preventive vaccine and limited access to medical care in developing countries. In developed countries, antiretroviral therapy is available, but it can not eliminate the virus, implying that life-long therapy is necessary. Therefore, it is important that other strategies such as therapeutic vaccination will be developed to control virus replication or even eliminate the virus. The major obstacles towards such a strategy are the huge variability of the virus and the profound HIV-induced immune dysfunction. In this review we focus on dendritic cell based immunotherapies against HIV. To develop an efficient immunotherapy, several elements should be taken into account such as which antigen and loading strategy to use, how to deliver the immunogen, how to optimize the interaction between antigenic peptide and T cells and avoid tolerance. Clearly, to develop an immunotherapy to complement the effect of HAART, it is not sufficient to enhance T cell responses against a consensus sequence or against the prevailing plasma virus. Broad and potent immune response are needed to suppress the entire quasispecies, including the latent reservoir, and to prevent any escape

Simultaneous activation of viral antigen-specific memory CD4+ and CD8+ T-cells using mRNA-electroporated CD40-activated autologous B-cells

Recently, it has become obvious that not only CD8 T-cells, but also CD4 T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 and CD8 T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-gamma-producing CD4 and CD8 autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 and CD8 T-cells. More detailed analysis of the activated interferon-gamma-producing CMV pp65 tetramer positive CD8 T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 and CD8 (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases

Lipoplexes carrying mRNA-encoding Gag protein modulate dendritic cells to stimulate HIV-specific immune responses

Aim: Cationic lipids (Lipofectamine [Invitrogen, Merelbeke, Belgium] and 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanol amine) and polymers (jetPEI and in vivo-jetPEI [Polyplus-transfection, Illkirch, France]) were evaluated for their potential to deliver mRNA to monocyte-derived dendritic cells. Materials & methods: Lipoplexes and polyplexes, containing mRNA-encoding GFP or Gag protein, were incubated with human monocyte-derived dendritic cells and transfection efficiencies were assessed by flow cytometry. Results: Lipofectamine was by far the most efficient in mRNA delivery, therefore it was used in further experiments. Incubation of monocyte-derived dendritic cells isolated from HIV-1-positive donors with mRNA-encoding Gag protein complexed to Lipofectamine resulted in 50% transfection. Importantly, coculture of these Gag-transfected dendritic cells with autologous T cells induced an over tenfold expansion of IFN-gamma- and IL-2-secreting CD4(+) and CD8(+) T cells. Conclusion: Cationic lipid-mediated mRNA delivery may be a useful tool for therapeutic vaccination against HIV-1. This approach can be applied to develop vaccination strategies for other infectious diseases and cancer. Original submitted 26 January 2012; Revised submitted 17 April 2012