Induction of liver-resident memory T cells and protection at liver-stage malaria by mRNA-containing lipid nanoparticles.

Recent studies have suggested that CD8+ liver-resident memory T (TRM) cells are crucial in the protection against liver-stage malaria. We used liver-directed mRNA-containing lipid nanoparticles (mRNA-LNPs) to induce liver TRM cells in a murine model. Single-dose intravenous injections of ovalbumin mRNA-LNPs effectively induced antigen-specific cytotoxic T lymphocytes in a dose-dependent manner in the liver on day 7. TRM cells (CD8+ CD44hi CD62Llo CD69+ KLRG1-) were induced 5 weeks after immunization. To examine the protective efficacy, mice were intramuscularly immunized with two doses of circumsporozoite protein mRNA-LNPs at 3-week intervals and challenged with sporozoites of Plasmodium berghei ANKA. Sterile immunity was observed in some of the mice, and the other mice showed a delay in blood-stage development when compared with the control mice. mRNA-LNPs therefore induce memory CD8+ T cells that can protect against sporozoites during liver-stage malaria and may provide a basis for vaccines against the disease.

Effect of Cholesterol Content of Lipid Composition in mRNA-LNPs on the Protein Expression in the Injected Site and Liver After Local Administration in Mice.

Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

Biochemical and Structural Characterization of l-2-Keto-3-deoxyarabinonate Dehydratase: A Unique Catalytic Mechanism in the Class I Aldolase Protein Superfamily.

l-2-Keto-3-deoxyarabinonate (l-KDA) dehydratase (AraD) catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde in the nonphosphorylative l-arabinose pathway from bacteria and belongs to the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein superfamily. All members of this superfamily, including several aldolases for l-KDA, share a common catalytic mechanism of retro-aldol fission, in which a lysine residue forms a Schiff base with the carbonyl C2 atom of the substrate, followed by proton abstraction of the substrate by a tyrosine residue as the base catalyst. Only AraD possesses a glutamine residue instead of this active site tyrosine, suggesting its involvement in catalysis. We herein determined the crystal structures of AraD from the nitrogen-fixing bacterium Azospirillum brasilense and AraD in complex with ß-hydroxypyruvate and 2-oxobutyrate, two substrate analogues, at resolutions of 1.9, 1.6, and 2.2 Å, respectively. In both of the complexed structures, the ε-nitrogen of the conserved Lys171 was covalently linked to the carbonyl C2 atom of the ligand, which was consistent with the Schiff base intermediate form, similar to other DHDPS/NAL members. A site-directed mutagenic study revealed that Glu173 and Glu200 played important roles as base catalysts, whereas Gln143 was not absolutely essential. The abstraction of one of the C3 protons of the substrate (but not the O4 hydroxyl) by Glu173 was similar to that by the (conserved) tyrosine residues in the two DHDPS/NAL members that produce α-ketoglutaric semialdehyde (d-5-keto-4-deoxygalactarate dehydratase and Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase), indicating that these enzymes evolved convergently despite similarities in the overall reaction.