Respiratory viral infection is associated with various diseases, such as influenza caused by seasonal influenza infection. Although current influenza vaccines provide only moderate protective effectiveness (typically 40–60 % at the best, CDC), their annual reproduction is required due to both the rapid antigenic evolution of the virus and the short-lived nature of the vaccine-induced immune response. These limitations contribute to continued vulnerability of populations to severe seasonal outbreaks. [1] Pulmonary vaccines, which may elicit anti-influenza immunity in the lung or even in the respiratory mucus, are attractive for respiratory infectious diseases such as influenza. [2,3] Moreover, without the need to use syringe needles, pulmonary vaccines may be appealing to increase patient uptake of vaccines especially for those with needle phobia. Lastly, universal influenza vaccines hold great potential as highly effective influenza-strain-agnostic vaccines, regardless of the seasonal influenza mutations. [4] Ectodomain of the matrix protein 2 (M2e) is highly conserved in all human influenza A virus strains, making M2e an attractive antigen for universal influenza A vaccines. [[5], [6], [7]]

Emerging advancement in mRNA vaccine technology showcase considerable potential for influenza prevention through accelerated development timelines and potent immune activation, and has entered clinical trials. [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17]] However, despite encouraging results in preclinical and clinical studies, effective prevention from pulmonary diseases by mRNA vaccines remains challenging, presumably due to 1) suboptimal biostability that limits the efficiency and duration of antigen translation and immunomodulation, [18] 2) overly strong innate immunity elicited by long mRNA vaccines that compromises the prophylactic and therapeutic efficacies of vaccines and leads to immunotoxicity [19], and 3) the activation of protein kinase R (PKR) and 2′-5′-oligoadenylate synthetase (OAS) by relatively long dsRNA in structurally complex and bulky mRNA, both of which hamper antigen translation and immunomodulatory efficacy. [20] Moreover, current mRNA vaccines based on mRNA-loaded LNPs are highly inflammatory, and yet the respiratory system is highly sensitive to inflammatory responses; this results in relatively low tolerability and a low dose limit of current mRNA vaccines for pulmonary delivery. Addressing these limitations can be critical to advance the development of pulmonary mRNA vaccine.

In contrast to conventional linear mRNA, circular mRNA (circRNA) is an emerging class of mRNA that has shown great potential as a novel platform of RNA medicine for versatile applications. The lack of termini endows circRNA with high stability as it avoids exonuclease degradation or 5′-decapping, which are the primary mechanisms for the degradation of linear RNA such as mRNA. [21] Natural circRNA, which is generated often by the back-splicing of precursor mRNA [22], has various biological functions ranging from coding proteins or peptides to sponge for intracellular proteins or microRNA. [[23], [24], [25], [26]] Synthetic circRNA has emerged as a promising platform for developing next-generation therapeutics and vaccines, offering appealing advantages over conventional linear mRNA approaches that are often constrained by limited stability and transient expression. [21,[27], [28], [29], [30], [31], [32]] In contrast to traditional mRNA, our previous research successfully engineered small circRNA vaccines demonstrating exceptional biostability and sustained antigen production capabilities. These optimized vaccine constructs elicited potent and durable T-cell immune responses with a great safety profile, ultimately achieving superior antitumor efficacy in immunotherapy applications. [32]

Here, we report a pulmonary circRNA influenza vaccine based on pulmonary delivery of M2e-encoding small circRNA using ionizable LNPs (Scheme 1). Small circRNA-M2e is highly stable, allowing it to durably express M2e antigen. Without the need for nucleoside modification, highly stable small circRNA does not elicit potent proinflammatory responses that may otherwise cause immune reactogenicity in the body. Using dye-labeled small circRNA, we showed that pulmonary delivery of circRNA-loaded LNPs promoted the tissue retention of circRNA in the lung, and enhanced the uptake of circRNA in APCs that are critical for small circRNA vaccine to elicit adaptive immune responses. Upon pulmonary delivery, circRNA-M2e vaccine elicited robust and durable anti-M2e T cell responses in both young adult mice but also in immunosenescent aged mice (18 months) for the prevention of influenza infection. As a result, pulmonary delivery of circRNA-M2e in mice effectively prevents the infection of mouse-adapted influenza.