Background/Objectives: Omicron, the predominant variant of SARS-CoV-2, exhibits strong immune-evasive properties, leading to the reduced efficacy of existing vaccines. Consequently, the development of versatile vaccines is imperative. Intranasal mRNA vaccines offer convenient administration and have the potential to enhance mucosal immunity. However, delivering vaccines via the nasal mucosa requires overcoming complex physiological barriers. The aim of this study is to modify PEGylated lipids to enhance the mucosal immune efficacy of the vaccine. Methods: The PEGylated lipid component of lipid nanoparticle (LNP) delivery vectors was modified with chitosan or mannose to generate novel LNPs that enhance vaccine adhesion or targeting on mucosal surfaces. The impact of the mRNA encoding the receptor-binding domain of Omicron BA.4/BA.5 on the immune response was examined. Results: Compared to the unmodified LNP group, the IgG and IgA titers in the chitosan or mannose-modified LNP groups showed an increasing trend. The chitosan-modified group showed better effects. Notably, the PEGylated lipid with 1.5 mol% of chitosan modification produced high levels of IgG1 and IgG2a antibodies, promoting Th1/Th2 responses while also generating high levels of IgA, which can induce stronger cellular immunity, humoral immunity, and mucosal immunity. Conclusions: The 1.5 mol% of chitosan-modified LNPs (mRNA-LNP-1.5CS) can serve as a safe and effective carrier for intranasal mRNA vaccines, offering a promising strategy for combating the Omicron variant.

Lipid nanoparticles (LNP) have emerged at the forefront of the delivery of RNA molecules during the COVID-19 pandemic, leading to a giant leap in RNA therapies. Despite their great success, the long-term storage and transportation of mRNA vaccines without ultra-low temperatures is still challenging due to their poor stability. Here, we demonstrated that LNP-mRNA could be lyophilized via a simple freeze‒drying process. This process produced a dry powder formulation that could maintain the physicochemical properties of LNP-mRNA after storage at 4 °C for at least two months. However, the shear forces generated during the lyophilization process may disrupt the structure of the LNP, affecting the efficacy of the vaccine. Therefore, a cholesterol analogue, β-sitosterol, and a type of phospholipid, DOPE, were utilized instead of cholesterol and DSPC to improve the transfection efficiency after freeze-drying. The optimized formulation of LNP exhibited an enhanced transfection effect both in vitro and in vivo. Additionally, intratracheal administration of reconstituted lyophilized LNPs could induce innate cellular, humoral and mucosal immunity in vivo, indicating that our LNP-mRNA may serve as an effective vaccine against COVID-19. In summary, our study revealed that lyophilization of LNPs could increase their stability and maintain their ability to be transfected both in vitro and in vivo, inducing strong immune responses.

Mucosal vaccines offer potential benefits over parenteral vaccines for they can trigger both systemic immune protection and immune responses at the predominant sites of pathogen infection. However, the defense function of mucosal barrier remains a challenge for vaccines to overcome. Here, we show that surface modification of exosomes with the fragment crystallizable (Fc) part from IgG can deliver the receptor-binding domain (RBD) of SARS-CoV-2 to cross mucosal epithelial layer and permeate into peripheral lung through neonatal Fc receptor (FcRn) mediated transcytosis. The exosomes F-L-R-Exo are generated by genetically engineered dendritic cells, in which a fusion protein Fc-Lamp2b-RBD is expressed and anchored on the membrane. After intratracheally administration, F-L-R-Exo is able to induce a high level of RBD-specific IgG and IgA antibodies in the animals' lungs. Furthermore, potent Th1 immune-biased T cell responses were also observed in both systemic and mucosal immune responses. F-L-R-Exo can protect the mice from SARS-CoV-2 pseudovirus infection after a challenge. These findings hold great promise for the development of a novel respiratory mucosal vaccine approach.