Oskar Thofte

Messenger RNA (mRNA) vaccines based on lipid nanoparticles (LNPs) are stabilized with cholesterol, which is thought to be a critical LNP component because it is essential for membrane integrity and endosomal escape. Here, it is shown that cholesterol in LNPs can be replaced with an immunopotentiating lipid, i.e., a synthetic analogue of the C‐type lectin receptor agonist monomycoloyl glycerol (MMG‐1), without compromising physicochemical properties, in vivo transfection efficiency, and immunogenicity of the mRNA‐loaded LNPs (mRNA‐LNPs). Replacement of cholesterol with MMG‐1 results in LNPs that mediate intracellular delivery of mRNA, which is translated into high levels of protein in vivo. Replacement of cholesterol with MMG‐1 in LNPs improves the transfection efficiency in T cells, B cells, and macrophages in the spleen and lymph nodes of mice. In mice, MMG‐1‐based LNPs loaded with mRNA encoding the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) induce S‐specific CD8 ⁺ T‐cell responses and virus‐neutralizing antibody responses, which are on par with the responses induced by cholesterol‐based LNPs. Upon vaccination and a subsequent SARS‐CoV‐2 challenge of Syrian golden hamsters, replacement of cholesterol with MMG‐1 in mRNA‐LNPs enhances S‐specific immunoglobulin G titers and reduces the SARS‐CoV‐2 load in the nasal cavity. These findings provide insights for improved design of LNPs for mRNA vaccine delivery.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing. Key Points Exposure of P5 at the surface of NTHi positively correlates with C4BP binding. C4BP bound to the bacterial surface retains its complement inhibitory capacity. C4BP binding to P5 is important for NTHi serum resistance.