Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes

Abstract

Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structures of these formulations are still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and the determination of the intra-LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well-characterized PEGylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size and envelope lipid composition to Doxil; however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of ¹⁴C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction imply that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilipid layers. Negative staining TEM shows 2-5 nm electron-dense spots in the LNP's interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply cross-link-stabilized RNA fragments. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the possibility of hydrogen bonding between mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of the ionizable lipid in Comirnaty, ALC-0315, displaying free ═O and -OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These structural features of mRNA-LNP may be important for the vaccine's activities in vivo.

Keywords: Doxil liposomes; SARS-CoV-2; atomic force microscopy; cryo-electron microscopy; dynamic light scattering; lipid nanoparticles; phospholipid membranes.

Figure 1

AFM height-contrast images (A–D) and corresponding 3D reconstructions (E–H) of Comirnaty vaccine and Doxil immobilized on a glass surface. (A) and (E) show a freshly diluted Comirnaty sample representing the jab inoculated into the deltoid muscle; (B) and (F) show a 1-day-old sample stored at 4 °C, representing the unused leftover; (C) and (G) show a refrozen sample, representing unintended acceleration of fragmentation by refreezing the leftover vaccine; (D) and (H) show a freshly opened Doxil sample.

Figure 2

Force spectroscopy of Comirnaty. (A) Force–distance curve of a particle indentation (red curve) and RNA extraction (blue curve). The numbered schematics along the curves illustrate the different stages of measurements, with the red and blue tips pointing to the peak “force points”, i.e., the distance where sudden transitions occur. (B) Force–distance curve of RNA stretching and its related schematics. Black dashed line shows worm-like chain model fit (persistence length = 345 pm; contour length = 83 nm). (C/1) Region of interest of RNA extraction force–distance curves superimposed (n = 20). (C/2) A selected RNA extraction trial with multiple RNA structural transition events. (D) Histogram of peak forces detected in RNA extraction curves (peak forces are labeled with red stars in (A)).

Figure 3

Cryo-TEM images of Comirnaty (A, C) and of Doxil (D). Comirnaty samples for cryo-TEM (A–C) were processed immediately after thawing the vaccine vial from −80 °C and dilution with saline, as instructed by the manufacturer for human application. Orange arrows show the bilayer coating of the NPs. Blue arrows point at bicompartmental NPs with an mRNA-containing bleb. (D) Cryo-TEM image of Doxil showing the rod-like doxoribicin-sulfate crystal inside the intra-liposome aqueous phase.

Figure 4

TEM images of a Comirnaty sample stored for 7 days in the refrigerator, diluted in water, and stained with acidic uranyl acetate as described in the Methods. Blue arrows in (A) point at the fusion of nanoparticles. (B) Higher magnification image of some LNPs. (C) Zoom-in of one of the elongated helical-like structures indicated by red arrows in (A).

Figure 5

Schematic current models of nucleic acid containing LNPs illustrating the variety of concepts. (A, B) “Multilamellar vesicle model” with siRNA sandwiched among the bilayers.^, In (A) there is an IPC lipid/cholesterol core, while in (B), the lipids are not differentiated. (C) Phospholipid monolayer-covered inverted micelle core. The siRNA is shown within the surface monolayer and intermicellar space. (D) Phospholipid-monolayer-coated inverted IPC micelles with siRNA inside the aqueous core of micelles. (E) Phospholipid-monolayer-coated inverted micelles with the siRNA randomly distributed. (F) PEG-lipid-coated random assembly of lipids and siRNA. (G) Phospholipid-monolayer-coated assembly of inverted IPC lipids. The mRNA is attached to the inner layer of inverted micelles. (H) Phospholipid-bilayer-coated assembly of mRNA, covered with IPC lipids. (I) Same as (H), except that the outer membrane is a monolayer. (J) mRNA randomly distributed in an amorphous lipid matrix with heterogeneous bilayer surface coat. (K) mRNA thread ball with no identifiable lipid and membrane components. (L) Bilayer-coated mRNA thread ball with no identifiable lipids.

Figure 6

(A) Scheme of a Comirnaty-specific LNP model wherein the stability of yarn ball-like mRNA lumps (gray) is secured by IPC lipid cross-links and clusters (green and red dots). The NP is surrounded by a phospholipid bilayer (double line), monolayer (single line), or no membrane (dotted line). (B) Chemical structure of DML, containing a diketopiperazine core, two methyl ester end-groups, and two linoleic ethyl ester end-groups. (C) Chemical structure of ALC-0315, the ionizable cationic lipid in Comirnaty [(4-hydroxybutyl)azanediyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate (Table S1 and ref (50)). (D) A four-nucleotide section of the mRNA chain in Comirnaty showing the N and O atoms available for hydrogen bonding, ψ, pseudouridine. (E) The Rissanau et al. model of DML lipid cluster–mRNA complexation shows a 30-nucleotide-containing short mRNA meandering along 2 DML clusters. (F) Molecular dynamics simulation of a 642-nucleotide-containing mRNA complexed with multiple DML clusters that protect the mRNA from hydrolysis. (E) and (F) are reprinted from ref (7) with permission from the Royal Society of Chemistry.