Optimising a mRNA Vaccine platform against HIV-1 : from the in vitro transcription design to envelope protein expression ; Optimisation d'une plateforme de vaccin ARNm contre le VIH-1 : de la conception de la transcription in vitro à l'expression des protéines d'enveloppe

The development of the first mRNA vaccine against the coronavirus pandemic of 2020 is now part of the annals of vaccinology. However, the overall reactogenicity of this first generation of mRNA vaccines is far to be perfect. The aim of this thesis concerns the design and optimization of the potency of an mRNA vaccine against HIV-1, starting from the mRNA sequence, production, purification, to the antigen selection and the in vivo proof of concept. Therefore, we designed and optimized the sequence of a standard mRNA backbone based on the untranslated region of the human β-globin. First, we evaluated in vitro the impact of different poly-A tails, and continue modifying the 5’UTR sequence with synthetic high ribosome loading sequences, demonstrating the benefit of a long poly-A tail of 148 adenines and the use of the synthetic UTR4. Once optimised the sequences, we focused on the production and purification of mRNA by-products generated during the in vitro transcription reaction. We capped the mRNA using antireverse cap analogs and enzymatic vaccinia capping, to later treat and purify the mRNA with cellulose, phosphatase or using a double purification. The results showed up to a 300% increased in eGFP expression in vitro. Once optimised the mRNA platform, we applied these advantages into the design of an mRNA candidate against HIV-1. We selected 7 different antigenic sequences to encode them into our platform. Among the antigens, we fused some of the antigens to a novel multimeric protein, looking forward to improving the immune response. Moreover, the mRNAs were formulated into lipid nanoparticles (MC3: DSPC: Cholesterol: DMG-PEG-2000) by microfluidics. The formulations were characterised by dynamic light scattering (for size, Z-potential and polydispersity) and by fluorometric assay to measure the mRNA encapsulation. The results demonstrated an overall high encapsulation efficiency, small particle size and quasi-neutral charge. All the mRNAs expression was validated in vitro using by immunofluorescence. These ...