Intralymphatic Administration of Naked <scp>mRNA</scp> Induces Allergen‐Specific <scp>IgG</scp>, Promotes Th1 Responses and Reduces Anaphylactic Potential
Authors: Marta Paolucci, Frida A. M. Nilsson, Agathe Duda, Marina Tusup, Mustafa Diken, Thomas M. Kündig, Pål Johansen
Journal: Allergy (2025)

Allergen immunotherapy (AIT) is the gold standard for treating allergies. However, due to the use of native proteins and the prolonged treatment duration, AIT has long faced challenges in minimising allergen-triggered side effects and ensuring patient compliance [1]. Here, we propose to pursue intralymphatic immunotherapy (ILIT) with mRNA. In traditional subcutaneous immunotherapy, only a small fraction of the allergenic protein reaches the lymph nodes. In contrast, ILIT allows direct allergen injections to the lymph nodes, triggering faster and stronger immune responses with lower and fewer allergen doses [2]. Additionally, mast cells and basophils are rare in lymph nodes, making ILIT less likely to trigger adverse events due to the degranulation of effector cells [3]. mRNA encodes the allergen in a highly purified form, unlike crude allergen extracts used in AIT. mRNA has demonstrated the ability to stimulate functional IgG antibodies (cf. COVID-19 vaccines) and Th1 and Treg cells, counterbalancing the Th2-dominated allergy responses [4, 5]. The rapid degradation of mRNA ensures that protein expression is transient [6], minimising the risk of adverse reactions caused by protein binding to or stimulation of allergen-specific IgE [1, 4, 5]. This study had two primary objectives. Firstly, it aimed to determine if mRNA-based AIT could elicit allergen-specific and potentially beneficial B- and T-cell responses in naïve mice. Secondly, it tested the safety potential of allergen-encoding mRNA in allergen-sensitised mice. To this end, naked mRNA (BioNTech) encoding ovalbumin (mRNA-OVA) was administered via intralymphatic (IL), intradermal (ID), or intramuscular (IM) routes to naïve mice on days 0, 14 and 28, followed by a boost with OVA protein on day 49. The utilised D1-capped mRNA was modified with an anti-reverse cap analogue that creates increased resistance to enzymatic degradation [7], hence, enhanced mRNA stability and translational efficiency. Blood samples were collected on days 15, 30, 48 and 90 for serological analysis, and splenocytes were collected on day 91 for in vitro restimulation with OVA (Figure 1A). To assess the safety of mRNA-OVA in allergic mice, naïve mice were sensitised via five weekly intraperitoneal (IP) injections of 1 μg OVA protein on adjuvant alum, followed by an IP challenge on day 42 with protein-OVA or mRNA-OVA (Figure 1B). Administration of mRNA-OVA did not induce allergic sensitisation as measured by allergen-specific IgE production in mice, while IgE production was evident after protein-OVA injection (data not shown). IL and high-dose ID administration induced strong anti-OVA IgG1 responses by day 48, with IL showing superior immunogenicity (Figure 1C). High-dose IL also produced the highest IgG2a levels (Figure 1D), indicating a Th1-skewed response. Seroconversion was observed as early as on day 15 and day 30 in mice receiving the highest mRNA-OVA dose via IL and ID routes, respectively. When mice received a subcutaneous injection with protein-OVA on day 49, a boosting of IgG1 antibody responses on day 90 was observed in mice primed by ID or IM administration of mRNA-OVA and of IgG2a in mice that received IL mRNA-OVA priming. IL mRNA-OVA also stimulated significant IFN-γ production from splenocytes in vitro (Figure 1E), suggesting that IL mRNA can effectively promote Th1 activation. Additionally, IL-2 production was observed in both the IL and ID groups, while low IL-10 levels were detected exclusively in the IL group. Notably, IL-4 was undetectable in mRNA-immunised mice, further supporting the Th1-skewing effect of mRNA-immunisation (data not shown). The potential safety risk of AIT with mRNA-OVA was assessed in a murine anaphylaxis model using OVA-sensitised mice. While a challenge with OVA protein produced immediate anaphylaxis with hypothermia, challenge with mRNA-OVA did not induce hypothermia within 2 h (Figure 1F) or clinical signs of systemic allergic reactions when monitoring for the symptoms orbital tightening, piloerection, hunched back and lack of activity 2, 6 and 24 h post challenge (data not shown). Altogether, this result supports the use of IL mRNA administration as a safe AIT alternative to protein. Biodistribution studies using mRNA-luciferase showed protein expression across all injection sites 2 days after IL, ID and IM administration (Figure 2). After IL administration, expression persisted for 4 days, suggesting that IL mRNA sustains protein expression longer than other administration routes. No protein expression was determined on day eight (not shown). While studies have shown that mRNA vaccines require adjuvant vehicles, for example, liposomes or lipid nanoparticles, our study demonstrates that IL administration of naked mRNA-OVA is immunogenic, induces a favourable Th1 responses, and results in prolonged protein expression while avoiding allergenicity or the safety risks associated with protein AIT. Hence, IL mRNA-based AIT could show promise for safer and more effective allergy management by leveraging the transient nature of mRNA and the localised immune responses promoted by lymph node delivery. Further studies are needed to validate these findings across murine models and allergens and to assess the therapeutic potential of the induced immune responses. M.P. analysed data and wrote the first version of the paper. F.A.M.N. performed data analysis. A.D. and M.T. performed animal experiments. M.D. designed and provided mRNA. T.M.K. provided funds. P.J. designed and supervised the study and edited the manuscript. All authors contributed to data discussion and review of the manuscript. The cantonal veterinary office in Zurich approved all experimental procedures, which were conducted in compliance with Swiss animal welfare laws and regulations (authorization: ZH 052/2016). M.D. is an employee of BioNTech who provided mRNA and has an interest in the therapeutic use of mRNA. The other authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.