DLin-MC3-Containing mRNA Lipid Nanoparticles Induce an Antibody Th2-Biased Immune Response Polarization in a Delivery Route-Dependent Manner in Mice

Abstract

mRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.

Optimizing mRNA-Loaded Lipid Nanoparticles as a Potential Tool for Protein-Replacement Therapy

“…This observation was made during the comparison between LM1-LNP (ALC-0315) and LM4-LNP (DLin-MC3-DMA), where the substitution of ALC-0315 with Dlin, an immunogenic ionizable lipid, led to reduced mRNA expression. This heightened reactogenicity was consistent with our measurements of cytokine secretion as well [ 29 ]. In another scenario, the replacement of the helper DSPC lipid with DOPE (in LM1 and LM2, respectively) may lead to differences in expression and biodistribution after intramuscular injection.…”

“…The mRNA for mouse administration was around 7 µg, which was higher than other reports (4 µg/mice [ 8 ]; 3 µg/mice [ 29 ]), but well under other doses tested for metabolic diseases: 20 µg/mice for hereditary tyrosinemia 1 [ 5 ], 60 μg/mice for phenylketonuria [ 6 ], or the 10 µg/mice doses used in LNP inflammation studies [ 30 ]. Similarly to the approach with PBMCs, high doses of mRNA may be related to high doses of lipids, which represents a suitable situation to study the effect of the lipids on the immune response.…”

COVID-19 vaccine adverse events: Evaluating the pathophysiology from an undersulfated and degraded glycocalyx perspective

“…Although COVID-19 vaccines are administered intramuscularly 10 , Friedensohn et al found a higher seroconversion rate with accidental subcutaneous administration 340 . This is in line with the study by Yavuz et al, who observed ionizable LNPs induced Ab Th2-biased immunity when subcutaneously administered 70 . Direct intramuscular and intradermal injections of LNP-mRNA were shown to offer the best levels of antigen expression and duration of effect, with the production of antigen protein peak at 4 hours, and maintained locally 8-10 days after injection, depending on the dose.…”

“…Therefore, ionizable cationic lipids are deprotonated under neutral conditions and positively charged under low pH conditions, thus below the acid dissociation constant (pKa) of the lipid [66][67][68][69] . The LNPs used in the Pfizer and Moderna COVID-19 vaccines contain an ionizable lipid, a structural phospholipid, cholesterol, and a PEG-lipid in molar ratio 70 . The ionizable lipid used in the Pfizer vaccine is ALC-0315, while SM-102 was used in the Moderna vaccine 71 .…”

“…Although the addition of PEG 2000 might seem to abolish toxicity to some degree, the presence of pH-sensitive lipids in ionizable LNPs can enhance cationic LNP toxicity by destabilizing the endosomal membrane, releasing cationic lipids into the cytoplasm 61 . Yavuz et al demonstrated that, independently of the type of ionizable lipid used to formulate LNPs, intramuscularly immunized mice induced Th1-biased polarization 70 . This aligns with the Pfizer vaccine that caused a Th1cellular-biased innate immune response, with the secretion of IL-6, TNFα and IFNγ 86,87 .…”