We reported that RNA condensed on protamine is protected from RNase-mediated degradation and can be used for vaccination. Here, we show that such complexes are also danger signals that activate mouse cells through a MyD88-dependent pathway. Moreover, mRNA-protamine complexes stimulate human blood cells. They strongly activate DC and monocytes, leading to TNF-a and IFN-a secretion. In addition, protamine-RNA complexes directly activate B cells, NK cells and granulocytes. The detailed analysis of the activated cell types, the study of the cytokines released from PBMC cultured with protamine-RNA complexes and recently published results suggest that TLR-7 and TLR-8 may be involved in the recognition of protamine-stabilized RNA. Our data indicate that protamine-stabilized RNA, which may be similar to RNA condensed in the nucleocapsids of RNA viruses, is a strong danger signal. Thus, similarly to plasmid DNA, protamine-RNA combines antigen production and non-specific immunostimulation. The studies presented here explain the capacity of protamine-RNA to act as a vaccine, and pave the way towards the development of safe and efficient mRNA-based immunotherapies.

In the context of developing a safe genetic vaccination strategy we tested and studied globin-stabilized mRNA-based vaccination in mice. This vaccination strategy has the advantages of genetic vaccination (easy production, adaptability to any disease and inexpensive storage when lyophilized), but not the drawbacks of DNA vaccination (long-term uncontrolled expression of a transgene, possibility of integration into the host genome and possible induction of anti-DNA antibodies). We report here that injection of naked beta-globin untranslated region (UTR)-stabilized mRNA coding for beta-galactosidase is followed by detectable translation in vivo. In addition, we show that such a vaccination strategy primes a T helper 2 (Th2) type of response which can be enhanced and shifted to a Th1-type immune response by application of recombinant granulocyte/macrophage colony-stimulating factor 1 day after mRNA injection. Our data demonstrate that the administration of globin UTR-stabilized mRNA is a versatile vaccination strategy that can be manipulated to fit the requirement of antiviral, antibacterial or antitumor immunity.

The efficiency of test vaccines needs to be evaluated by quantification of the triggered cellular immune response. Usually, for these assays, autologous target cells expressing the vaccine antigen are required. In the context of messenger RNA (mRNA)-based vaccinations, the target cells used for the read-out are mRNA-transfected monocyte-derived dendritic cells (Mo-DCs). Their production typically requires samples of 100 ml blood from the patients, and limits the number of assays that can be performed. We show here that fresh peripheral blood mononuclear cells (PBMCs) can be transfected with mRNA by electroporation. Such cells are as efficient as mRNA-transfected Mo-DCs for their ability to activate memory T cells in vitro. Thus, mRNA-transfected PBMCs are a convenient replacement of mRNA-transfected Mo-DCs for the in vitro monitoring of natural or vaccine-induced immune responses.

Background: Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator™ SMART™ cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis.

The definition of an optimal siRNA results from the in vitro testing of several siRNA designed to specifically target a gene. Usually, such in vitro tests consist in the transfection of the several siRNA duplexes in a cell expressing stably the gene of interest. When a siRNA specific for a mRNA coding toxic proteins (certain transcription factors, transporters, toxins, cell cycle controlling proteins, etc.) must be tested, the generation of a target cell is difficult. Here we report a quick method to test the efficiency of a siRNA through its co-transfection with the targeted mRNA. This technique can be used as a fast method to test siRNA even when they target genes that cannot be stably expressed in the cells of interest.