Treatment of influenza virus with beta-propiolactone alters viral membrane fusion.

Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2-1000µM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.

Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer.

Lipid nanoparticles (LNPs) for RNA and DNA delivery have attracted considerable attention for their ability to treat a broad range of diseases and to vectorize mRNA for COVID vaccines. LNPs are produced by mixing biomolecules and lipids, which self-assemble to form the desired structure. In this domain, microfluidics shows clear advantages: high mixing quality, low-stress conditions, and fast preparation. Studies of LNPs produced in micromixers have revealed, in certain ranges of flow rates, a degradation in performance in terms of size, monodispersity and encapsulation efficiency. In this study, we focus on the ring micromixer, which is well adapted to high throughput. We reveal three regimes, side-by-side, transitional and highly mixed, that control the mixing performance of the device. Furthermore, using cryo-TEM and biochemical analysis, we show that the mixing performances are strongly correlated to the characteristics of the LNPs we produce. We emphasize the importance of the flow-rate ratio and propose a physical criterion based on the onset of temporal instabilities for producing LNPs with optimal characteristics in terms of geometry, monodispersity and encapsulation yield. These criteria are generally applicable.

An imidazole modified lipid confers enhanced mRNA-LNP stability and strong immunization properties in mice and non-human primates.

The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current "best in class". Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group.

Characterization of different strains of poliovirus and influenza virus by differential scanning calorimetry.

Vaccines against poliomyelitis and influenza contain inactivated forms of poliovirus and influenza virus. These antigens are generated on an industrial scale from the purified active viruses that have been analysed in this study by DSC (differential scanning calorimetry). Multiple unfolding transitions are seen for influenza virus A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) and B/Shangdong/7/97. These data, combined with previously reported data on other influenza viruses, indicates that each influenza virus strain has a characteristic unfolding behaviour. Only minor changes were seen in the thermogram of betaPL (beta-propiolactone)-inactivated influenza virus, which is consistent with the proposition that betaPL reacts mainly with the nucleotide fraction of the virus. We demonstrate that a peak annotation of the thermogram of the native virus is possible using bromelain-treated virus and virosomes. At pH 1.5-2.5, poliovirus of type I unfolds in a single unfolding event with respective Tm (midpoint of protein unfolding transition) values between 34 and 45 degrees C. At pH 2, polioviruses of type II unfold equally in a single event, but, compared with the type I virus, with a Tm value increased by 3.7 degrees C. At neutral pH, the DSC thermogram of type I poliovirus was very 'noisy'. Data obtained offer the possibility of precisely characterizing and identifying different viral strains.