Immunogenicity and biodistribution of lipid nanoparticle formulated self-amplifying mRNA vaccines against H5 avian influenza.

This study reports on the immunogenicity and biodistribution of H5 hemagglutinin (HA)-based self-amplifying (sa) mRNA vaccines in mice. Four sa-mRNA vaccines encoding either a secreted full-length HA, a secreted HA head domain, a secreted HA stalk domain, or a full-length membrane-anchored HA were investigated. All vaccines elicited an adaptive immune response. However, the full-length HA sa-RNA vaccines demonstrated superior performance compared to head and stalk domain vaccines. The antibody titers positively correlated with the vaccine dose. Cellular immune responses and antigen-specific IgA antibodies in the lungs were also observed. The comparison of the sa-mRNA vaccines encoding the secreted and membrane-anchored full-length HA revealed that anchoring of the HA to the membrane significantly enhanced the antibody and cellular responses. In addition to the injection site, the intramuscularly injected sa-mRNA-LNPs were also detected in the draining lymph nodes, spleen, and to a lesser extent, in the lung, kidney, liver, and heart.

Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs.

Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.