Tailored Monoacyl Poly(2-oxazoline)- and Poly(2-oxazine)-Lipids as PEG-Lipid Alternatives for Stabilization and Delivery of mRNA-Lipid Nanoparticles.

The successful use of lipid nanoparticles (LNPs) for clinical development of the COVID-19 mRNA vaccines marked a breakthrough in mRNA-LNP therapeutics. As one of the vital components of LNPs, poly(ethylene glycol)-lipid conjugates (PEG-lipids) influence particle biophysical properties and stability, as well as interactions within biological environments. Reports suggesting that anti-PEG antibodies can be detected quite commonly within the human population raise concerns that PEG content in commercial LNP products could further stimulate immune responses to PEG. The presence of anti-PEG antibodies has been linked to accelerated clearance of LNPs, potentially a source of variability in the biological response to mRNA-LNP products. This motivated us to explore potential PEG alternatives. Herein, we report physicochemical and biological properties of mRNA-LNPs assembled using poly(2-oxazoline) (POx)- and poly(2-oxazine) (POz)-based polymer-lipid conjugates. Notably, we investigated monoacyl lipids as alternatives to diacyl lipids. mRNA-LNPs produced using monoacyl POx/POz-lipids displayed comparable biophysical characteristics and cytocompatibility. Delivery of reporter mRNA resulted in similar transfection efficiencies, in both adherent and suspension cells, and in mice, compared to PEG-lipid equivalents. Our results suggest that monoacyl POx/POz-lipid-containing LNPs are promising candidates for the development of PEG-free LNP-based therapeutic products.

Interim results from a phase I randomized, placebo-controlled trial of novel SARS-CoV-2 beta variant receptor-binding domain recombinant protein and mRNA vaccines as a 4th dose booster.

BACKGROUND: SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG1 Fc-fusion protein, and an mRNA encoding a membrane-anchored RBD. METHODS: 76 healthy adults aged 18-64 y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45 µg, N = 32), mRNA vaccine (10, 20, or 50 µg, N = 32), or placebo (saline, N = 12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29. CLINICALTRIALS: govNCT05272605. FINDINGS: No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4+ and CD8+ T cell activation. INTERPRETATION: There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains. FUNDING: Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors.

Delivery and Expression of mRNA in the Secondary Lymphoid Organs Drive Immune Responses to Lipid Nanoparticle-mRNA Vaccines after Intramuscular Injection.

Lipid nanoparticles (LNPs) are the prime delivery vehicle for mRNA vaccines. Previous hypotheses suggested that LNPs contribute to innate reactogenicity and lead to the establishment of a vaccine adaptive response. It has not been clear whether LNP adjuvancy in the muscle is the prime driver of adaptive immune responses or whether delivery to secondary lymphatic organs is necessary to induce strong adaptive responses. To address this, we formulated reporter gene (NLuc) or OVA mRNA into LNP or coadministered the mRNA with empty LNP. After IM injection, we correlated the delivery with adaptive immune responses. Additionally, we investigated humoral responses to modified mRNA encoding the SARS-CoV-2 spike protein. Compared to unformulated mRNA encoding nanoluciferase, with or without co-administered empty LNPs, LNP-formulated mRNA resulted in high levels of nanoluciferase in the secondary lymphoid organs. Similarly, LNP-mRNA encoding ovalbumin led to a cellular immune response against OVA while free mRNA, with or without empty adjuvanted LNPs, caused little or no immune response. Finally, only mice injected with LNP-formulated mRNA encoding SARS-CoV-2 spike protein elicited robust cellular and humoral immune responses. Our results suggest that the mRNA delivery and transfection of secondary lymphatic organs, not LNP adjuvancy or RNA expression in muscle, are the main drivers for adaptive immune response in mice. This work informs the design of next-generation mRNA delivery systems where better delivery to secondary lymphatic organs should lead to a better vaccine response.