Abstract by Melike Ongun

The respiratory tract serves as the first line of defense and is continuously exposed to inhaled allergens, pollutants, and pathogens, e.g., respiratory viruses. The coordination of innate and adaptive immune responses within the respiratory tract is critical for protection against invading respiratory pathogens.

The success of vaccines based on messenger RNA (mRNA) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has demonstrated the promising potential of mRNA therapeutics as safe, effective, and widely applicable tools for enhancing public health on a global scale. Current mRNA vaccines based on lipid nanoparticles (LNPs) are primarily administered via intramuscular (i.m.) injection, which effectively induces systemic immunity, but provides limited mucosal immunity. However, waning immunity and the ongoing evolution of more transmissible and immune-evasive viral variants have resulted in decreased vaccine effectiveness. Vaccines have also become less effective at preventing transmission, likely due to insufficient induction of mucosal immunity in the respiratory tract. Robust mucosal immunity can block infections at mucosal surfaces and prevent further transmission, and mitigating the potential for future pandemics. Notably, the mucosal immunity elicited by potential mRNA vaccines remains largely unexplored, with limited studies addressing this aspect.

The aims of the PhD project were: i) to investigate the effect of polyethylene glycol (PEG)-lipid content of mRNA-loaded LNPs and route of administration (pulmonary versus nasal) on lung delivery (protein expression) in mice, and ii) to investigate whether nasal and pulmonary administration of mRNA-LNPs can mediate induction of antigen-specific mucosal immune responses in the respiratory mucosa and assess whether different prime-boost immunization strategy based on administration route can induce immune responses in respiratory tract.

Lipid nanoparticles loaded with mRNA were formulated with different PEG-lipid content. The colloidal stability of mRNA-LNPs after spraying with the PennCentury microsprayer was assessed by characterizing their hydrodynamic diameter and polydispersity index (PDI) using dynamic light scattering, along with evaluating mRNA entrapment efficiency and in vitro protein expression. The morphology was investigated using cryogenic transmission electron microscopy and internal structure using small angle X-ray scattering to further characterize the structure of mRNA-LNPs with different PEG-lipid contents. The results showed that increasing the PEG-lipid content improved the colloidal stability during the aerosolization process but negatively affected transfection efficiency in vitro and protein expression in vivo. Additionally, the protein expression levels in the lungs of mice declined more rapidly after nasal administration than after pulmonary administration.

Further investigation showed that parenteral immunization with mRNA-LNPs elicited strong antigen-specific CD8⁺ and CD4⁺ T-cell responses in the spleen and high IgG levels in the serum. Mucosal prime-boost immunization via local pulmonary or nasal administration of mRNA-loaded C12-200 LNPs failed to induce antigen-specific systemic and mucosal immune responses. In contrast, pulmonary pull immunization following an i.m. prime immunization with mRNA-loaded C12-200 LNPs improved systemic immunity by increasing IgG1 levels in serum. However, this prime-pull immunization strategy using mRNA-loaded C12-200 LNPs enhanced IgG1 responses in the lungs but still was insufficient to induce mucosal IgA responses.

These results suggest that formulations designed for parenteral administration, such as currently approved mRNA-LNP vaccines technology, are not optimal for inducing mucosal immunity. Further optimization is needed, focusing on the use of non-toxic, highly tolerable materials to improve delivery systems tailored for the respiratory mucosa. The prime-pull immunization strategy shows promise for inducing mucosal immunity. However, additional refinements, such as optimizing the interval between prime and pull doses or incorporating additional pull dose to provide more antigen in respiratory mucosa, could enhance the effectiveness of this approach.