A lipid nanoparticle platform incorporating trehalose glycolipid for exceptional mRNA vaccine safety

Abstract

The rapid development of messenger RNA (mRNA) vaccines formulated with lipid nanoparticles (LNPs) has contributed to control of the COVID-19 pandemic. However, mRNA vaccines have raised concerns about their potential toxicity and clinical safety, including side effects, such as myocarditis, anaphylaxis, and pericarditis. In this study, we investigated the potential of trehalose glycolipids-containing LNP (LNP S050L) to reduce the risks associated with ionizable lipids. Trehalose glycolipids can form hydrogen bonds with polar biomolecules, allowing the formation of a stable LNP structure by replacing half of the ionizable lipids. The efficacy and safety of LNP S050L were evaluated by encapsulating the mRNA encoding the luciferase reporter gene and measuring gene expression and organ toxicity, respectively. Furthermore, mice immunized with an LNP S050L-formulated mRNA vaccine expressing influenza hemagglutinin exhibited a significant reduction in organ toxicity, including in the heart, spleen, and liver, while sustaining gene expression and immune efficiency, compared to conventional LNPs (Con-LNPs). Our findings suggest that LNP S050L, a trehalose glycolipid-based LNP, could facilitate the development of safe mRNA vaccines with improved clinical safety.

Keywords: Immunogenicity; Lipid nanoparticle; Toxicity; Trehalose glycolipid; mRNA vaccine.

Graphical abstract

Fig. 1

Schematic illustration of novel LNP S050L based on trehalose glycolipids. A partial substitution of ionizable lipids with trehalose glycolipids maintained high efficacy in mRNA expression and immune response. The toxicity to the heart and liver was reduced compared to Con-LNPs and the mRNAs encapsulated within LNP S050L were traced in the lymph node and spleen.

Fig. 2

In vivo optimization of trehalose glycolipids-containing LNPs. The optimal LNPs exhibiting superior efficiency was subsequently cherry-picked, considering the interplay between helper lipids and the molar ratio of ionizable lipids. The luminescence signal of R/L at 6 and 24 h after injection of R/L mRNA⊂LNPs was expressed on a logarithmic scale. (A) A proof of concept was conducted with a primary optimizing group (LNP D100C, D050C, D050L, and T100L). (B) A cone-shaped SM-102 (LNP S050L) was replaced DLin-MC3-DMA (LNP D050L). (C) Steroid lipid structure was optimized by changing alkyl groups in steroids (LNP S050L, S050C, S050L′, and S050L″). (D) Optimization of the substitution ratio of TDO to ionizable lipid (SM-102) was investigated by changing the ratio (LNP T100L, S025L, S050L, S075L, and S100L). (E) Optimization of N/P ratio of LNP S050L. N/P ratio: moles of cationizable nitrogen in ionizable lipids divided by moles of phosphodiesters in mRNAs. Nil: the group injected with saline. RNA: the group injected with only mRNA. Data are represented as the mean ± standard deviation (SD). Statistical significance was analyzed using two-way ANOVA. Statistically significant differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3

Characterization of LNP S050L compared to Con-LNP. (A) Size distributions, (B) TEM images, (C) SAXS scans, and (D) mRNA encapsulation efficiencies of LNP S050L and Con-LNP.

Fig. 4

Bio-distribution of LNP S050L. Real-time whole-body imaging of LNP S050L at different time points post-injection. Mice were injected with 5 μg of mRNA. (A) In vivo distribution patterns of F/L mRNA⊂LNP S050L were evaluated after I.M. injections. The maximum F/L mRNA expression was observed at 6 h. (B, C) In vivo bio-distribution analysis of the I.M. injection revealed F/L bioluminescence signal in injection site and spleen. (D) Ex vivo bio-distribution in the I.M. injection group. F/L bioluminescence signal remained at the injection site and migrated toward to the right side of the draining lymph node. AxLN: Axillary lymph node, InLN: Inguinal lymph node, PoLN: Popliteal Lymph node. All data are presented as the mean ± SD of values from independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by a two-tailed Student's t-test.

Fig. 5

In vivo toxicity of LNP S050L and Con-LNP. Mice were immunized with saline or R/L mRNA⊂LNPs to evaluate the state of the major organs and the toxicity of the LNPs. (A, B) Overview of the experimental groups and immunization schedules. (C) Representative images of the heart, spleen, and lymph nodes from immunized mice captured during euthanization. Pericardial calcification was observed in the group immunized with con-LNPs (G3). (D) Hematological safety profiles of serum samples collected from immunized mice during euthanization. The results showed an increase in alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and troponin I activity and a decrease in blood urea nitrogen (BUN) levels in the serum. Data are represented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA. Statistically significant differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6

Histological analysis. Representative H&E-stained images of the hearts, spleens, and livers of immunized mice during euthanization. Data are represented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA. Statistically significant differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 7

In vivo immune response of the LNP S050L and Con-LNP. Mice were immunized with saline and HA mRNA⊂LNPs to compare the immune responses to the two distinct LNPs. (A, B) Overview of the experimental groups and immunization schedules. (C) Serum samples were collected from mice 2 weeks after the first immunization with mRNA-LNPs, and the levels of IgG1 and IgG2a were measured using ELISA. (D) Serum samples were collected from mice 2 weeks after the second immunization with mRNA⊂LNPs, and the levels of IgG1 and IgG2a were measured using ELISA. (E). HI titer against vaccine strains measured by HI assay using serum collected 2weeks after boost. (F) The numbers of HA peptide-Specific IFN-γ cells in splenocytes were measured using ELISPOT. (G) The concentration of TNF-α, IL-2, and IFN-γ was measured with ELISA in splenocyte culture supernatants. Data are represented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA. Statistically significant differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.