An mRNA-delivered consensus allergen induces a neutralizing IgG response against food and pollen allergens

Domain: neuroscience
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
Cross-allergies affect a significant proportion of the population, and contribute to detrimental health and socioeconomic impacts, yet allergen immunotherapies often target a single allergen source disregarding cross-reactive allergens from other sources. Here we introduce an immunization approach developed for improved desensitization in cross-allergic patients using a consensus allergen (cnsLTP1), which contains orthologous non-specific lipid transfer proteins (nsLTP) derived from relevant fruit and pollen allergens. In BALB/c mice, vaccination via either mRNA-lipid nanoparticle (LNP) vehicle or traditional protein formulation induces cnsLTP1-specific IgGs capable of recognizing and binding to multiple nsLTPs. These IgGs block allergen binding by patient serum IgEs and prevent humanized rat basophil degranulation in vitro. Meanwhile, in an allergic mouse model, the mRNA-LNP formulation is tolerated and induces allergen-specific IgG responses but does not ameliorate subsequent allergen challenge responses. Regardless, this cross-allergen mRNA-LNP-based immunotherapy may have translation value once route of administration, formulation and/or dosing are optimized.

Introduction
Allergy is a chronic condition affecting 10–30% of the global population, with increasing incidence1. Cross-reactivity occurs when structurally similar proteins from different sources share epitopes, leading to immune recognition and reactions induced by multiple allergens2–4. The management of food allergy is complicated by an abundance of homologous, cross-reactive proteins in edible foods and aeroallergens5,6. This results in patients having allergic sensitization to many biologically related foods or cross-reactivity between pollen and plant foods4.

The short-term treatment of food allergy involves either avoiding the allergen source or using nonspecific acute treatments, such as antihistamines for milder cases and adrenaline for severe systemic cases (anaphylaxis)7. Besides avoidance measures, the only approach for mitigating the effects of food allergy is food allergy immunotherapy (AIT) (through oral (OIT), sublingual (SLIT), or epicutaneous (EPIT) routes) either alone or in combination with an anti-IgE monoclonal antibody, such as omalizumab8. However, these therapies are often associated with frequent and potentially severe adverse reactions.

Pollen AIT has proven effective for the treatment of respiratory symptoms, but prospective studies are needed to establish its role in the prevention and moderation of symptoms upon food exposure in cases of pollen-food allergy syndrome (PFAS)2. Over time, AIT modulates the immune response, shifting the production of allergen-specific antibodies from IgE to IgG subtypes, while inducing a tolerant T H1- and regulatory T (T reg)-cell phenotype9,10. These allergen-specific IgG antibodies protect against allergic reactions by blocking the allergen, thereby reducing allergen sensitivity11–14.

Even though AIT may effectively reduce the frequency and risk of severe food allergic reactions, its main limitations include the risk of severe adverse reactions and the duration of treatment. In the case of OIT, long-term tolerance may require continued ingestion of a maintenance dose. For pollen AIT, the treatment lasts from three to five years with frequent clinical follow-ups and variable efficacy among patients11. Moreover, traditional AIT relies on protein extracts where the specific allergens crucial for desensitization are combined with other proteins at undetermined concentrations. Each mixture therefore not only contains the target allergen, but also other unrelated proteins that might restrict the immune response against the triggering allergen15. Particularly for patients with multiple food allergies due to genuine and cross-reactive allergens, desensitization to a specific allergen isoform in the extract might not induce protection against other related allergens found in other food and pollen sources16–18. This lack of paraspecificity, coupled with lengthy treatment durations contributes to high discontinuation rates observed in AIT for respiratory allergens like grass pollen19–21. Specifically, studies report discontinuation rates of up to 77% for SCIT and 36% for SLIT22.

In this study, we explore a new therapeutic approach for mitigating the effect of extensive cross-reactive food allergies by combining mRNA-based immunization with a designed consensus allergen-a single engineered protein that closely resembles multiple related natural allergens. Successful delivery of natural allergens encoded by mRNA has already been achieved in the context of AIT23–26. Therefore, we hypothesized that an approach relying on a consensus allergen would enable simultaneous treatment of multiple allergies. Further, delivery via mRNA encapsulated in a lipid nanoparticle (mRNA-LNP) can ensure predominantly intracellular expression, thereby reducing extracellular allergen presence and availability for binding by preexisting IgEs. With this approach, we aim to provide a treatment that alleviates many of the adverse effects associated with conventional AIT.

To explore the utilization of the abovementioned strategy for the cross-protection against different allergies, we focused on widely cross-reactive allergens: non-specific lipid transfer proteins (nsLTPs). nsLTPs are allergens belonging to a highly conserved protein family found in multiple plant-derived foods, such as peaches, apples, and nuts, as well as in pollen from olive tree, plane tree, pellitory, and mugwort. LTP syndrome is more prevalent in Mediterranean countries and is often linked to peach as the primary sensitizing food. Due to the broad cross-reactivity of nsLTPs, patients develop symptoms upon consumption of fruits, nuts, and vegetables, and symptoms can range from mild oral reactions to severe anaphylaxis18,25. Commonly, patients with nsLTP allergies are polysensitized to several nsLTPs, potentially hampering the clinical efficacy of traditional AIT protocols.

To evaluate our immunization approach, we immunise naive mice with either mRNA or protein-based formulations of the consensus allergen and characterize the cross-binding properties of serum antibodies to a panel of recombinant and natural nsLTPs, as well as their capacity to prevent IgE binding to allergens and basophil degranulation, demonstrating the IgG blocking capacity. In addition, we test our formulation in an allergic mouse model. The mRNA-LNP formulation is well tolerated upon injection, while inducing allergen-specific IgG responses. Taken together, our results provide proof of concept for a new treatment strategy based on mRNA-LNP technology and consensus allergens, as well as showcase the utility of this approach on nsLTPs involved in LTP syndrome.

Results
Consensus allergen retains nsLTP folding and binds IgE
For the design of the consensus allergen (cnsLTP1), nsLTP amino acid sequences from different plant botanical families, constituting food and pollen allergens, were included to broadly represent the ubiquitous nsLTP family. While the designed consensus allergen did not mirror any specific natural sequence, it exhibited the highest sequence identity to food nsLTPs, with Mal d 3 (apple) being the closest related, showing over 81% sequence identity, followed by Pru p 3 (peach) with 75% sequence identity. In contrast, the least identity was observed with pellitory pollen nsLTPs, to the consensus allergen sharing less than 30% identity with Par j 2 (Supplementary Fig. 1). This broad range of sequence diversity, from high identity with Mal d 3 to low identity with Par j 2, highlights the ability of the cnsLTP1 to encompass epitopic features across diverse nsLTP variants, supporting its utility in eliciting cross-reactive immune responses.

For characterization and further comparison of the cnsLTP1 to other nsLTPs, we designed a panel of recombinant allergens. This panel included a variety of nsLTP allergens from both food (like fruit, nuts, and legumes) and pollen sources. These allergens were chosen for their significant differences in amino acid sequences, as shown in the sequence identity matrix (Fig. 1c). The selected allergens have varying degrees of identity to the cnsLTP1, ranging from 29.35% (Par j 2 and Ole e 7) to 74.73% (Pru p 3), as detailed in Fig. 1c. Despite considerable sequence variability across the nsLTP family, structural comparison revealed high identity between pollen and food nsLTPs, as well as with the consensus allergen (Fig. 1a), suggesting that all nsLTP variants and the consensus allergen share a common structural scaffold. The far-UV circular dichroism (CD) spectrum of the cnsLTP1 (Fig. 1b) indicated a predominant α-helical structure, fitting well with its predicted three-dimensional conformation. When heated to 85 °C, the protein did not fully denature but retained a spectrum indicative of an α-helix-rich structure. The original three-dimensional structure was restored after cooling the sample to 25 °C.

The panel of recombinant allergens, appearing with a molecular weight between 10 and 15 kDa on SDS-PAGE, were all recognized by a commercial Mal d 3 polyclonal antibody in their reduced but not in their native form, indicating a change in structure upon reduction and the presence of hidden epitopes (Supplementary Fig. 2). We hypothesize that the anti-Mal d 3 antibody has been raised against an unfolded Mal d 3 and, therefore, is restricted to linear epitopes that are hidden when the protein is not completely reduced.

To ascertain the clinical relevance of the designed cnsLTP1, we assessed its recognition by IgEs present in the serum of patients diagnosed with nsLTP cross-allergies. Our analysis included 10 patients presenting severe oral allergy syndrome, who were recruited after systemic reactions to nsLTP-containing foods and were sensitized to various nsLTPs but exhibited distinct sensitization patterns (Supplementary Table 2). The patients did not show any other relevant co-sensitization beyond nsLTPs. Western blotting demonstrated that IgE antibodies from all tested patients recognized the cnsLTP1 (Supplementary Fig. 3). These findings substantiate that the cnsLTP1 contains relevant IgE epitopes, underscoring its clinical relevance in the context of nsLTP cross-allergies.

cnsLTP1 IgG induction by mRNA-LNP and protein immunization
To evaluate the immunogenicity of the cnsLTP1, we introduced cnsLTP1, Pru p 3, and Par j 2 by intramuscular administration to naive BALB/c mice through an mRNA-LNP. The cnsLTP1 was also tested by a conventional protein immunization with incomplete Freund’s adjuvant. Additionally, we evaluated the effect of the route of administration by testing the cnsLTP1 mRNA formulation and the protein formulation with poly(I:C) or Freund’s adjuvant via subcutaneous injection. All formulations were administered three times at three-week intervals (Fig. 2a). No signs of discomfort or fever were observed in the animals throughout the study.

Both methods of immunization increased antibody titers across all isotypes, except IgA, compared to PBS-treated controls (Fig. 2b). Protein immunization resulted in significantly increased IgG1, whereas mRNA immunization significantly increased IgG2a and IgM compared to the PBS-treated controls, indicating a more IgG2a- and IgM-driven response in the mRNA-immunized mice (Fig. 2b). The mice immunized with either the cnsLTP1 or the Pru p 3 mRNA-LNP formulation produced low levels of cnsLTP1-binding antibodies after a single dose (day 21) (Fig. 2c). In contrast, the protein formulation necessitated a booster dose to elicit a comparable response. Both mRNA and protein formulation demonstrated increased allergen-specific antibody titers after the first (day 42) and second (day 63) booster doses, with increased IgG1 for both protein and mRNA immunization, whereas only mRNA immunization significantly increased cnsLTP1-binding IgG2a titers (Figs. 2d and 3). Equivalent results were found using subcutaneous administration (Supplementary Fig. 4a–c).

Thus, a distinct trend emerged, where cnsLTP1-binding IgG2a titers in mRNA-immunized mice were higher than IgG1 titers, while the opposite was observed in protein-immunized mice, where the titers of IgG1 were higher than those of IgG2a (Figs. 2c and 3). In the context of allergen neutralization, the human isotype IgG4 is responsible for neutralizing allergens and preventing the binding to IgE27. Such a role is performed by IgG1 and IgG2a isotypes in mice28.

To evaluate the induction of TH1/TH2 cells in response to mRNA or protein immunization, we restimulated T cells from the spleen and draining inguinal lymph nodes (iLNs) (Supplementary Figs. 5, 6). Following intramuscular immunization, spleens of mice subjected to mRNA immunization had slightly lower percentages of IFNgamma (IFNg)-producing TH1 cells and higher IL4-producing TH2 cells, resulting in a lower TH1/TH2 ratio, whereas the cnsLTP1 protein formulation was similar to PBS (Supplementary Fig. 5a, b). The draining iLNs, however, had similar or higher percentages of TH1 cells following mRNA immunization compared to protein immunization or PBS, with no significant differences observed for TH2 cells and TH1/TH2 ratios (Supplementary Fig. 5c, d). Following subcutaneous immunization, no significant differences were observed across neither the spleen nor iLNs (Supplementary Fig. 5e–h).

We also assessed the induction of IgE to compare the potential of the two immunization formulations for triggering an allergic reaction. No allergen-specific IgE was detected in mice immunized intramuscularly with either the mRNA-LNP or protein formulation. However, in the subcutaneous administration of the protein formulation, the inclusion of an adjuvant, specifically poly(I:C), significantly increased the production of allergen-specific IgE compared to PBS-treated mice (Supplementary Fig. 4d, e).

Cross-reactivity of IgGs with recombinant nsLTP
To assess whether antibodies produced in response to immunization with the cnsLTP1 recognized natural allergens, we conducted an IgG1 and IgG2a ELISA with a panel of recombinantly expressed allergens. Antibodies elicited against the cnsLTP1 demonstrated binding capabilities across all tested allergens except Ole e 7, with significantly stronger recognition than antibodies derived from Pru p 3 or Par j 2 immunizations. Specifically, mice immunized with the cnsLTP1 mRNA formulation showed broader cross-reactivity, recognizing all allergens tested, including Pru p 3, Cor a 8, Ara h 9, Par j 2, Pla a 3, and Tri a 14. In contrast, mice immunized with Pru p 3 mRNA recognized Pru p 3, Cor a 8, Ara h 9, and Pla a 3, but not Tri a 14 or Par j 2. Similarly, mice immunized with Par j 2 mRNA primarily recognized Par j 2 and Pla a 3, but showed no significant recognition of the other allergens (Fig. 4).

A comparison of cnsLTP1 mRNA and protein formulations revealed that the former induce significant IgG1 recognition of Pru p 3, Cor a 8, and Par j 2 (Fig. 3a). Regarding IgG2a, the antibodies induced using the mRNA formulation demonstrated significant binding to all the allergens compared to the antibodies derived using the protein formulation (Fig. 3b). This enhanced recognition was accompanied by higher titers of allergen-specific IgG2a. Notably, the intensity of recognition observed in the ELISA correlated with the percentage sequence identity shared with the cnsLTP1, with the strongest signal observed for Pru p 3 (74.73% sequence identity) and the weakest for Par j 2 and Ole e 7 (29.35% sequence identity).

Murine antibodies block allergen-IgE interaction
To investigate the ability of vaccine-induced antibodies to block relevant human IgE epitopes, we evaluated whether murine antibodies induced by immunization with the cnsLTP1 could block the interaction between allergens and human IgEs from patient sera using two complementary assays: an IgE ELISA (Fig. 5) and a degranulation assay with hRBL-2H3 cells (Fig. 6). Initial tests revealed that the patient sera exhibited varied sensitization patterns across the panel of recombinant allergens, including the cnsLTP1, Pru p 3, Cor a 8, Ara h 9, Par j 2, Tri a 14, and Pla a 3. These patterns were consistent between the IgE ELISA and the degranulation assay, confirming the utility of both approaches for evaluating allergen-IgE interactions (Figs. 5a and 6a).

Blocking experiments demonstrated that antibodies from both mRNA-LNP and protein-immunized mice could effectively inhibit IgE binding to all recombinant allergens in a dose-dependent manner (Fig. 5c). Furthermore, this blocking translated into functional inhibition, as reduced degranulation of hRBL-2H3 cells was observed when the allergens were preincubated with mouse serum prior to being introduced to the cells pre-sensitized with patient serum (Fig. 6c). Even though serum from mRNA-LNP-immunized mice showed stronger blocking potency compared to serum from protein-immunized mice, as evidenced by lower IC50 values, the differences were not statistically significant (Figs. 5b, 6b; Supplementary Table 3). These findings indicate that the cnsLTP1 in the mRNA-LNP and protein formulations induced a robust and effective antibody response capable of disrupting allergen-IgE interactions and strongly reducing cellular degranulation in response to a wider range of nsLTPs originating from pollen and food sources.

mRNA-LNP cnsLTP1 induces allergen-specific antibodies in an allergic mouse model
To assess the therapeutic potential of the cnsLTP1 mRNA-LNP formulation in a sensitized setting, we established an allergic anaphylactic mouse model using intranasal Pru p 3 and LPS (Fig. 7a). After sensitization, mice were monitored after each injection of the mRNA-LNP formulation, and no clinical signs of allergy or anaphylaxis were observed. Following intraperitoneal challenge with Pru p 3, body temperature in the treated animals did not differ significantly from that of sensitized but untreated mice. Thus, with the employed experimental setup and small cohorts of mice, the utility of the immunization approach for reducing the level of anaphylaxis could not be established. Measurement of mMCP-1, a marker of mast cell degranulation, did not reveal significant differences between groups (Fig. 7c). Analysis of Pru p 3–specific antibodies in the serum of the mice revealed that levels of IgE were comparable between treated and untreated sensitized groups (Fig. 7g). Allergen-specific IgG1 levels were comparable for non-treated and treated mice for Pru p 3, Cor a 8, and Ara h 9 (Fig. 7d). However, the levels of Par j 2, Tri a 14, and Pla a 3-specific IgG2a were significantly higher in treated mice, demonstrating the induction of cross-reactive antibody populations caused by the cnsLTP1-mRNA treatment. Following the same tendency, mice immunized with cnsLTP1 mRNA-LNP exhibited a significant increase in IgG2a and IgG2b levels for all the nsLTPs tested (Fig. 7e-f).

Although the therapeutic utilization of induced antibodies in the allergic mouse model could not be established in the current setup (Fig. 7b), their induction demonstrates the capacity of the consensus allergen to elicit an antigen-specific antibody response. We therefore speculate that changing the route of administration and further optimizing formulation and/or dosing could lead to protective efficacy.

Methods
This study complies with all relevant ethical regulations and is approved by the Danish Animal Experiments Inspectorate, Institutional Animal Care and Use Committee of IBIMA and by the Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible of the Junta de Andalucía. A summary table with all the antibodies, catalogue numbers and dilutions can be found in Supplementary Table 4.

Design of consensus allergen
nsLTPs were selected based on phylogenetic clustering of sequences and different allergen families26. For each cluster or allergen family, at least a single nsLTP was chosen. Amino acid sequences were retrieved from UniProt and aligned using the Clustal Omega algorithm, accessed through the European Bioinformatics Institute (EMBL-EBI) web server (https://www.ebi.ac.uk/Tools/msa/clustalo/) with default parameters46,47. Only sequences of ~90 amino acids in length were selected to be included in the consensus design. The consensus sequence was designed based on the selection of the most conserved or physicochemical-relevant amino acid for each position. For each position, the most frequent amino acid was kept, and in case of a tie, the most abundant amino acid within the most present physicochemical property was used: acid (E, D), basic (K, R), polar (S, T, Y, N, Q), and nonpolar (G, A, V, L, I, M, W, F). P and C formed their own group. The final sequence is in Supplementary Table 5.

Expression of nsLTP allergens
Recombinant allergens were generated by cloning and heterologous expression. Codon-optimized consensus allergen cDNA (Eurofins) was subcloned into the pPICZαA expression vector (#V19520, Invitrogen) and introduced into Komagataella phaffii KM71H (#C18200, Invitrogen) by electroporation47. Colonies were selected by their ability to grow in the presence of YPDS (20 g/L peptone, 10 g/L yeast extract, 20% (w/v) dextrose, 182.2 g/L sorbitol, 20 g/L agar) supplemented with Zeocin 1000 µg/mL plates. Individual colonies grew at 30 °C and 220 rpm for 24 h in BMGY (10 g/L yeast extract, 20 g/L peptone, 0.1 M potassium phosphate pH 6.0, 1.34% (w/v) YNB, 0.04 µg/mL biotin, 1% (v/v) glycerol), and expression was induced over 72 h in BMMY (10 g/L yeast extract, 20 g/L peptone, 0.1 M potassium phosphate pH 6.0, 1.34% (w/v) YNB, 0.04 µg/mL biotin, 0.5% (v/v) methanol) with addition of 1% (v/v) methanol every 24 h. The supernatant was collected and filtered through a 0.22 µm membrane and dialyzed against Bis-Tris 20 mM pH 6.0 in a 3.5 K MWCO nitrocellulose membrane. Dialyzed samples were purified by cation exchange on a UNOSphere S (BioRad) column. Elutions corresponding to the correct molecular weight according to SDS-PAGE analysis were pooled, dialyzed against 50 mM ammonium bicarbonate pH 7.0, lyophilized, and stored at −80 °C.

Characterization of consensus allergen
Purified proteins (2 µg/well) were mixed with 3X loading dye, with or without 1 mM DTT, and boiled for 10 min at 95 °C47. Proteins were transferred to a pre-activated PVDF membrane (#LC2002, Novex) using a SureLock Minicell at 30 V for 1 h. Subsequently, the membrane was blocked overnight in a solution of PBS containing 0.1% Tween 20 (v/v) (PBST) and 5% (w/v) skim milk. After blocking, the membrane underwent three washes with PBST, followed by incubation with a primary rabbit anti-Mal d 3 polyclonal antibody (Catalog #abx300086, Abbexa) for 1 h. This step was succeeded by another series of washes. The membrane was then incubated with a secondary HRP-conjugated goat anti-rabbit antibody (#31460, Invitrogen) for 1 h, concluded by a final wash. Detection of bound antibodies was achieved using Clarity ECL Substrate (#1705061, BioRad), with chemiluminescence signals measured thereafter.

The secondary structure of recombinant proteins and thermal stability was analyzed with far-UV CD spectroscopy using a Jasco J-715 spectropolarimeter equipped with a Neslab RTE-111 thermostat using a 0.1 cm pathlength thermostated cuvette. Consensus allergens were dissolved in 10 mM HEPES, 0.1 M NaCl to avoid pH changes during heating. CD spectra were recorded at 25 °C and 85 °C, after 0.5 °C/min heating while registering changes in ellipticity at 208 nm. Reverse cooling was performed at the same rate, and final spectra were recorded after cooling down to 25 °C.

Sera from allergic patients
Patients were selected due to their history of cross-reactivity to multiple nsLTPs confirmed by skin prick test and ImmunoCAP (Thermo Fisher). All studies involving human material were reviewed and approved prior to initiation by the Comitè d’Ètica de la Investigació amb Medicaments (CEIm) de l’Hospital Universitari Vall d’Hebron and by the Comitè d’Ètica de la Investigació amb Medicaments (CEIm) de l’Hospital Clínic de Barcelona, as applicable. All participants were recruited in accordance with the approved protocols and provided written informed consent prior to sample collection.

IgE recognition of consensus allergen in patients
IgE recognition of the cnsLTP1 was tested with human sera from 10 food nsLTP allergic patients. Purified protein was transferred onto a nitrocellulose membrane after SDS-PAGE (200 ng/strip) and an indirect Western blot was performed with patients’ sera to test for IgE recognition. After 1 h of incubation with blocking buffer, strips were incubated with individual patient sera (1/5 diluted in blocking buffer) for 2 h at room temperature (RT) and later washed three times with PBST. Peroxidase-labeled anti-IgE antibody (#9160-05, Southern Biotech) was incubated (1/1000) for 1 h, and after the final three washes, bound IgE was detected with Clarity ECL Substrate (Bio-Rad) using chemiluminescence detector Fujifilm LAS3000.

mRNA design
The cnsLTP1 mRNA sequence was designed for MHC-II presentation through targeted lysosomal delivery mechanisms by fusing the cnsLTP1 coding sequence to a lysosomal transmembrane domain (LAMP1) and a C-terminal lysosomal translocation sequence48. The mRNA encoding for cnsLTP1 was codon-optimized for humans and single-point synonymous modifications were included to avoid secondary structure formation in the mRNA. The mRNA encoding the consensus allergen was purchased from RiboPro, including a 150 nt poly-A-tail and a Cap1 sequence.

Consensus allergen mRNA-LNP preparations
The mRNA was encapsulated in LNP based on the Pfizer/BioNTech vaccine Comirnaty (BNT162b2), but with the PEGylated lipid ALC-0159 exchanged with DMG-PEG2000, resulting in a formulation consisting of ALC0315:Cholesterol:DSPC:DMG-PEG2000 (molar ratio 46.3:42.7:9.4:1.6, N/P ratio 6 corresponding to 39 nmol lipid per µg mRNA)49,50. All lipids were acquired from Avanti Polar Lipids. Lipids were dissolved in ethanol and mRNA in 100 mM sodium acetate (pH 4), and particles were formed by mixing in 1:3 ratios using the NanoAssemblr Ignite microfluidic mixer (Precision Nanosystems) at a final concentration of 100 µg/mL mRNA. Particles were buffer exchanged to HBS (25 mM HEPES, 150 mM NaCl, pH 7.4) by discontinuous diafiltration using Amicon Ultracel spin filters (100 kDa cutoff) and upconcentrated to 0.35 µg/µL. Characterization of mRNA-LNPs was done with dynamic light scattering (DLS) and M3-PALS using a Zetasizer Nano ZS (Malvern Instruments) to determine size, polydispersity, and zeta potential and found to have a size (Z-Ave) of 76.4 ± 5.6 nm and a PDI of 0.11 ± 0.04 (mean values for four batches prepared). Encapsulation efficiency and final yield of mRNA were determined with the RiboGreen Assay, by comparing the RiboGreen emission for LNPs in Tris buffer to LNPs disassembled by incubating with 0.1% Triton X-100 at 60 °C for 30 min. The particles were slightly smaller and more monodisperse than those in BNT162b250. The zeta potential of −12.0 ± 5.0 mV is comparable to what has been reported for BNT162b250. The mRNA encapsulation efficiency was high, reaching 91.7 ± 3.6%. Particles were diluted in 5% sucrose to the desired concentrations and volumes and stored at −80 °C until use. The size (Z-Ave), polydispersity, zeta potential, and encapsulation efficiency were determined after thaw to confirm LNP stability (Supplementary Table 1).

Consensus allergen protein preparations
The consensus allergen protein was dissolved in sterile PBS containing 50 µg/mL of polyinosinic–polycytidylic acid sodium salt (poly(I:C) #P1530, Sigma-Aldrich) or mixed 50% (v/v) with incomplete Freund’s adjuvant (#F5506, Sigma-Aldrich).

Immunization schedule for naïve mice
The studies were approved by the Danish Animal Experiments Inspectorate (approval #2020-15-0201-00748). The animals were co-housed in the DTU BioFacility which includes SPF-status barrier breeding unit, a laboratory for sample processing and facilities for surgical procedures. Animals were maintained on a 12 h light/12 h dark cycle, with ambient temperature kept at 20–22 °C and relative humidity maintained at 45–65%. Food and water were provided ad libitum, and animals were group-housed with environmental enrichment. The immunization was performed in two independent groups of mice, subcutaneously or intramuscularly. For the subcutaneous immunization, 4-to-5-week-old BALB/cAnNRj female mice (#0003 Janvier Labs) were immunized by subcutaneous injection three times at three-week intervals with 9 µg mRNA-LNP (n=6), 24 µg protein-poly(I:C) (n=3), 24 µg protein-incomplete Freund’s adjuvant (n=3), or PBS (n=7). For the intramuscular immunization, four-to-five-week-old BALB/cAnNRj female mice were immunized by intramuscular injection three times at three-week intervals with 7.5 µg mRNA-LNP (n=5), 20 µg protein-incomplete Freund’s adjuvant (n=5), or PBS (n=5). Mice were euthanised by cervical dislocation. The specific amounts of mRNA-LNP or protein were determined in titration experiments comprising three mice each, and the responses were evaluated by measuring the presence of total Igs and allergen-specific IgG1 and IgG2a. The specific doses were determined in previous titration experiments, and the range was established42,51–53.

Establishment of allergic anaphylactic mouse model and immunization schedule
Four-to five-week-old BALB/cAnNRj mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France) and housed at the IBIMA Plataforma BIONAND animal facility, which includes SPF-status barrier breeding unit, a laboratory for sample processing and facilities for surgical procedures (Registration No. ES 290670001687). All animal experiments were formally reviewed and authorized prior to initiation by the Institutional Animal Care and Use Committee of IBIMA and by the Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible of the Junta de Andalucía, under approved protocol number 14/05/2025/051. All procedures were conducted in full compliance with Spanish and European legislation regulating animal experimentation (RD1201/2005, Law 32/2007, Directive 2010/63/EU, and RD53/2013) and adhered to the principles of Replacement, Reduction, and Refinement (3Rs). Animals were maintained on a 12 h light/12 h dark cycle, with ambient temperature kept at 20–22 °C and relative humidity maintained at 45–65%. Food and water were provided ad libitum. Mice were divided into three co-housed groups: non-sensitized controls (n=8), sensitized non-treated (n=8), and sensitized mRNA-LNP treated (n=8). In all groups 3 male and 5 female mice were included. Sensitization was performed intranasally under isoflurane anesthesia using 20 µg of natural Pru p 3 (Bial Laboratory, Zamudio, Spain) combined with 20 ng of LPS (InvivoGen, San Diego, CA) in a final volume of 12 µL PBS. Control mice received 12 µL PBS following the same schedule54,55. Starting at week 6, mice in the treatment group received intramuscular injections of 7.5 µg cnsLTP1 mRNA-LNP in 30 µL once weekly for four consecutive weeks, while control and sensitized non-treated groups received 30 µL PBS according to the same schedule. One week after the last treatment dose, sensitized mice were challenged intraperitoneally with 100 µg of Pru p 3 to assess systemic reactions. Anaphylaxis was evaluated within 30–40 min after challenge by monitoring rectal temperature and recording clinical symptoms according to an established scoring system56. Immediately after the last rectal temperature measurement, mice were bled from the retro-orbital plexus under ketamine-xylazine intraperitoneal anesthesia, and sera were stored at –20 °C for humoral analyses. Following blood extraction, mice were euthanized by cervical dislocation, and spleens were aseptically removed to prepare single-cell suspensions for cellular assays.

Total antibodies evaluation by ELISA
Total antibodies were measured according to the manufacturer’s instructions (#88-50630-88, Invitrogen), using 1/5000 diluted mouse serum.

Mouse Igs recognition of cnsLTP1 and natural nsLTPs by ELISA
Two types of ELISAs were performed to quantify mouse antibody responses against cnsLTP1, recombinant nsLTPs, and Pru p 3. Unless otherwise stated, all assays were performed in duplicate and washed thoroughly between steps using PBS containing 0.05% (v/v) Tween-20 (PBST).

Assays were carried out using the Mouse Ig Isotyping Uncoated ELISA Kit (#88-50630-88, Invitrogen) with minor modifications. Half-area 96-well plates (#675061, Greiner) were coated overnight at 4 °C with 500 ng/well of either cnsLTP1 or recombinant nsLTPs. Plates were washed with PBST and PBS and blocked with 125 µL blocking buffer for 2 h at room temperature (RT). A mixture of 25 µL assay buffer (PBS containing 0.05% Tween-20 and 0.5% BSA) and 25 µL of diluted mouse serum (1:1000 or 1:5000) was added and incubated for 1 h at RT. After washing, wells were incubated with anti-mouse isotype-specific antibodies for 1 h, followed by a goat anti-rat IgG (H+L) HRP-conjugated secondary antibody (#A10549, Invitrogen; 1:2000, 1 h at RT). Bound antibodies were detected using 50 µL/well TMB substrate, and the reaction was stopped with 1 M H2SO4 before reading absorbance at 450 nm.

To quantify Pru p 3–specific antibodies (IgG, IgG1, IgG2a, and IgG2b, and IgE), high-binding 96-well ELISA plates (#439454, Thermo Scientific) were coated overnight at 4 °C with purified Pru p 3. After blocking with BSA-containing buffer, mouse sera were added at 1:8 dilution for IgE detection and 1:50 dilution for IgG isotypes and incubated overnight at 4 °C. Plates were washed and incubated with biotinylated rat anti-mouse isotype-specific antibodies, followed by avidin–horseradish peroxidase (HRP). Detection was performed with TMB substrate, and the reaction was stopped with H2SO4 prior to reading absorbance at 450 nm.

Mouse IgE recognition of cnsLTP1 and natural nsLTPs by DELFIA
Half-area 96-well plates were coated overnight at 4 °C with 2.5 µg/mL anti-mouse IgE antibody (MA5-16779, Invitrogen). Following coating, the plates were washed three times with PBST and three times with PBS before being blocked with 125 µL of blocking buffer for 2 h at RT. To each well, 25 µL of assay buffer (PBS with 0.05% Tween-20 and 0.5% BSA) and 25 µL of diluted mouse serum (1/1000 or 1/5000) were added and incubated for 1 h at RT. The plates were then washed, and 50 µL of biotinylated cnsLTP1 (10 µg/mL) was added to each well. After five washes with PBST and three washes with PBS, europium-labeled streptavidin (#1244-360, Revvity) was added at a 1/500 dilution in assay buffer and incubated at 19–21 °C for 30 min. The plates were washed again with PBST and PBS, followed by the addition of DELFIA enhancement solution (#4001-0010, Revvity) for 5 min. Finally, time-resolved fluorescence (TRF) was measured at 615 nm after excitation at 340 nm using a VICTOR Nivo plate reader (PerkinElmer).

Inhibition of IgE binding to natural nsLTPs by ELISA
ELISA plates were coated, blocked, and washed as described. Following the addition of mouse serum in increasing serial dilutions, the plate was incubated for 1 h at RT and washed three times with PBST and three times with PBS. Human serum from four allergic patients with proven IgE-mediated peach allergies was pooled and added in 1/10 dilutions to wells following 1 h incubation. goat anti-human IgE secondary antibody, HRP (#A18793, Invitrogen) was diluted (1/500) and added to wells and incubated for 1 h. Bound antibodies were detected with 50 µL/well TMB substrate, the reaction was stopped with 1 M H2SO4, and the plates were read at 450 nm.

mMCP-1 assay
To obtain an additional readout of systemic anaphylaxis, serum levels of mouse mast cell protease-1 (mMCP-1) were measured following allergen challenge. Blood was collected from the retro-orbital plexus 1 h after intraperitoneal administration of Pru p 3, allowed to clot at room temperature, and centrifuged at 10,000 rpm g for 10 min at 4 °C. Sera were stored at –20 °C until analysis. Quantification of mMCP-1 was performed using a commercial ELISA kit (88-7503-88, Invitrogen) according to the manufacturer’s instructions. Absorbance was read at 450 nm with wavelength correction at 570 nm using a microplate reader. Concentrations of mMCP-1 were calculated from a standard curve generated with recombinant mMCP-1 supplied in the kit.

TH1/TH2 cell polarization following immunization
The right iLN and spleen were harvested from the mice on the day of takedown and kept in PBS. Cells were released from the tissues by mashing through a 70 µm cell strainer, and the spleen cells were subjected to RBC lysis buffer (#00-4333-57, eBioscience) to remove red blood cells. Cells were activated in RPMI 1640 (#61870044, Thermo Fisher Scientific), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin, with 1X Leukocyte Activation Cocktail, with GolgiPlug (#550583, BD Bioscience) for 6 h in a humidified 37 °C incubator. Cells were washed in PBS and 2% FBS (FACS buffer) and stained at 4 °C for 25 min in staining buffer containing anti-B220-BV605 (#563708, BD Horizon), anti-CD3e-PerCP (#561089, BD Horizon), anti-CD44-BV480 (#566200, BD Horizon), anti-CD62L-PECy7 (#560516, BD Horizon), anti-CD4-FITC (#100406, Biolegend), and LD-near infrared (#L34976A, Invitrogen). Cells were washed twice in FACS buffer and fixed for 15 min at RT in BD Cytofix (#554714, BD Bioscience). Cells were washed twice and incubated for 15 min in 1X Cytoperm buffer (#554714, BD Bioscience) at RT. Cells were spun down and resuspended in Cytoperm containing anti-IFN-γ-PE (#554412, BD Pharmingen), anti-IL4-BV421 (#562915, BD Horizon), and anti-IL13-APC (#159405, Biolegend) and stained for 30 min at RT. Cells were washed twice in Cytoperm, resuspended in FACS buffer, and acquired on a FACSymphony S6 (BD Bioscience). For gating strategy, see Supplementary Fig. 6.

Inhibition of allergen-induced degranulation in RBL-2H3
RBL-2H3 cells (#ACC312, DSMZ), originating from rat basophilic leukemia, were transfected using TurboFectin 8.0 (#TF81001, OriGene) with DNA encoding the human high-affinity IgE receptor α-unit (hFcεRIα) (RC203321, OriGene), resulting in their humanization (hRBL-2H3)57. These cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (#36253, Stemcell) supplemented with 5% (v/v) FBS (# F4135, Sigma-Aldrich), 300 µg/ml L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. Cultures were grown in 25 cm² flasks (#156367, Thermo Fisher) at 37 °C within a humidified environment containing 5% (v/v) CO2. Cells were subcultured twice weekly using 0.25% (w/v) trypsin with 10 mM EDTA (#25200056, Fisher Scientific). To evaluate allergen-induced degranulation, the release of N-acetyl-β-D-hexosaminidase was measured. Cells were first IgE-coated overnight in complete medium containing 5% (v/v) serum from allergic patients. The next day, the medium was discarded, and the cells were stimulated with 50 nM of allergen diluted in Tyrode’s solution (#J67607-AP, Alpha Aesar). The enzymatic activity was assessed using a colorimetric assay, as described by Vogel et al53. 1 h after stimulation, 30 µL of cell supernatant was transferred to a 96-well plate and combined with 50 µL of a substrate solution containing 40 mM citric acid, 0.35% (w/v) p-nitrophenyl-N-acetyl-β-D-glucosaminide, pH 4.5. The mixture was incubated for 1 h at 37 °C. The reaction was terminated by adding 100 µL of 0.4 M glycine (pH 10.7) to each well, and absorbance was measured at 405 nm using a VICTOR Nivo plate reader (PerkinElmer). For total β-hexosaminidase activity, cells in unstimulated control wells were lysed with compound C48/80 (C2313, Sigma-Aldrich) at 100 µg/mL58. A 30 µL aliquot of the lysate was used for the enzymatic reaction. For the inhibition experiment, mouse serum in increasing serial dilutions was preincubated for 1 h at 37 °C with the allergen prior to addition to the cells preincubated with 5% (v/v) serum from allergic patients.

Data and statistical analysis
We analyzed data and performed statistics in GraphPad Prism (Graphpad, v10.4.1). Flow cytometry analysis was performed using FlowJo (BD, v10.10.0). When comparing multiple groups to control, we conducted two-way ANOVA analyses with Dunnett’s multiple comparison tests comparing the groups to the PBS controls. When comparing two groups, we used unpaired two-tailed Student’s T-tests. For the anaphylactic model we conducted one-way ANOVA analyses assuming normal (Gaussian) distribution. We considered P ≤ 0.05 to be statistically significant and indicated P values with * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, and **** for P ≤ 0.0001. For the murine serum-induced blocking of human IgEs, we fitted non-linear regressions using a three-parameter inhibition versus response equation (Y=Bottom+(Top−Bottom)/(X/IC50)) using least squares regression.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General\nIn the manuscript by Møiniche et al., the authors present an allergy immunotherapy for treatment of pollen-food allergy\nsyndrome. The authors approach’ involved designing a consensus allergen that represents a broad spectrum of non-specific\nlipid transfer protein (cnsLTP-1) allergens. Naïve mice were subsequently immunized with cnsLTP-1 using a traditional\nprotein formulation (with poly(I:C) as adjuvant) or using lipid nanoparticle technology containing mRNA (LNP-mRNA)\nencoding the consensus allergen. Immunization with either formulation induced cnsLTP-1-specific IgG production, with\nLNP-mRNA avoiding allergen-specific IgE production. The induced antibodies were shown to bind various nsLTPs, and\nblocked binding of allergens by serum IgEs derived from allergic patients using an ELISA setup. In my opinion, this is a well-\nwritten article that would be of interest broad for a broad readership.\nSpecific\n- Page 4 line 102/103: Upon mentioning the use of mRNA-based vaccination, it would be appropriate to refer to previous\nreports using LNP-mRNA technology for allergy applications (e.g. Xu et al., Use of a Liver-Targeting Immune-Tolerogenic\nmRNA Lipid Nanoparticle Platform to Treat Peanut-Induced Anaphylaxis by Single- and Multiple-Epitope Nucleotide\nSequence Delivery. ACS Nano. 2023 Mar 14;17(5):4942-4957, Jitthamstaporn et al, Nucleoside-modified mRNA vaccines\nyield robust blocking antibody responses against major house dust mite allergens. Allergy. 2023 Jan;78(1):315-318,\nThanasarnthungcharoen et al., Immunotherapeutic effects of specific and nonspecific mRNA-lipid nanoparticles in a mouse\nmodel of HDM-induced airway inflammation. Allergy. 2024 Feb 8. doi: 10.1111/all.16049, and Yin et al., Allergen-specific\nimmunotherapy with Der p 1/2 mRNA vaccines alleviates house dust mite-induced allergic airway inflammation in a mouse\nmodel. Research Square. 2024 https://dx.doi.org/10.21203/rs.3.rs-3865185/v1).\n- Page 6 section 2.5.2: When referring to Comirnaty (BNT162b2), it would be appropriate to also refer to the primary literature\ndescribing its development (e.g. Polack et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med.\n- Page 7 line 222-224: Did the authors also determine the LNP-mRNA physicochemical parameters after thawing, given the\nfreeze-thaw process can affect these? It would be valuable to add a Table to the Supplementary Information summarizing\nthe LNP-mRNA physicochemical parameters before and after freeze-thawing.\n- Page 7 line 232: How was the dosing regimen for the LNP-mRNA (3 x 0.6 ug, 3-week intervals) established and why was\nthe subcutaneous administration route chosen (rather than for example i.m.)? Please include references or additional\ninformation in the main text.\n- Figure 2: Perhaps the Western Blots raw data can be added to the Supplementary Information. Figure 1 and 2 can be\nmerged.\n- Figure 3: Given the varying antibody titers induced by immunization and PBS (Figure 3B), it would have strengthened the\nmanuscript if the authors had taken along a non-coding poly-cytosine mRNA as control, also considering the\nimmunostimulatory effects of mRNA and the ionizable cationic material in the LNP formulation. Have the authors taken this\ninto account?\n- Figure 4: Figure 4 can be merged with Figure 3.\n- Figure 5: Figure 5 and Figure 6 can be merged (and potentially also include IC50 values summarized in Table S2).\n- Table S2: Please review the Table’s number of significant digits.\n- Page 13 line 433-437: It would have strengthened the manuscript if data was included to demonstrate the Th1 response,\ne.g. by using splenocyte restimulations as demonstrated by Jitthamstaporn et al.,Allergy, 2023.\n- Page 14 Limitations section: As the authors indicate themselves, it would be valuable to demonstrate the effects of the\nLNP-mRNA immunotherapy in a therapeutic setting using mouse models with prior allergen sensitization.", "reviewer_2": "The manuscript by Moiniche et al. describes preliminary evauation of the potential of a designed consensus sequence\nmRNA for nsLTPs for application in AIT. Two aspects are potentially novel: mRNA in nanoparticles and the consensus\nsequence approach. Having said that I have some concerns and doubts.\n1) The authors designate LTP allergy as a PFAS. In some instances this may be true, but the consensus is that LTP allergy\nis mainly driven by peach LPT (Pru p 3) as primary sensitizer. Primary sensitization by pollen LTPs is more exception than\nrule. So, LTP allergy is not dominantly a PFAS. Since it is very cross-reactive between foods, the approach would still\npotentially be relevant.\n2) As is illustrated in table S1, the ten patients are having their strongest IgE responses against Pru p 3 and Mal d 3,\nconfirming my point above: Pru p 3 (and possibly Mal d 3) are primary sensitizers, the rest is cross-reactivity. When this is\ntrue, the whole consensus approach is unnecessary: just take mRNA for Pru p 3. That will do the trick. A comparison\nbetween mRNA for Pru p 3 and for cnsLTP should have been included if added value is claimed (which the authors do).\n3) The approach for designing the cnsLTP sequence is not well described. This should be spelled out in more detail.\n4) The human serum samples should also have been tested for IgE against pollen LTPs (Art v / Par j / Ole e).\n5)How do the authors explain that the Mal d 3-specific antibody reacts to reduced but not to unreduced LTP? There is good\nevidence that reduction and alkylation makes LTPs unreactive with IgE.\n6) How was IgE measured in mouse serum? It is well established that IgG in mice immunized with allergens blocks\nrecognition of IgE, so it is better to measure IgE in a capturing ELISA format coating plates with anti-IgE, and using a\nbiotimylated allergen.\n7) The diagnostic potential claimed in line 414-417 is not really appropriate. There are no data to support such a claim. A\nreal comparison should then be made between individual LTPs from different foods and the cnsLTP.\n8) Fig 3E in line 428 shows IgG2a not IgE.\n9) Line 437: do we really want proinflammatory antibody responses in AIT. Yes if accompanied by Treg but perhaps not if no\nTregs?\n10) Also for the other potenial applications, such as for PR-10 and profilins, just using mRNA for the primary sensitizer would\nprobably be sufficient. The 2S albumins are also mentioned but they are not very cross-reactive, so that will definitely not\nwork.\n11) The background on which the authors claim that AIT for PFAS with normal extracts does not work is based on birch\npollen AIT, not on LTP allergies. It has never really been tested whether Pru p 3 is effective for cross-reactive foods as well.\n12) The claim that mRNA approaches would require less immunizations (than the 3-5 years for conventional AIT) is not\nsubstatiated by any proof.", "reviewer_3": "Comments on “mRNA-delivered consensus allergens induce a neutralizing IgG response against food and pollen allergens”\nby Rivera-de-Torre et al.\nGeneral comments:\nThis manuscript, by Rivera-de-Torre et al., is an experimental mouse study aiming to test a new allergen immunotherapy\n(AIT) approach with a consensus allergen and furthermore to test mRNA vaccine technology or traditional method with\nprotein formula injection for immunization in naïve mice. They found good recognition of the consensus allergen and\ninduction of IgG and in the mRNA vaccine treated mice also avoidance of specific IgE production.\nThe novel technique used is interesting, although we hesitate to call it AIT since the experimental mice were naïve, but there\nare some question marks about some of the methods used and about conclusions drawn by the authors.\nIntroduction:\nGeneral comment:\nAfter reading the introduction section, it is still unclear to us what is known and what is not known in this research field. Is the\nnew AIT approach completely unique or are there other publications in the field? Can you be more precise about the\nknowledge gap?\nSpecific comments:\nLines 67-76: About description of PFAS. First of all, authors must disentangle between LTP-related PFAS which can lead to\nsystemic reactions and PR-10-related PFAS known only to cause mild oral symptoms, except from rare case reports of\nsystemic reaction following intake of large amount of soy protein Gly m 4. Treatment with corticosteroids or adrenaline for\nPFAS is generally not used, especially not for PR-10 related reactions.\nA better description of PFAS is needed since it constitutes both LTPS and PR-10 proteins but are highly different in their\nclinical presentation and structure and might not be equally suitable for this kind of treatment. SLIT for the treatment of nsLTP\nallergy with Pru p 3 as the allergen have shown positive results to both peach and cross-reactive allergens (1). There is a\nneed to focus more on LTPS (lipid transfer protein syndrome) rather than the much wider definition PFAS.\nLines 97-101: The claim that 90% of discontinuation of AIT needs to be explained. Is it really true for all forms of AIT\nincluding SCIT? We doubt that. And that patient with PFAS do not consider AIT can be because PFAS is not an indication\nfor AIT and the suffering from PFAS for most patients, at least PR-10 related OAS, is limited.\nLines 102-110: However, the idea of a new AIT approach using mRNA vaccine technique and engineered proteins is\ninteresting.\nMethod:\nGeneral comments:\nFirst of all, we are primarily clinical researchers and not experts on laboratory methods. We leave to other reviewers more\nskilled in this area to evaluate the laboratory methods used in this study. However, some thoughts about the methods:\nTo further study the effect of protein immunization and mRNA vaccine the study needs to add a recall assay to study the T\ncell response of the different methods of immunization. And the study also lacks the analysis of autoantibodies which needs\nto be analysed in vaccine studies as it could be a side effect that is crucial to avoid. Further, the sequence of the pre-ordered\nmRNA used in the experiments needs to be presented in the method-section.\nSpecific comment:\nLines 225-228: Why use the adjuvant poly(I:C) in these experiments? The adjuvant poly(I:C) to the protein immunization\nactivates primarily TLRs leading to interferon responses and previous studies have shown that it increases IgE and IgG1 (2),\nwhich makes it maybe less suitable for comparison with LNPs. A suggestion would instead be a comparison with an\nadjuvant for protein immunization - a mineral oil/water emulsion (as incomplete Freunds adjuvant) like in Montanide ISA-51\nor similar.\nLines 229-238: How many mice were included in the immunization experiment?\nResults:\nSpecific comments:\nLines 282-290 and 292-302: The differences between the two paragraphs is not clear. The most interesting comparison from\na clinical point of view is with peach Pru p 3, the LTP most widely used as an LTP proxy in clinical diagnostics. It would be\ninteresting to understand why the consensus allergen is more useful. The 79% similarity with Mal d 3 is not convincing.\nClinical apple allergy is normally PR-10 related and mild. Mal d 3-related Apple allergy is to our knowledge very uncommon.\nLines 325-330: How many mice were included in the immunization experiment?\nFigure 3 B and C have likely been mixed up. The IgE response to protein immunization could be explained by the adjuvant\nand not the superiority of the mRNA vaccine. This is also mentioned briefly in the discussion but needs to be further\naddressed and further studies on the T cell response should have been/needs to be done to further study this.\nFigure 4. The effect on the adjuvant used in the protein immunization could also affect the IgG response with increased IgG1\nand less IgG2a, see above. Needs to be further addressed.\nFigure 5/6. Through inhibition of human serum IgE-binding to nsLTP it is suggested that mRNA might be better than protein\nimmunization in blocking IgE-binding (due to a trend in the figures). However, the variability is high and the number of\nindividuals is low, making the statistical power too low to make any conclusions.\nTable S2. What were the systemic reactions the patients exhibited? Are there any other allergies/sensitizations than to the\npresented LTP?\nDiscussion:\nGeneral comments:\nThe authors needs to acknowledge the difference in LTP and PR-10 proteins before proposing that this kind of treatment\ncould be suitable for birch-apple allergy. The authors should focus on LTPS throughout the manuscript.\nTo draw the conclusion of alleviation of symptoms using a handful of mRNA-LNP doses, doesn’t long-term studies need to\nbe conducted first?\nSpecific comment:\nLines 415-417: See first comment under results. Why is the consensus LTP more useful than Pru p 3?\nConclusion:\nGeneral comments:\nMotivate why the next step is clinical trials as proposed. The next step could also be further studies with the use of more\nnaïve mice, then immunized for example using an adjuvant triggering an allergic response like alum, analyse the T-cell\nresponse to these different methods and check for the presence of autoantibodies after treatment.\nAlso, the authors conclude that their mRNA-LNP formulation is superior over protein immunization; this statement cannot be\nproven with the experiments done. The variability is too big, given the small number of mice, and the adjuvant used might\nitself explain the better results of the mRNA-LNP formulation in these experiments.\n1. Gomez F, Bogas G, Gonzalez M, et al. The clinical and immunological effects of Pru p 3 sublingual immunotherapy on\npeach and peanut allergy in patients with systemic reactions. Clin Exp Allergy. 2017; 47: 339-350\n2. Wan H, Yang H, Wei M, Chen W. Polyinosinic:polycytidylic acid aggravates calcipotriol-induced atopic dermatitis-like skin\nlesions in mice by increasing the expression of thymic stromal lymphopoietin. Ann Transl Med. 2022 Feb;10(4):209. doi:\n10.21037/atm-22-282. PMID: 35280398; PMCID: PMC8908153.", "reviewer_4": "The overall goal is to develop a novel form of alleren-specific immunotherapy for the cross-reactive LTP food and pollen\nallergens. Accordingly, the authors present an interesting approach by developing a consensus LTP allergen sequence,\nthen formulating it into mRNA-LNP for subcutaneous administration. The work clearly shows induction of IgG1 and IgG2a\nantibodies against LTPs in naive mice, which is a starting point in the development of a novel allergy immunotherapy\napproach. However, I have some concerns with the manuscript in its current form:\nMajor concerns:\n1. The work is designed towards developing a novel molecule, cnsLTP-1, for allergy immunotherapy. However, the authors\ndo not assess the molecule in allergic mice. Rather, all in vivo studies were conducted in naive animals. The authors should\nstrongly consider sensitizing mice to LTPs, then using the cnsLTP-1 mRNA or protein formulations as immunotherapy. This\nwill provide much needed efficacy data in an allergic mouse model. The data currently shown are not convincing that the\napproach provides any therapeutic benefit to allergic animals.\n2. Similarly, the authors must assess safety of the cnsLTP-1. Of course it is \"safe\" in naive animals, but what happens when\na mouse is allergic and primed to have reactions due to existing LTP-specific IgE? The authors should sensitize mice to\nLTPs and then inject the mRNA and protein formulations to look for symptoms of allergy. This safety data is mandatory\nbefore considering human clinical trials.\n3. The IgE inhibition studies are nice, but this doesn't exactly predict the efficacy of IgG in a therapeutic context. The authors\nshould use cell based assays (human basophil activation tests, or RBL cell lines with human IgE) to better define whether\nthe cns-LTP IgG can interfere with IgE function.\n4. I would strongly recommend including T cell readouts. Does the cnsLTP alter T cell cytokine responses in an allergic (or\neven naive) mouse.\n5. The sample sizes for the mouse studies are very low (n=3 per treatment group). More mice should be included for\nreproducibility and rigor. Similarly, for Figures 3 and 4, there appears to be trends in differences between mRNA and\nProtein-treated mice, however, the sample sizes are too small to detect these differences.\nMinor Concerns:\nLine 83 - Xolair (Omalizumab) is now also approved for treating food allergies.\nLine 367 - Figure 4C is referenced but did not appear in the submitted version of the manuscript.\nFigure 1A - why is the cnsLTP-1 a mixture of proteins? Shouldn't it be a single band like the other LTPs shown in the SDS-\nPAGE gels?", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Response: We thank the reviewer for their time and for the constructive comments to our manuscript,\nwhich we have done our best to implement.\nSpecific\n- Page 4 line 102/103: Upon mentioning the use of mRNA-based vaccination, it would be appropriate to\nrefer to previous reports using LNP-mRNA technology for allergy applications (e.g. Xu et al., Use of a\nLiver-Targeting Immune-Tolerogenic mRNA Lipid Nanoparticle Platform to Treat Peanut-Induced\nAnaphylaxis by Single- and Multiple-Epitope Nucleotide Sequence Delivery. ACS Nano. 2023 Mar\n14;17(5):4942-4957, Jitthamstaporn et al, Nucleoside-modified mRNA vaccines yield robust blocking\nantibody responses against major house dust mite allergens. Allergy. 2023 Jan;78(1):315-318,\nThanasarnthungcharoen et al., Immunotherapeutic effects of specific and nonspecific mRNA-lipid\nnanoparticles in a mouse model of HDM-induced airway inflammation. Allergy. 2024 Feb 8. doi:\n10.1111/all.16049, and Yin et al., Allergen-specific immunotherapy with Der p 1/2 mRNA vaccines\nalleviates house dust mite-induced allergic airway inflammation in a mouse model. Research Square.\n2024 https://dx.doi.org/10.21203/rs.3.rs-3865185/v1).\nResponse: We have updated the text and included the suggested references (lines 91-92)\n- Page 6 section 2.5.2: When referring to Comirnaty (BNT162b2), it would be appropriate to also refer to\nthe primary literature describing its development (e.g. Polack et al. Safety and Efficacy of the BNT162b2\nmRNA Covid-19 Vaccine. N Engl J Med. 2020 Dec 31;383(27):2603-2615) in addition to the review (ref.\n30) currently cited.\nResponse: Implemented\n- Page 7 line 222-224: Did the authors also determine the LNP-mRNA physicochemical parameters after\nthawing, given the freeze-thaw process can affect these? It would be valuable to add a Table to the\nSupplementary Information summarizing the LNP-mRNA physicochemical parameters before and after\nfreeze-thawing.\nResponse: We have now performed a characterization of the physicochemical properties of the LNPs\nafter thaw, which confirmed the stability of the particles. We have included a table in the\nsupplementary material (Supplementary table S1) with the specific parameters.\n- Page 7 line 232: How was the dosing regimen for the LNP-mRNA (3 x 0.6 ug, 3-week intervals)\nestablished and why was the subcutaneous administration route chosen (rather than for example i.m.)?\nPlease include references or additional information in the main text.\nResponse: Dosing was based on previous literature\n(https://pmc.ncbi.nlm.nih.gov/articles/PMC9538380/) and a preliminary titration experiment, which is\nnow emphasized in the manuscript. Subcutaneous injections may lead to slower absorption of the\nvaccine, potentially resulting in a more prolonged exposure of the immune system to the antigen.\nStill, we have considered the reviewer comment and have repeated the mouse experiments using the\nsame immunisation agenda and intramuscular administration as a supplement to subcutaneous\ninjections. Materials and Methods section 2.5.4 Immunization schedule has been modified to include\nthe experiment details (lines 2020-221)\n- Figure 2: Perhaps the Western Blots raw data can be added to the Supplementary Information. Figure 1\nand 2 can be merged.\nResponse: Figure 1 has been updated and figure 2 has been moved to supplementary information (now\nSupplementary figure 3).\n- Figure 3: Given the varying antibody titers induced by immunization and PBS (Figure 3B), it would have\nstrengthened the manuscript if the authors had taken along a non-coding poly-cytosine mRNA as\ncontrol, also considering the immunostimulatory effects of mRNA and the ionizable cationic material in\nthe LNP formulation. Have the authors taken this into account?\nResponse: After including extra mice for the subcutaneous immunisation and doing the experiment with\nintramuscular immunisation, we have not found significant changes in antibody titers between the mice\ninjected with PBS and the mice immunised with either mRNA or protein formulations (Figure 1 and\nFigure 4)\nWe decided not to include a control with non-coding poly-cytosine mRNA as the literature already\ndescribes that the LNP composition used in this work does not affect the total antibody levels\n(https://www.nature.com/articles/s12276-023-01086-x). We have included the citation in line 187.\n- Figure 4: Figure 4 can be merged with Figure 3.\nResponse: Figure 3 and 4 have been merged in the new figure 2.\n- Figure 5: Figure 5 and Figure 6 can be merged (and potentially also include IC50 values summarized in\nTable S2).\nResponse: Figure 5 and 6 has been merged (Figure 3) and we have included a figure displaying the\nspecific IC values from Supplementary table 3.\n50\n- Table S2: Please review the Table’s number of significant digits.\nResponse: The IC values have been revisited and are updated in the Supplementary Table 3.\n50\n- Page 13 line 433-437: It would have strengthened the manuscript if data was included to demonstrate\nthe Th1 response, e.g. by using splenocyte restimulations as demonstrated by Jitthamstaporn et\nal.,Allergy, 2023.\nResponse: Following the reviewer’s advice, we performed a splenocyte restimulation following the\nanimal experiment, in which the mice were immunised intramuscularly. We did not observe major\ndifferences between Th1 and Th2 populations. This is now included in the manuscript Supplementary\nFigure 5.\n- Page 14 Limitations section: As the authors indicate themselves, it would be valuable to demonstrate\nthe effects of the LNP-mRNA immunotherapy in a therapeutic setting using mouse models with prior\nallergen sensitization.\nResponse: We acknowledge the necessity to test the concept presented in this manuscript in an\nappropriate allergy model. However, securing ethical approvals and completing the sensitization\nprotocols was not feasible within the timeframe of this revision.We have included blocking assays in\nhumanised RBL-2H3 cells, which resemble the degranulation caused in basophiles upon exposure to the\nallergens. The results summarized in Figure 3 demonstrate that the antibodies induced in the naïve\nanimals not only block binding, but also that such binding is functionally relevant to avoid the activation\nof cells involved in the allergic response.\nWe would like to emphasize the novel advance of this work is the use of the consensus design and the\ninduction of broadly-neutralising antibodies that can block binding and avoid the degranulation of\neffector cells. There are other publications that have already proven that animals immunised with\nallergens using an mRNA-LNP formulation protects them against anaphylaxis\n(https://pmc.ncbi.nlm.nih.gov/articles/PMC10019335/), and while we would have liked to include such\nadditional in vivo studies, these are beyond what we are able to do currently.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Moiniche et al. describes preliminary evauation of the potential of a designed\nconsensus sequence mRNA for nsLTPs for application in AIT. Two aspects are potentially novel: mRNA in\nnanoparticles and the consensus sequence approach. Having said that I have some concerns and doubts.\nResponse: We thank the reviewer for their time taken to review our manuscript. We have attempted to\nimplement all the reviewer’s comments to the best of our ability.\n1) The authors designate LTP allergy as a PFAS. In some instances this may be true, but the consensus is\nthat LTP allergy is mainly driven by peach LPT (Pru p 3) as primary sensitizer. Primary sensitization by\npollen LTPs is more exception than rule. So, LTP allergy is not dominantly a PFAS. Since it is very cross-\nreactive between foods, the approach would still potentially be relevant.\nResponse: We have modified the manuscript to refer to LTP as a “cross-reactive allergy” instead of\nPFAS, and we acknowledge that LTP is mainly a food allergy. Examples can be found in lines 53, 89, 99.\n2) As is illustrated in table S1, the ten patients are having their strongest IgE responses against Pru p 3\nand Mal d 3, confirming my point above: Pru p 3 (and possibly Mal d 3) are primary sensitizers, the rest\nis cross-reactivity. When this is true, the whole consensus approach is unnecessary: just take mRNA for\nPru p 3. That will do the trick. A comparison between mRNA for Pru p 3 and for cnsLTP should have been\nincluded if added value is claimed (which the authors do).\nResponse: We thank the reviewer for the suggestion. We have performed a new animal experiment, in\nwhich we have immunised mice with mRNA encoding Pru p 3 and Par j 2, and we have compared the\nantibody cross-reactivity with the one obtained when immunising mice with cnsLTP1.\nWe have observed that the animals treated with the cnsLTP1 can bind to all the allergens included in the\npanel (including both food and pollen allergens). However, the animals immunised with either Pru p 3 or\nPar j 2 have a limited cross-reactivity profile restricted to very similar allergens (Figure 2). Therefore, we\ndo believe that the claimed added value is warranted.\n3) The approach for designing the cnsLTP sequence is not well described. This should be spelled out in\nmore detail.\nResponse: Details have been included in the Materials and method section “Design of consensus\nallergen” and we have included the full sequence in the Supplementarty materials.\n4) The human serum samples should also have been tested for IgE against pollen LTPs (Art v / Par j / Ole\ne).\nResponse: We have included Par j 2, Pla a 3, and Ole e 7 in the allergen panel. We tried producing Art v\n3, but the protein was not correctly folded according to its circular dichroism spectra, and we decided to\ninclude Pla a 3 instead as it is one of the most medically-relevant allergens in the Mediterranean area.\n5)How do the authors explain that the Mal d 3-specific antibody reacts to reduced but not to unreduced\nLTP? There is good evidence that reduction and alkylation makes LTPs unreactive with IgE.\nResponse: We hypothesize that Mal d 3-specific antibody is raised against an unfolded protein and\ntherefore it can only recognize linear epitopes that are hidden when the protein is not completely\nreduced. We have added this statement in lines 343-344\n6) How was IgE measured in mouse serum? It is well established that IgG in mice immunized with\nallergens blocks recognition of IgE, so it is better to measure IgE in a capturing ELISA format coating\nplates with anti-IgE, and using a biotimylated allergen.\nResponse: We thank the reviewer for their suggestions. Following these suggestions, we biotinylated\nthe allergens and detected using Strp-Eu in a DELFIA assay. We tested a different adjuvant (complete\nFreund’s adjuvant instead of poly I:C) and confirmed that the IgE induction in mice immunised with\nprotein formulation was an adjuvant dependent effect and not due to the protein formulation\n(Supplementary Figure 4).\n7) The diagnostic potential claimed in line 414-417 is not really appropriate. There are no data to\nsupport such a claim. A real comparison should then be made between individual LTPs from different\nfoods and the cnsLTP.\nResponse: We have eliminated the claim.\n8) Fig 3E in line 428 shows IgG2a not IgE.\nResponse: Figures have been merged and the annotation in the text has been modified\n9) Line 437: do we really want proinflammatory antibody responses in AIT. Yes if accompanied by Treg\nbut perhaps not if no Tregs?\nResponse: We thank the reviewer for this comment. We have edited the text to accommodate.\n10) Also for the other potenial applications, such as for PR-10 and profilins, just using mRNA for the\nprimary sensitizer would probably be sufficient. The 2S albumins are also mentioned but they are not\nvery cross-reactive, so that will definitely not work.\nResponse: We have included a new animal experiment, in which using the natural allergens in mRNA\nformat induces a restricted cross-reactive response in comparison with the consensus design,\nhighlighting the potential of the consensus design approach in other cross-reactive syndromes. Even\nthough the extent of albumin 2s is small in comparison with PR-10 and profilins, it has been described to\nbe an important allergen in the cross-reactivity between peanuts, tree nuts and sesame seeds in the so-\ncalled pistachio-cashew syndrome or hazelnut-walnut syndrome\n(https://pmc.ncbi.nlm.nih.gov/articles/PMC8035160/).\n11) The background on which the authors claim that AIT for PFAS with normal extracts does not work is\nbased on birch pollen AIT, not on LTP allergies. It has never really been tested whether Pru p 3 is\neffective for cross-reactive foods as well.\nResponse: There are reports in the literature confirming that immunotherapy with a single allergen in\ncross-reactive patients might not induce protection against all the allergens the patients react to\n(https://ctajournal.biomedcentral.com/articles/10.1186/s13601-020-00325-y,\nhttps://onlinelibrary.wiley.com/doi/10.1111/cea.12901). We have included the reference on lines 83-\n84.\n12) The claim that mRNA approaches would require less immunizations (than the 3-5 years for\nconventional AIT) is not substatiated by any proof.\nResponse: While direct data on the use of mRNA technology in allergen immunotherapy are limited, the\nrapid and durable immunogenicity observed in mRNA vaccine trials suggests that fewer doses may be\nsufficient to achieve a protective immune profile. For instance, Wu et al. (2021)\n(https://pubmed.ncbi.nlm.nih.gov/33730471/) reported robust antibody titers and functional immune\nresponses following the administration of two doses of mRNA COVID-19 vaccines. This stands in stark\ncontrast to the prolonged treatment timelines of conventional AIT, which span years. Still, as the\nreviewer points out, this is an speculation not tested in out case and therefore we have modified our\nwork in in the Discussion section (line 502-504).\nReviewer #3 (Remarks to the Author):\nComments on “mRNA-delivered consensus allergens induce a neutralizing IgG response against food and\npollen allergens” by Rivera-de-Torre et al.\nResponse: We thank the reviewer for their time, for the attention to detail, and for the constructive\ncomments to our manuscript, which we have done our best to implement.\nGeneral comments:\nThis manuscript, by Rivera-de-Torre et al., is an experimental mouse study aiming to test a new allergen\nimmunotherapy (AIT) approach with a consensus allergen and furthermore to test mRNA vaccine\ntechnology or traditional method with protein formula injection for immunization in naïve mice. They\nfound good recognition of the consensus allergen and induction of IgG and in the mRNA vaccine treated\nmice also avoidance of specific IgE production.\nThe novel technique used is interesting, although we hesitate to call it AIT since the experimental mice\nwere naïve, but there are some question marks about some of the methods used and about conclusions\ndrawn by the authors.\nResponse: We acknowledge the necessity in regards to the use of the term ”AIT”. We have corrected\nthe wording to “therapeutic approach” in the introduction and the discussion sections. (Lines 88 and\n524)\nIntroduction:\nGeneral comment:\nAfter reading the introduction section, it is still unclear to us what is known and what is not known in\nthis research field. Is the new AIT approach completely unique or are there other publications in the\nfield? Can you be more precise about the knowledge gap?\nResponse: The use of mRNA-LNP-based therapies in allergy research has been successfully explored in\nprevious studies, which we have now clarified in the updated introduction (lines 91-92). However, to the\nbest of our knowledge, these efforts have been limited to delivering mRNA encoding natural allergens,\nsuch as Der p 1, Ara h 2, and Phl p 5. The novel aspect of our work lies in the use of consensus allergens\ndesigned to address cross-reactivity. In our opinion, this approach represents an innovative step\nforward, aiming to overcome the limitations of natural allergens by targeting a broader spectrum of\nrelated allergens, which has not been previously reported.\nSpecific comments:\nLines 67-76: About description of PFAS. First of all, authors must disentangle between LTP-related PFAS\nwhich can lead to systemic reactions and PR-10-related PFAS known only to cause mild oral symptoms,\nexcept from rare case reports of systemic reaction following intake of large amount of soy protein Gly m\n4. Treatment with corticosteroids or adrenaline for PFAS is generally not used, especially not for PR-10\nrelated reactions.\nResponse: We appreciate the comment and have modified the manuscript to refer to LTP as a cross-\nreactive allergy instead of PFAS, and we have acknowledged that LTP is mainly a food allergy. Examples\ncan be found in lines 53, 89, 99.\nA better description of PFAS is needed since it constitutes both LTPS and PR-10 proteins but are highly\ndifferent in their clinical presentation and structure and might not be equally suitable for this kind of\ntreatment. SLIT for the treatment of nsLTP allergy with Pru p 3 as the allergen have shown positive\nresults to both peach and cross-reactive allergens (1).\nResponse: We thank the reviewer for their valuable feedback. We agree that PFAS encompasses both\nnsLTP and PR-10 proteins, which differ significantly in their clinical presentation and structure, and these\ndifferences may influence their suitability for this treatment approach. Regarding SLIT with Pru p 3,\nwhile it has shown positive results for peach and cross-reactive allergens, there is evidence in the\nliterature indicating that immunotherapy with a single allergen in cross-reactive patients may not\nprovide protection against all allergens to which they react\n(https://ctajournal.biomedcentral.com/articles/10.1186/s13601-020-00325-y). We have included this\nreference in the introduction (line 104). To further evaluate the potential of the consensus allergen in\ndriving a cross-reactive response against natural allergens, we conducted new experiments immunizing\nmice with Pru p 3 and Par j 2 in mRNA format. Our findings demonstrate that antibodies induced by the\nconsensus allergen can bind to all tested allergens except Ole e 7, whereas Pru p 3 mRNA immunization\nonly induced binding to Pru p 3, Cor a 8, Ara h 9, and Pla a 3, and Par j 2 mRNA immunization primarily\ninduced binding to Par j 2 and Pla a 3 (Figure 2). These results underscore the broader cross-reactivity\nelicited by the consensus design compared to individual allergens, highlighting its potential usefulness\nfor addressing cross-reactive allergies comprehensively.\nThere is a need to focus more on LTPS (lipid transfer protein syndrome) rather than the much wider\ndefinition PFAS.\nResponse: We have focused on LTPs and have restricted our discussion about cross-reactivity and PR-10\nto the discussion section as an interesting perspective to pursue in the future (lines 505-512).\nLines 97-101: The claim that 90% of discontinuation of AIT needs to be explained. Is it really true for all\nforms of AIT including SCIT? We doubt that. And that patient with PFAS do not consider AIT can be\nbecause PFAS is not an indication for AIT and the suffering from PFAS for most patients, at least PR-10\nrelated OAS, is limited.\nResponse: We thank the reviewer for highlighting this point. We appreciate the opportunity to clarify\nand provide additional context. The discontinuation rates cited in the manuscript refer specifically to AIT\nfor respiratory allergens, such as grass pollen, rather than food allergies due to the lack of extensive\nadherence studies for such indications. For example, adherence to subcutaneous immunotherapy (SCIT)\nhas been reported to range from 23% to 89%, while adherence to sublingual immunotherapy (SLIT)\nranges from 64% to over 95% in some studies. However, despite these promising rates, a survey in Italy\nreported a concerning discontinuation rate of up to 90% for SLIT three years after prescription for grass\npollen allergy. We have updated the manuscript (lines 76-78) to specify that these statistics are based\non grass pollen allergy and have revised the description of adherence rates to better reflect the nuanced\nchallenges of AIT adherence. Additional references have also been included to support these points.\nLines 102-110: However, the idea of a new AIT approach using mRNA vaccine technique and engineered\nproteins is interesting.\nResponse: We thank the reviewer for their positive remark.\nMethod:\nGeneral comments:\nFirst of all, we are primarily clinical researchers and not experts on laboratory methods. We leave to\nother reviewers more skilled in this area to evaluate the laboratory methods used in this study.\nHowever, some thoughts about the methods:\nTo further study the effect of protein immunization and mRNA vaccine the study needs to add a recall\nassay to study the T cell response of the different methods of immunization. And the study also lacks the\nanalysis of autoantibodies which needs to be analysed in vaccine studies as it could be a side effect that\nis crucial to avoid. Further, the sequence of the pre-ordered mRNA used in the experiments needs to be\npresented in the method-section.\nResponse: We thank the reviewer for their thoughtful feedback. We appreciate the opportunity to\naddress these important points.\n T cell response: We analyzed the T cell populations (Th1 and Th2) in the spleen and draining\nlymph nodes of mice immunized via both intramuscular and subcutaneous routes\n(Supplementary Figure 5). Statistically significant differences were observed only in the spleens\nof mice immunized intramuscularly: mice immunized with cnsLTP-1 mRNA and Par j 2 mRNA\nexhibited a slightly increased Th2 population in the spleen compared to the PBS group.\nHowever, as these experiments were conducted in naïve mice, and the significance does not\ncorrelate in the lymph nodes, definitive conclusions cannot be drawn in a non-allergenic\ncontext.\n Analysis of autoantibodies: The potential induction of autoantibodies is indeed a critical\nconsideration for mRNA-LNP-based formulations. While this study did not include autoantibody\nanalysis, this aspect will be carefully evaluated in subsequent studies during the later stages of\npreclinical and clinical development. As is correctly noted, mRNA-LNP formulations carry a\ntheoretical risk of inducing autoantibodies, particularly with repeated dosing. However, nsLTPs,\nincluding the consensus allergen cnsLTP-1, do not have known paralogues in mice or humans.\nWe performed protein BLAST searches for all nsLTPs used in this study, including cnsLTP-1, and\ndid not identify any significant similarity with human or murine proteins. Thus, the risk of\nautoantibody induction against the consensus allergen is expected to be very low. We have\nadded this information to the manuscript to address this concern.\n mRNA sequence: we have added the sequence information in the Supplementary information in\nFASTA format to facilitate reuse, transparency, and reproducibility.\nSpecific comment:\nLines 225-228: Why use the adjuvant poly(I:C) in these experiments? The adjuvant poly(I:C) to the\nprotein immunization activates primarily TLRs leading to interferon responses and previous studies have\nshown that it increases IgE and IgG1 (2), which makes it maybe less suitable for comparison with LNPs. A\nsuggestion would instead be a comparison with an adjuvant for protein immunization - a mineral\noil/water emulsion (as incomplete Freunds adjuvant) like in Montanide ISA-51 or similar.\nResponse: We thank the reviewer for their comment and suggestion regarding the use of poly(I:C) as an\nadjuvant in these experiments. We initially selected poly(I:C) to mimic the nucleotide adjuvant effect of\nmRNA, as it also stimulates TLR3 and is commonly used to model similar immune responses. However,\nwe acknowledge that the choice of adjuvant significantly impacts antibody responses, and your\nsuggestion for a mineral oil/water emulsion such as incomplete Freund’s adjuvant is well taken.\nTo address this, we repeated the experiment using incomplete Freund’s adjuvant instead of poly(I:C) in\nboth subcutaneous and intramuscular immunizations. We observed that while allergen-specific IgG1 and\nIgG2a responses were maintained, the use of incomplete Freund’s adjuvant did not induce the\nproduction of IgE (Figure 2 and Supplementary figure 4). These findings provide further insight into the\nrole of adjuvants in shaping the immune response and highlight the potential of Freund’s adjuvant for\navoiding IgE induction. We have updated the results section accordingly and included the comparison in\nFigure S4D and E.\nLines 229-238: How many mice were included in the immunization experiment?\nResponse: Section 2.5.4 has been updated to specify the number of mice used per group (lines 221–\n229). Briefly, for subcutaneous immunization, four- to five-week-old BALB/c female mice were\nimmunized three times at three-week intervals with 9 µg mRNA-LNP (n=6), 24 µg protein-Poly(I:C) (n=3),\n24 µg protein-incomplete Freund’s adjuvant (n=3), or PBS (n=7). For intramuscular immunization, four-\nto five-week-old BALB/c female mice were immunized three times at three-week intervals with 7.5 µg\nmRNA-LNP (n=5), 20 µg protein-incomplete Freund’s adjuvant (n=5), or PBS (n=5).\nResults:\nSpecific comments:\nLines 282-290 and 292-302: The differences between the two paragraphs is not clear. The most\ninteresting comparison from a clinical point of view is with peach Pru p 3, the LTP most widely used as\nan LTP proxy in clinical diagnostics. It would be interesting to understand why the consensus allergen is\nmore useful. The 79% similarity with Mal d 3 is not convincing. Clinical apple allergy is normally PR-10\nrelated and mild. Mal d 3-related Apple allergy is to our knowledge very uncommon.\nResponse: Thank you for the opportunity to clarify this point. We have used the identity shared between\nnatural allergens and our consensus design to specify that we are not accidentally replicating a natural\nnsLTP variant. Mal d 3 sharing a 79% and Par j 2 sharing less than 30% is a way to determine that our\nconsensus allergen is covering the diversity contained in this range. Our study demonstrated variable IgE\nrecognition patterns among patients (Supplementary Table 2), reflecting individual sensitization profiles.\nHowever, Western blot analysis (Supplementaru figure 3) showed consistent recognition of cnsLTP-1 by\npatient IgEs, regardless of variability in individual sensitization patterns. This uniform recognition\nhighlights the potential of cnsLTP-1 as a diagnostic screening tool for nsLTP-related allergies, offering a\nstandardized approach to detect sensitization in patients with diverse allergen recognition profiles.\nThe advantage of cnsLTP-1 over natural allergens like Pru p 3 is further highlighted by the immunization\nexperiments using natural allergens in mRNA format. These experiments demonstrated that cnsLTP-1\nelicited a broader antibody response compared to Pru p 3 or Par j 2, emphasizing its superior coverage\nand utility in addressing cross-reactive allergy management. We have added a clarification in lines 319-\n321 and updated the results with Pru p 3 and Par j 2 immunisation antibody responses (Figure 2).\nLines 325-330: How many mice were included in the immunization experiment?\nResponse: The text has been updated to specify the number of mice used per group (lines 209–221).\nEach point in figures 2 and S4 corresponds to an individual mouse.\nFigure 3 B and C have likely been mixed up. The IgE response to protein immunization could be\nexplained by the adjuvant and not the superiority of the mRNA vaccine. This is also mentioned briefly in\nthe discussion but needs to be further addressed and further studies on the T cell response should have\nbeen/needs to be done to further study this.\nResponse: The figures have been reorganized to improve clarity. The new Figure 2 now includes all data\nrelated to antibody characterization in mice immunized intramuscularly, while Figure S4 contains the\ndata for mice immunized subcutaneously, including the comparison of IgE responses with different\nadjuvants. These results confirm that the observed IgE induction is due to the adjuvant effect rather\nthan differences between the protein and mRNA formulations. Additionally, T cell populations were\nanalyzed (Supplementary Figure 5); however, the results related to population distribution are\ninconclusive in this non-allergenic context. Further studies will be required to comprehensively evaluate\nthe T cell responses under conditions that better mimic allergic sensitization.\nFigure 4. The effect on the adjuvant used in the protein immunization could also affect the IgG response\nwith increased IgG1 and less IgG2a, see above. Needs to be further addressed.\nResponse: While IgE induction was shown to be dependent on the adjuvant used, the levels of allergen-\nspecific IgG1 and IgG2a were comparable between the protein formulations, regardless of the adjuvant\nemployed (Supplementary Figure 4). This suggests that the choice of adjuvant did not significantly\ninfluence the IgG subclass distribution in the protein immunization.\nFigure 5/6. Through inhibition of human serum IgE-binding to nsLTP it is suggested that mRNA might be\nbetter than protein immunization in blocking IgE-binding (due to a trend in the figures). However, the\nvariability is high and the number of individuals is low, making the statistical power too low to make any\nconclusions.\nResponse: We have increased the number of animals used from 3 to 5, which has reduced variability\nand allowed for a more robust analysis of the differences between protein and mRNA-LNP formulations\nin both binding (ELISA) and cell degranulation (hRBL-2H3) assays. While no statistically significant\ndifferences were observed in the IC50 values between the mRNA and protein formulations, the IC50\nvalues for mRNA formulations were generally lower, indicating a potential trend towards greater\nefficacy.\nTable S2. What were the systemic reactions the patients exhibited? Are there any other\nallergies/sensitizations than to the presented LTP?\nResponse: The patients presented with severe oral allergy syndrome, characterized by itchiness and\nredness upon contact with peach. No other relevant co-sensitizations were observed beyond the\npresented LTP allergens. We have added this information in lines 349-350.\nDiscussion:\nGeneral comments:\nThe authors needs to acknowledge the difference in LTP and PR-10 proteins before proposing that this\nkind of treatment could be suitable for birch-apple allergy. The authors should focus on LTPS throughout\nthe manuscript.\nResponse: We agree that the primary focus of our manuscript is on nsLTPs, and we have revised the text\nto clarify this distinction. Specifically, we have removed references to PR-10-related syndromes and\nadjusted the discussion to focus on the potential applications of our consensus allergen design for\naddressing cross-reactive related allergies, in which cross-reactivity is driven by structurally conserved\nfamilies (lines 505-512).\nTo draw the conclusion of alleviation of symptoms using a handful of mRNA-LNP doses, doesn’t long-\nterm studies need to be conducted first?\nResponse: We acknowledge the complexity of foreseeing the long-term effect of novel immunisation\nstrategies and have modified our statement appropriately in the discussion (lines 502-504).\nSpecific comment:\nLines 415-417: See first comment under results. Why is the consensus LTP more useful than Pru p 3?\nResponse: As mentioned previously, we have clarified this point by immunising animals with Pru p 3, Par\nj 2, and cnsLTP-1 in mRNA format and comparing the cross-reactivity of the raised Igs. cnsLTP-1 elicits a\nbroader cross-reactivity than Pru p 3, binding to all tested nsLTPs, whereas Pru p 3 shows limited\nrecognition beyond closely related allergens (Figure 2). This broader coverage highlights its potential for\naddressing cross-reactive allergies comprehensively.\nConclusion:\nGeneral comments:\nMotivate why the next step is clinical trials as proposed. The next step could also be further studies with\nthe use of more naïve mice, then immunized for example using an adjuvant triggering an allergic\nresponse like alum, analyse the T-cell response to these different methods and check for the presence of\nautoantibodies after treatment.\nResponse: This is an excellent suggestion, and we have included it in our discussion as a roadmap for\nnext steps.\nAlso, the authors conclude that their mRNA-LNP formulation is superior over protein immunization; this\nstatement cannot be proven with the experiments done. The variability is too big, given the small\nnumber of mice, and the adjuvant used might itself explain the better results of the mRNA-LNP\nformulation in these experiments.\nResponse: in light of our complete experiments with a bigger sample, we have made better assessment\nof the differences between mRNA and protein, and so far, in the assayed context, we cannot conclude a\nclear advantage of any of the formulation tested for cnsLTP-1. The conclusions text has been modified\naccordingly.\n1. Gomez F, Bogas G, Gonzalez M, et al. The clinical and immunological effects of Pru p 3 sublingual\nimmunotherapy on peach and peanut allergy in patients with systemic reactions. Clin Exp Allergy. 2017;\n47: 339-350\n2. Wan H, Yang H, Wei M, Chen W. Polyinosinic:polycytidylic acid aggravates calcipotriol-induced atopic\ndermatitis-like skin lesions in mice by increasing the expression of thymic stromal lymphopoietin. Ann\nTransl Med. 2022 Feb;10(4):209. doi: 10.21037/atm-22-282. PMID: 35280398; PMCID: PMC8908153.\nReviewer #4 (Remarks to the Author):\nThe overall goal is to develop a novel form of alleren-specific immunotherapy for the cross-reactive LTP\nfood and pollen allergens. Accordingly, the authors present an interesting approach by developing a\nconsensus LTP allergen sequence, then formulating it into mRNA-LNP for subcutaneous administration.\nThe work clearly shows induction of IgG1 and IgG2a antibodies against LTPs in naive mice, which is a\nstarting point in the development of a novel allergy immunotherapy approach. However, I have some\nconcerns with the manuscript in its current form:\nResponse: We thank the reviewer for their time and for the valuable feedback on our manuscript. We\nhave done our best to implement the reviewer’s suggestions.\nMajor concerns:\n1. The work is designed towards developing a novel molecule, cnsLTP-1, for allergy immunotherapy.\nHowever, the authors do not assess the molecule in allergic mice. Rather, all in vivo studies were\nconducted in naive animals. The authors should strongly consider sensitizing mice to LTPs, then using\nthe cnsLTP-1 mRNA or protein formulations as immunotherapy. This will provide much needed efficacy\ndata in an allergic mouse model. The data currently shown are not convincing that the approach\nprovides any therapeutic benefit to allergic animals.\nResponse: We acknowledge the importance of testing cnsLTP-1 in an allergic mouse model to evaluate\nits therapeutic efficacy. However, obtaining the necessary ethical approvals and completing the\nsensitization protocols was not feasible within the timeframe of this revision. To address this, we have\nemphasized throughout the manuscript that the current study focuses on the prophylactic potential of\ncnsLTP-1 rather than therapeutic applications and we have included a functional assay with hRBL-2H3\ncells reinforces the neutralisation capacity of the induced antibodies upon immunisation. Additionally,\nother studies in the literature have demonstrated the efficacy of mRNA-LNP formulations in allergic\nmodels, including their ability to protect against anaphylaxis (e.g., Xu et al., 2023; Jitthamstaporn et al.,\n2023). Future studies will prioritize testing cnsLTP-1 in sensitized models to evaluate its therapeutic\nbenefits, which has been emphasized in lines 517-519.\n2. Similarly, the authors must assess safety of the cnsLTP-1. Of course it is \"safe\" in naive animals, but\nwhat happens when a mouse is allergic and primed to have reactions due to existing LTP-specific IgE?\nThe authors should sensitize mice to LTPs and then inject the mRNA and protein formulations to look for\nsymptoms of allergy. This safety data is mandatory before considering human clinical trials.\nResponse: We agree that assessing safety in sensitized models is critical for eventual clinical translation.\nWhile our study demonstrates the absence of adverse reactions in naïve animals, safety in allergic mice\nwith pre-existing LTP-specific IgE will need to be evaluated in future studies. Notably, mRNA-LNP\nformulations have shown a favorable safety profile in sensitized models in prior studies (Xu et al., 2023).\nWe have acknowledged the safety concern and highlighted it in lines 490-491 of the discussion.\n3. The IgE inhibition studies are nice, but this doesn't exactly predict the efficacy of IgG in a therapeutic\ncontext. The authors should use cell based assays (human basophil activation tests, or RBL cell lines with\nhuman IgE) to better define whether the cns-LTP IgG can interfere with IgE function.\nResponse: We thank the reviewer for this suggestion, which has significantly strengthened our\nmanuscript. We included functional assays using hRBL-2H3 cells sensitized with human IgE. Our results\ndemonstrate that antibodies induced by cnsLTP-1 immunization effectively block allergen-induced\ndegranulation, highlighting the functional relevance of IgG responses beyond IgE binding inhibition.\nThese findings are summarized in Figure 3 and the discussion section.\n4. I would strongly recommend including T cell readouts. Does the cnsLTP alter T cell cytokine responses\nin an allergic (or even naive) mouse.\nResponse: We analyzed T cell populations (Th1 and Th2) in the spleen and draining lymph nodes of\nimmunized mice (Supplementary figure 5). While we observed a modest increase in Th2 cells in the\nspleens of intramuscularly mRNA-immunized mice, there were no significant differences in the lymph\nnodes, nor in the subcutaneous injections, and the results were inconclusive in this non-allergenic\ncontext. This highlights the need for further studies in sensitized models to assess T cell responses more\ncomprehensively. This has been emphasized in the discussion in lines 485-491.\n5. The sample sizes for the mouse studies are very low (n=3 per treatment group). More mice should be\nincluded for reproducibility and rigor. Similarly, for Figures 3 and 4, there appears to be trends in\ndifferences between mRNA and Protein-treated mice, however, the sample sizes are too small to detect\nthese differences.\nResponse: We have increased the number of mice in our immunization experiments (n=5 per group) and\nconducted additional experiments using both intramuscular and subcutaneous routes with different\nadjuvants. This improved the statistical power of our analyses and allowed for more robust comparisons\nbetween protein and mRNA formulations. These updates are reflected in Figures 2 and Supplementary\nfigure 4.\nMinor Concerns:\nLine 83 - Xolair (Omalizumab) is now also approved for treating food allergies.\nResponse: updated in the introduction in lines 63-64\nLine 367 - Figure 4C is referenced but did not appear in the submitted version of the manuscript.\nResponse: we have reorganised the figures for clarity.\nFigure 1A - why is the cnsLTP-1 a mixture of proteins? Shouldn't it be a single band like the other LTPs\nshown in the SDS-PAGE gels?\nResponse: The multiple bands observed for cnsLTP-1 in SDS-PAGE (Supplementary figure S2) can be\nexplained by the presence of multiple isoforms with shuffled disulfide bonds which might undergo\nvariable reduction in the assayed conditions. This phenomenon has been observed with other nsLTPs\ndue to their extensive disulfide bonding. We have clarified this point in the manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and input and hope that our manuscript served as a\ngood training ground."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I commend the authors for comprehensively addressing my comments in full, and revising the manuscript by making textual\nchanges and clarifications, adding references, and including additional data. It has certainly strengthened the manuscript\nand I have no further comments.", "reviewer_2": "I want to thank the authors for performing the suggested additional experiments that demonstrate that their consensus\napproach seems to work better than using the primary sensitizer. This is an important contribution to the field of AIT for cross-\nreactive allergies.", "reviewer_3": "The authors have responded well and in detail and have made further experiments based on the comments. I am satisfied\nwith the answers.", "reviewer_4": "I appreciate the authors' efforts to address my concerns related to showing data from mice with an established allergy and\nevaluating safety and efficacy. Safety appears fine. Efficacy isn't complete as mice treated with the LTP mRNA still have\nanaphylaxis and MMCP-1 release. However, it is reassuring that IgG2a and 2b increase substantially indicating a desired\neffect of the therapy.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nGeneral\nIn the manuscript by Møiniche et al., the authors present an allergy immunotherapy for treatment of\npollen-food allergy syndrome. The authors approach’ involved designing a consensus allergen that\nrepresents a broad spectrum of non-specific lipid transfer protein (cnsLTP-1) allergens. Naïve mice were\nsubsequently immunized with cnsLTP-1 using a traditional protein formulation (with poly(I:C) as\nadjuvant) or using lipid nanoparticle technology containing mRNA (LNP-mRNA) encoding the consensus\nallergen. Immunization with either formulation induced cnsLTP-1-specific IgG production, with LNP-\nmRNA avoiding allergen-specific IgE production. The induced antibodies were shown to bind various\nnsLTPs, and blocked binding of allergens by serum IgEs derived from allergic patients using an ELISA\nsetup. In my opinion, this is a well-written article that would be of interest broad for a broad readership.\nResponse: We thank the reviewer for their time and for the constructive comments to our manuscript,\nwhich we have done our best to implement.\nSpecific\n- Page 4 line 102/103: Upon mentioning the use of mRNA-based vaccination, it would be appropriate to\nrefer to previous reports using LNP-mRNA technology for allergy applications (e.g. Xu et al., Use of a\nLiver-Targeting Immune-Tolerogenic mRNA Lipid Nanoparticle Platform to Treat Peanut-Induced\nAnaphylaxis by Single- and Multiple-Epitope Nucleotide Sequence Delivery. ACS Nano. 2023 Mar\n14;17(5):4942-4957, Jitthamstaporn et al, Nucleoside-modified mRNA vaccines yield robust blocking\nantibody responses against major house dust mite allergens. Allergy. 2023 Jan;78(1):315-318,\nThanasarnthungcharoen et al., Immunotherapeutic effects of specific and nonspecific mRNA-lipid\nnanoparticles in a mouse model of HDM-induced airway inflammation. Allergy. 2024 Feb 8. doi:\n10.1111/all.16049, and Yin et al., Allergen-specific immunotherapy with Der p 1/2 mRNA vaccines\nalleviates house dust mite-induced allergic airway inflammation in a mouse model. Research Square.\n2024 https://dx.doi.org/10.21203/rs.3.rs-3865185/v1).\nResponse: We have updated the text and included the suggested references (lines 92-93)\n- Page 6 section 2.5.2: When referring to Comirnaty (BNT162b2), it would be appropriate to also refer to\nthe primary literature describing its development (e.g. Polack et al. Safety and Efficacy of the BNT162b2\nmRNA Covid-19 Vaccine. N Engl J Med. 2020 Dec 31;383(27):2603-2615) in addition to the review (ref.\n30) currently cited.\nResponse: Implemented\n- Page 7 line 222-224: Did the authors also determine the LNP-mRNA physicochemical parameters after\nthawing, given the freeze-thaw process can affect these? It would be valuable to add a Table to the\nSupplementary Information summarizing the LNP-mRNA physicochemical parameters before and after\nfreeze-thawing.\nResponse: We have now performed a characterization of the physicochemical properties of the LNPs\nafter thaw, which confirmed the stability of the particles. We have included a table in the\nsupplementary material (Supplementary table S1) with the specific parameters.\n- Page 7 line 232: How was the dosing regimen for the LNP-mRNA (3 x 0.6 ug, 3-week intervals)\nestablished and why was the subcutaneous administration route chosen (rather than for example i.m.)?\nPlease include references or additional information in the main text.\nResponse: Dosing was based on previous literature\n(https://pmc.ncbi.nlm.nih.gov/articles/PMC9538380/) and a preliminary titration experiment, which is\nnow emphasized in the manuscript. Subcutaneous injections may lead to slower absorption of the\nvaccine, potentially resulting in a more prolonged exposure of the immune system to the antigen.\nStill, we have considered the reviewer comment and have repeated the mouse experiments using the\nsame immunisation agenda and intramuscular administration as a supplement to subcutaneous\ninjections. Materials and Methods Immunization schedule has been modified to include the experiment\ndetails (lines 220-227)\n- Figure 2: Perhaps the Western Blots raw data can be added to the Supplementary Information. Figure 1\nand 2 can be merged.\nResponse: Figure 1 has been updated and figure 2 has been moved to supplementary information (now\nSupplementary figure 3).\n- Figure 3: Given the varying antibody titers induced by immunization and PBS (Figure 3B), it would have\nstrengthened the manuscript if the authors had taken along a non-coding poly-cytosine mRNA as\ncontrol, also considering the immunostimulatory effects of mRNA and the ionizable cationic material in\nthe LNP formulation. Have the authors taken this into account?\nResponse: After including extra mice for the subcutaneous immunisation and doing the experiment with\nintramuscular immunisation, we have not found significant changes in antibody titers between the mice\ninjected with PBS and the mice immunised with either mRNA or protein formulations (Figure 2 and\nFigure S4)\nWe decided not to include a control with non-coding poly-cytosine mRNA as the literature already\ndescribes that the LNP composition used in this work does not affect the total antibody levels\n(https://www.nature.com/articles/s12276-023-01086-x). We have included the citation in line 196.\n- Figure 4: Figure 4 can be merged with Figure 3.\nResponse: Figure 3 and 4 have been merged in the new figure 2.\n- Figure 5: Figure 5 and Figure 6 can be merged (and potentially also include IC50 values summarized in\nTable S2).\nResponse: Figure 5 and 6 has been merged (Figure 3) and we have included a figure displaying the\nspecific IC values from Supplementary table 3.\n50\n- Table S2: Please review the Table’s number of significant digits.\nResponse: The IC values have been revisited and are updated in the Supplementary Table 3.\n50\n- Page 13 line 433-437: It would have strengthened the manuscript if data was included to demonstrate\nthe Th1 response, e.g. by using splenocyte restimulations as demonstrated by Jitthamstaporn et\nal.,Allergy, 2023.\nResponse: Following the reviewer’s advice, we performed a splenocyte restimulation following the\nanimal experiment, in which the mice were immunised intramuscularly. We did not observe major\ndifferences between Th1 and Th2 populations. This is now included in the manuscript Supplementary\nFigure 5.\n- Page 14 Limitations section: As the authors indicate themselves, it would be valuable to demonstrate\nthe effects of the LNP-mRNA immunotherapy in a therapeutic setting using mouse models with prior\nallergen sensitization.\nResponse: In this revised version, we have now included data from a Pru p 3–sensitized (allergic)\nmouse model, which provides the first in vivo assessment of the consensus allergen in an anaphylactic\ncontext (Figure 4). The treatment was well tolerated upon injection, indicating the safety of the mRNA-LNP\nformulation (and absence of antigen leakage), and immunized animals displayed increased antigen-\nspecific IgG and IgG responses, indicating the induction of a humoral response even in sensitized\n2a 2b\nhosts, although we could not demonstrate that our approach could prevent/reduce anaphylaxis under\nthe tested regimen (with small cohorts of mice and very highly individual variation in responses).\nAdditionally, we performed the blocking assays using humanized RBL-2H3 cells, which confirm that\nantibodies induced in naïve animals can functionally inhibit IgE-mediated degranulation. Together, the in\nvivo and in vitro findings demonstrate both the safety and immunological activity of the mRNA-LNP\nconsensus allergen formulation.\nMoreover, previous studies have already demonstrated that mRNA-LNP–based allergen immunization\ncan protect against anaphylaxis in other models (https://pmc.ncbi.nlm.nih.gov/articles/PMC10019335/).\nOur findings complement these by establishing a broadly cross-reactive, consensus-based approach,\nwhich represents a conceptual advance. Achieving full therapeutic protection, however, will likely\nrequire further optimization of formulation and delivery parameters, which is beyond the scope of the\npresent manuscript.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Moiniche et al. describes preliminary evauation of the potential of a designed\nconsensus sequence mRNA for nsLTPs for application in AIT. Two aspects are potentially novel: mRNA in\nnanoparticles and the consensus sequence approach. Having said that I have some concerns and doubts.\nResponse: We thank the reviewer for their time taken to review our manuscript. We have attempted to\nimplement all the reviewer’s comments to the best of our ability.\n1) The authors designate LTP allergy as a PFAS. In some instances this may be true, but the consensus is\nthat LTP allergy is mainly driven by peach LPT (Pru p 3) as primary sensitizer. Primary sensitization by\npollen LTPs is more exception than rule. So, LTP allergy is not dominantly a PFAS. Since it is very cross-\nreactive between foods, the approach would still potentially be relevant.\nResponse: We have modified the manuscript to refer to LTP as a “cross-reactive allergy” instead of\nPFAS, and we acknowledge that LTP is mainly a food allergy. Examples can be found in lines 59, 90, 100.\n2) As is illustrated in table S1, the ten patients are having their strongest IgE responses against Pru p 3\nand Mal d 3, confirming my point above: Pru p 3 (and possibly Mal d 3) are primary sensitizers, the rest\nis cross-reactivity. When this is true, the whole consensus approach is unnecessary: just take mRNA for\nPru p 3. That will do the trick. A comparison between mRNA for Pru p 3 and for cnsLTP should have been\nincluded if added value is claimed (which the authors do).\nResponse: We thank the reviewer for the suggestion. We have performed a new animal experiment, in\nwhich we have immunised mice with mRNA encoding Pru p 3 and Par j 2, and we have compared the\nantibody cross-reactivity with the one obtained when immunising mice with cnsLTP1.\nWe have observed that the animals treated with the cnsLTP1 can bind to all the allergens included in the\npanel (including both food and pollen allergens). However, the animals immunised with either Pru p 3 or\nPar j 2 have a limited cross-reactivity profile restricted to very similar allergens (Figure 2). Therefore, we\ndo believe that the claimed added value is warranted.\n3) The approach for designing the cnsLTP sequence is not well described. This should be spelled out in\nmore detail.\nResponse: Details have been included in the Materials and method section “Design of consensus\nallergen” and we have included the full sequence in the Supplementary materials.\n4) The human serum samples should also have been tested for IgE against pollen LTPs (Art v / Par j / Ole\ne).\nResponse: We have included Par j 2, Pla a 3, and Ole e 7 in the allergen panel. We tried producing Art v\n3, but the protein was not correctly folded according to its circular dichroism spectra, and we decided to\ninclude Pla a 3 instead as it is one of the most medically-relevant allergens in the Mediterranean area.\n5)How do the authors explain that the Mal d 3-specific antibody reacts to reduced but not to unreduced\nLTP? There is good evidence that reduction and alkylation makes LTPs unreactive with IgE.\nResponse: We hypothesize that Mal d 3-specific antibody is raised against an unfolded protein and\ntherefore it can only recognize linear epitopes that are hidden when the protein is not completely\nreduced. We have added this statement in lines 392-394\n6) How was IgE measured in mouse serum? It is well established that IgG in mice immunized with\nallergens blocks recognition of IgE, so it is better to measure IgE in a capturing ELISA format coating\nplates with anti-IgE, and using a biotimylated allergen.\nResponse: We thank the reviewer for their suggestions. Following these suggestions, we biotinylated\nthe allergens and detected using Strp-Eu in a DELFIA assay. We tested a different adjuvant (complete\nFreund’s adjuvant instead of poly I:C) and confirmed that the IgE induction in mice immunised with\nprotein formulation was an adjuvant dependent effect and not due to the protein formulation\n(Supplementary Figure 4).\n7) The diagnostic potential claimed in line 414-417 is not really appropriate. There are no data to\nsupport such a claim. A real comparison should then be made between individual LTPs from different\nfoods and the cnsLTP.\nResponse: We have eliminated the claim.\n8) Fig 3E in line 428 shows IgG2a not IgE.\nResponse: Figures have been merged and the annotation in the text has been modified\n9) Line 437: do we really want proinflammatory antibody responses in AIT. Yes if accompanied by Treg\nbut perhaps not if no Tregs?\nResponse: We thank the reviewer for this comment. We have edited the text to accommodate.\n10) Also for the other potenial applications, such as for PR-10 and profilins, just using mRNA for the\nprimary sensitizer would probably be sufficient. The 2S albumins are also mentioned but they are not\nvery cross-reactive, so that will definitely not work.\nResponse: We have included a new animal experiment, in which using the natural allergens in mRNA\nformat induces a restricted cross-reactive response in comparison with the consensus design,\nhighlighting the potential of the consensus design approach in other cross-reactive syndromes. Even\nthough the extent of albumin 2s is small in comparison with PR-10 and profilins, it has been described to\nbe an important allergen in the cross-reactivity between peanuts, tree nuts and sesame seeds in the so-\ncalled pistachio-cashew syndrome or hazelnut-walnut syndrome\n(https://pmc.ncbi.nlm.nih.gov/articles/PMC8035160/).\n11) The background on which the authors claim that AIT for PFAS with normal extracts does not work is\nbased on birch pollen AIT, not on LTP allergies. It has never really been tested whether Pru p 3 is\neffective for cross-reactive foods as well.\nResponse: There are reports in the literature confirming that immunotherapy with a single allergen in\ncross-reactive patients might not induce protection against all the allergens the patients react to\n(https://ctajournal.biomedcentral.com/articles/10.1186/s13601-020-00325-y,\nhttps://onlinelibrary.wiley.com/doi/10.1111/cea.12901 ). We have included the reference on lines 84-\n85\n12) The claim that mRNA approaches would require less immunizations (than the 3-5 years for\nconventional AIT) is not substatiated by any proof.\nResponse: While direct data on the use of mRNA technology in allergen immunotherapy are limited, the\nrapid and durable immunogenicity observed in mRNA vaccine trials suggests that fewer doses may be\nsufficient to achieve a protective immune profile. For instance, Wu et al. (2021)\n(https://pubmed.ncbi.nlm.nih.gov/33730471/) reported robust antibody titers and functional immune\nresponses following the administration of two doses of mRNA COVID-19 vaccines. This stands in stark\ncontrast to the prolonged treatment timelines of conventional AIT, which span years. Still, as the\nreviewer points out, this is a speculation not tested in our case and therefore we have modified our\nwork in in the Discussion section (line 518-521).\nReviewer #3 (Remarks to the Author):\nComments on “mRNA-delivered consensus allergens induce a neutralizing IgG response against food and\npollen allergens” by Rivera-de-Torre et al.\nResponse: We thank the reviewer for their time, for the attention to detail, and for the constructive\ncomments to our manuscript, which we have done our best to implement.\nGeneral comments:\nThis manuscript, by Rivera-de-Torre et al., is an experimental mouse study aiming to test a new allergen\nimmunotherapy (AIT) approach with a consensus allergen and furthermore to test mRNA vaccine\ntechnology or traditional method with protein formula injection for immunization in naïve mice. They\nfound good recognition of the consensus allergen and induction of IgG and in the mRNA vaccine treated\nmice also avoidance of specific IgE production.\nThe novel technique used is interesting, although we hesitate to call it AIT since the experimental mice\nwere naïve, but there are some question marks about some of the methods used and about conclusions\ndrawn by the authors.\nResponse: We acknowledge the necessity in regards to the use of the term ”AIT”. We have corrected\nthe wording to “therapeutic approach” and “immunization” in the introduction and the discussion\nsections. (Lines 89 and 533)\nIntroduction:\nGeneral comment:\nAfter reading the introduction section, it is still unclear to us what is known and what is not known in\nthis research field. Is the new AIT approach completely unique or are there other publications in the\nfield? Can you be more precise about the knowledge gap?\nResponse: The use of mRNA-LNP-based therapies in allergy research has been successfully explored in\nprevious studies, which we have now clarified in the updated introduction (lines 92-93). However, to the\nbest of our knowledge, these efforts have been limited to delivering mRNA encoding natural allergens,\nsuch as Der p 1, Ara h 2, and Phl p 5. The novel aspect of our work lies in the use of consensus allergens\ndesigned to address cross-reactivity. In our opinion, this approach represents an innovative step\nforward, aiming to overcome the limitations of natural allergens by targeting a broader spectrum of\nrelated allergens, which has not been previously reported.\nSpecific comments:\nLines 67-76: About description of PFAS. First of all, authors must disentangle between LTP-related PFAS\nwhich can lead to systemic reactions and PR-10-related PFAS known only to cause mild oral symptoms,\nexcept from rare case reports of systemic reaction following intake of large amount of soy protein Gly m\n4. Treatment with corticosteroids or adrenaline for PFAS is generally not used, especially not for PR-10\nrelated reactions.\nResponse: We appreciate the comment and have modified the manuscript to refer to LTP as a cross-\nreactive allergy instead of PFAS, and we have acknowledged that LTP is mainly a food allergy. Examples\ncan be found in lines 59, 90, 100.\nA better description of PFAS is needed since it constitutes both LTPS and PR-10 proteins but are highly\ndifferent in their clinical presentation and structure and might not be equally suitable for this kind of\ntreatment. SLIT for the treatment of nsLTP allergy with Pru p 3 as the allergen have shown positive\nresults to both peach and cross-reactive allergens (1).\nResponse: We thank the reviewer for their valuable feedback. We agree that PFAS encompasses both\nnsLTP and PR-10 proteins, which differ significantly in their clinical presentation and structure, and these\ndifferences may influence their suitability for this treatment approach. Regarding SLIT with Pru p 3,\nwhile it has shown positive results for peach and cross-reactive allergens, there is evidence in the\nliterature indicating that immunotherapy with a single allergen in cross-reactive patients may not\nprovide protection against all allergens to which they react\n(https://ctajournal.biomedcentral.com/articles/10.1186/s13601-020-00325-y). We have included this\nreference in the introduction (line 107). To further evaluate the potential of the consensus allergen in\ndriving a cross-reactive response against natural allergens, we conducted new experiments immunizing\nmice with Pru p 3 and Par j 2 in mRNA format. Our findings demonstrate that antibodies induced by the\nconsensus allergen can bind to all tested allergens except Ole e 7, whereas Pru p 3 mRNA immunization\nonly induced binding to Pru p 3, Cor a 8, Ara h 9, and Pla a 3, and Par j 2 mRNA immunization primarily\ninduced binding to Par j 2 and Pla a 3 (Figure 2). These results underscore the broader cross-reactivity\nelicited by the consensus design compared to individual allergens, highlighting its potential usefulness\nfor addressing cross-reactive allergies comprehensively.\nThere is a need to focus more on LTPS (lipid transfer protein syndrome) rather than the much wider\ndefinition PFAS.\nResponse: We have focused on LTPs and have restricted our discussion about cross-reactivity and PR-10\nto the discussion section as an interesting perspective to pursue in the future (lines 522-527).\nLines 97-101: The claim that 90% of discontinuation of AIT needs to be explained. Is it really true for all\nforms of AIT including SCIT? We doubt that. And that patient with PFAS do not consider AIT can be\nbecause PFAS is not an indication for AIT and the suffering from PFAS for most patients, at least PR-10\nrelated OAS, is limited.\nResponse: We thank the reviewer for highlighting this point. We appreciate the opportunity to clarify\nand provide additional context. The discontinuation rates cited in the manuscript refer specifically to AIT\nfor respiratory allergens, such as grass pollen, rather than food allergies due to the lack of extensive\nadherence studies for such indications. For example, adherence to subcutaneous immunotherapy (SCIT)\nhas been reported to range from 23% to 89%, while adherence to sublingual immunotherapy (SLIT)\nranges from 64% to over 95% in some studies. However, despite these promising rates, a survey in Italy\nreported a concerning discontinuation rate of up to 90% for SLIT three years after prescription for grass\npollen allergy. We have updated the manuscript (lines 86-88) to specify that these statistics are based\non grass pollen allergy and have revised the description of adherence rates to better reflect the nuanced\nchallenges of AIT adherence. Additional references have also been included to support these points.\nLines 102-110: However, the idea of a new AIT approach using mRNA vaccine technique and engineered\nproteins is interesting.\nResponse: We thank the reviewer for their positive remark.\nMethod:\nGeneral comments:\nFirst of all, we are primarily clinical researchers and not experts on laboratory methods. We leave to\nother reviewers more skilled in this area to evaluate the laboratory methods used in this study.\nHowever, some thoughts about the methods:\nTo further study the effect of protein immunization and mRNA vaccine the study needs to add a recall\nassay to study the T cell response of the different methods of immunization. And the study also lacks the\nanalysis of autoantibodies which needs to be analysed in vaccine studies as it could be a side effect that\nis crucial to avoid. Further, the sequence of the pre-ordered mRNA used in the experiments needs to be\npresented in the method-section.\nResponse: We thank the reviewer for their thoughtful feedback. We appreciate the opportunity to\naddress these important points.\n• T cell response: We analyzed the T cell populations (Th1 and Th2) in the spleen and draining\nlymph nodes of mice immunized via both intramuscular and subcutaneous routes\n(Supplementary Figure 5). Statistically significant differences were observed only in the spleens\nof mice immunized intramuscularly: mice immunized with cnsLTP-1 mRNA and Par j 2 mRNA\nexhibited a slightly increased Th2 population in the spleen compared to the PBS group.\nHowever, as these experiments were conducted in naïve mice, and the significance does not\ncorrelate in the lymph nodes, definitive conclusions cannot be drawn in a non-allergenic\ncontext.\n• Analysis of autoantibodies: The potential induction of autoantibodies is indeed a critical\nconsideration for mRNA-LNP-based formulations. While this study did not include autoantibody\nanalysis, this aspect will be carefully evaluated in subsequent studies during the later stages of\npreclinical and clinical development. As is correctly noted, mRNA-LNP formulations carry a\ntheoretical risk of inducing autoantibodies, particularly with repeated dosing. However, nsLTPs,\nincluding the consensus allergen cnsLTP-1, do not have known paralogues in mice or humans.\nWe performed protein BLAST searches for all nsLTPs used in this study, including cnsLTP-1, and\ndid not identify any significant similarity with human or murine proteins. Thus, the risk of\nautoantibody induction against the consensus allergen is expected to be very low. We have\nadded this information to the manuscript to address this concern.\n• mRNA sequence: we have added the sequence information in the Supplementary information in\nFASTA format to facilitate reuse, transparency, and reproducibility.\nSpecific comment:\nLines 225-228: Why use the adjuvant poly(I:C) in these experiments? The adjuvant poly(I:C) to the\nprotein immunization activates primarily TLRs leading to interferon responses and previous studies have\nshown that it increases IgE and IgG1 (2), which makes it maybe less suitable for comparison with LNPs. A\nsuggestion would instead be a comparison with an adjuvant for protein immunization - a mineral\noil/water emulsion (as incomplete Freunds adjuvant) like in Montanide ISA-51 or similar.\nResponse: We thank the reviewer for their comment and suggestion regarding the use of poly(I:C) as an\nadjuvant in these experiments. We initially selected poly(I:C) to mimic the nucleotide adjuvant effect of\nmRNA, as it also stimulates TLR3 and is commonly used to model similar immune responses. However,\nwe acknowledge that the choice of adjuvant significantly impacts antibody responses, and your\nsuggestion for a mineral oil/water emulsion such as incomplete Freund’s adjuvant is well taken.\nTo address this, we repeated the experiment using incomplete Freund’s adjuvant instead of poly(I:C) in\nboth subcutaneous and intramuscular immunizations. We observed that while allergen-specific IgG1 and\nIgG2a responses were maintained, the use of incomplete Freund’s adjuvant did not induce the\nproduction of IgE (Figure 2 and Supplementary figure 4). These findings provide further insight into the\nrole of adjuvants in shaping the immune response and highlight the potential of Freund’s adjuvant for\navoiding IgE induction. We have updated the results section accordingly and included the comparison in\nFigure S4D and E.\nLines 229-238: How many mice were included in the immunization experiment?\nResponse: The Materials and Methods section has been updated to specify the number of mice used\nper group (lines 221–229). Briefly, for subcutaneous immunization, four- to five-week-old BALB/c female\nmice were immunized three times at three-week intervals with 9 µg mRNA-LNP (n=6), 24 µg protein-\nPoly(I:C) (n=3), 24 µg protein-incomplete Freund’s adjuvant (n=3), or PBS (n=7). For intramuscular\nimmunization, four- to five-week-old BALB/c female mice were immunized three times at three-week\nintervals with 7.5 µg mRNA-LNP (n=5), 20 µg protein-incomplete Freund’s adjuvant (n=5), or PBS (n=5).\nResults:\nSpecific comments:\nLines 282-290 and 292-302: The differences between the two paragraphs is not clear. The most\ninteresting comparison from a clinical point of view is with peach Pru p 3, the LTP most widely used as\nan LTP proxy in clinical diagnostics. It would be interesting to understand why the consensus allergen is\nmore useful. The 79% similarity with Mal d 3 is not convincing. Clinical apple allergy is normally PR-10\nrelated and mild. Mal d 3-related Apple allergy is to our knowledge very uncommon.\nResponse: Thank you for the opportunity to clarify this point. We have used the identity shared between\nnatural allergens and our consensus design to specify that we are not accidentally replicating a natural\nnsLTP variant. Mal d 3 sharing a 79% and Par j 2 sharing less than 30% is a way to determine that our\nconsensus allergen is covering the diversity contained in this range. Our study demonstrated variable IgE\nrecognition patterns among patients (Supplementary Table 2), reflecting individual sensitization profiles.\nHowever, Western blot analysis (Supplementary figure 3) showed consistent recognition of cnsLTP-1 by\npatient IgEs, regardless of variability in individual sensitization patterns. This uniform recognition\nhighlights the potential of cnsLTP-1 as a diagnostic screening tool for nsLTP-related allergies, offering a\nstandardized approach to detect sensitization in patients with diverse allergen recognition profiles.\nThe advantage of cnsLTP-1 over natural allergens like Pru p 3 is further highlighted by the immunization\nexperiments using natural allergens in mRNA format. These experiments demonstrated that cnsLTP-1\nelicited a broader antibody response compared to Pru p 3 or Par j 2, emphasizing its superior coverage\nand utility in addressing cross-reactive allergy management. We have added a clarification in lines 371-\n374 and updated the results with Pru p 3 and Par j 2 immunisation antibody responses (Figure 2).\nLines 325-330: How many mice were included in the immunization experiment?\nResponse: The text has been updated to specify the number of mice used per group (lines 219-229).\nEach point in figures 2 and S4 corresponds to an individual mouse.\nFigure 3 B and C have likely been mixed up. The IgE response to protein immunization could be\nexplained by the adjuvant and not the superiority of the mRNA vaccine. This is also mentioned briefly in\nthe discussion but needs to be further addressed and further studies on the T cell response should have\nbeen/needs to be done to further study this.\nResponse: The figures have been reorganized to improve clarity. The new Figure 2 now includes all data\nrelated to antibody characterization in mice immunized intramuscularly, while Figure S4 contains the\ndata for mice immunized subcutaneously, including the comparison of IgE responses with different\nadjuvants. These results confirm that the observed IgE induction is due to the adjuvant effect rather\nthan differences between the protein and mRNA formulations. Additionally, T cell populations were\nanalyzed (Supplementary Figure 5); however, the results related to population distribution are\ninconclusive in this non-allergenic context. Further studies will be required to comprehensively evaluate\nthe T cell responses under conditions that better mimic allergic sensitization.\nFigure 4. The effect on the adjuvant used in the protein immunization could also affect the IgG response\nwith increased IgG1 and less IgG2a, see above. Needs to be further addressed.\nResponse: While IgE induction was shown to be dependent on the adjuvant used, the levels of allergen-\nspecific IgG1 and IgG2a were comparable between the protein formulations, regardless of the adjuvant\nemployed (Supplementary Figure 4). This suggests that the choice of adjuvant did not significantly\ninfluence the IgG subclass distribution in the protein immunization.\nFigure 5/6. Through inhibition of human serum IgE-binding to nsLTP it is suggested that mRNA might be\nbetter than protein immunization in blocking IgE-binding (due to a trend in the figures). However, the\nvariability is high and the number of individuals is low, making the statistical power too low to make any\nconclusions.\nResponse: We have increased the number of animals used from 3 to 5, which has reduced variability\nand allowed for a more robust analysis of the differences between protein and mRNA-LNP formulations\nin both binding (ELISA) and cell degranulation (hRBL-2H3) assays. While no statistically significant\ndifferences were observed in the IC50 values between the mRNA and protein formulations, the IC50\nvalues for mRNA formulations were generally lower, indicating a potential trend towards greater\nefficacy.\nTable S2. What were the systemic reactions the patients exhibited? Are there any other\nallergies/sensitizations than to the presented LTP?\nResponse: The patients presented with severe oral allergy syndrome, characterized by itchiness and\nredness upon contact with peach. No other relevant co-sensitizations were observed beyond the\npresented LTP allergens. We have added this information in lines 396-400.\nDiscussion:\nGeneral comments:\nThe authors needs to acknowledge the difference in LTP and PR-10 proteins before proposing that this\nkind of treatment could be suitable for birch-apple allergy. The authors should focus on LTPS throughout\nthe manuscript.\nResponse: We agree that the primary focus of our manuscript is on nsLTPs, and we have revised the text\nto clarify this distinction. Specifically, we have removed references to PR-10-related syndromes and\nadjusted the discussion to focus on the potential applications of our consensus allergen design for\naddressing cross-reactive related allergies, in which cross-reactivity is driven by structurally conserved\nfamilies (lines 522-524).\nTo draw the conclusion of alleviation of symptoms using a handful of mRNA-LNP doses, doesn’t long-\nterm studies need to be conducted first?\nResponse: We acknowledge the complexity of foreseeing the long-term effect of novel immunisation\nstrategies and have modified our statement appropriately in the discussion (lines 529-532).\nSpecific comment:\nLines 415-417: See first comment under results. Why is the consensus LTP more useful than Pru p 3?\nResponse: As mentioned previously, we have clarified this point by immunising animals with Pru p 3, Par\nj 2, and cnsLTP-1 in mRNA format and comparing the cross-reactivity of the raised Igs. cnsLTP-1 elicits a\nbroader cross-reactivity than Pru p 3, binding to all tested nsLTPs, whereas Pru p 3 shows limited\nrecognition beyond closely related allergens (Figure 2). This broader coverage highlights its potential for\naddressing cross-reactive allergies comprehensively.\nConclusion:\nGeneral comments:\nMotivate why the next step is clinical trials as proposed. The next step could also be further studies with\nthe use of more naïve mice, then immunized for example using an adjuvant triggering an allergic\nresponse like alum, analyse the T-cell response to these different methods and check for the presence of\nautoantibodies after treatment.\nResponse: This is an excellent suggestion, and we have included it in our discussion as a roadmap for\nnext steps.\nAlso, the authors conclude that their mRNA-LNP formulation is superior over protein immunization; this\nstatement cannot be proven with the experiments done. The variability is too big, given the small\nnumber of mice, and the adjuvant used might itself explain the better results of the mRNA-LNP\nformulation in these experiments.\nResponse: In light of our complete experiments with a bigger sample, we have made better assessment\nof the differences between mRNA and protein, and so far, in the assayed context, we cannot conclude a\nclear advantage of any of the formulation tested for cnsLTP-1. The conclusions text has been modified\naccordingly.\n1. Gomez F, Bogas G, Gonzalez M, et al. The clinical and immunological effects of Pru p 3 sublingual\nimmunotherapy on peach and peanut allergy in patients with systemic reactions. Clin Exp Allergy. 2017;\n47: 339-350\n2. Wan H, Yang H, Wei M, Chen W. Polyinosinic:polycytidylic acid aggravates calcipotriol-induced atopic\ndermatitis-like skin lesions in mice by increasing the expression of thymic stromal lymphopoietin. Ann\nTransl Med. 2022 Feb;10(4):209. doi: 10.21037/atm-22-282. PMID: 35280398; PMCID: PMC8908153.\nReviewer #4 (Remarks to the Author):\nThe overall goal is to develop a novel form of alleren-specific immunotherapy for the cross-reactive LTP\nfood and pollen allergens. Accordingly, the authors present an interesting approach by developing a\nconsensus LTP allergen sequence, then formulating it into mRNA-LNP for subcutaneous administration.\nThe work clearly shows induction of IgG1 and IgG2a antibodies against LTPs in naive mice, which is a\nstarting point in the development of a novel allergy immunotherapy approach. However, I have some\nconcerns with the manuscript in its current form:\nResponse: We thank the reviewer for their time and for the valuable feedback on our manuscript. We\nhave done our best to implement the reviewer’s suggestions.\nMajor concerns:\n1. The work is designed towards developing a novel molecule, cnsLTP-1, for allergy immunotherapy.\nHowever, the authors do not assess the molecule in allergic mice. Rather, all in vivo studies were\nconducted in naive animals. The authors should strongly consider sensitizing mice to LTPs, then using\nthe cnsLTP-1 mRNA or protein formulations as immunotherapy. This will provide much needed efficacy\ndata in an allergic mouse model. The data currently shown are not convincing that the approach\nprovides any therapeutic benefit to allergic animals.\nResponse: We acknowledge the importance of testing cnsLTP1 in an allergic mouse model to evaluate\nits therapeutic efficacy. In this revised version, we have now included data from a Pru p 3–sensitized\n(allergic anaphylactic) mouse model, which provides the first in vivo assessment of the consensus\nallergen in a relevant anaphylactic context (Figure 4). Treated mice developed increased antigen-specific\nIgG2a and IgG2b antibodies, confirming that a humoral response can be elicited even in sensitized hosts.\nAlthough we could not demonstrate the that these antibodies could prevent anaphylaxis under the\ntested conditions (small cohorts of mice and large individual variation), this first therapeutic experiment\nrepresents a key step toward establishing efficacy in allergic models by showing induction of the desired\nantibody response. We have included a functional assay with hRBL-2H3 cells reinforces the\nneutralisation capacity of the induced antibodies upon immunisation. Additionally, other studies in the\nliterature have demonstrated the efficacy of mRNA-LNP formulations in allergic models, including their\nability to protect against anaphylaxis (e.g., Xu et al., 2023; Jitthamstaporn et al., 2023). Based on our\ncurrent findings, achieving full protection against anaphylaxis will likely depend on further optimization\nof formulation composition and administration parameters, which is beyond the scope of this\nmanuscript focused on cross-reactivity.\n2. Similarly, the authors must assess safety of the cnsLTP-1. Of course it is \"safe\" in naive animals, but\nwhat happens when a mouse is allergic and primed to have reactions due to existing LTP-specific IgE?\nThe authors should sensitize mice to LTPs and then inject the mRNA and protein formulations to look for\nsymptoms of allergy. This safety data is mandatory before considering human clinical trials.\nResponse: The cnsLTP1 mRNA-LNP formulation was administered to Pru p 3–sensitized (allergic\nanaphylactic) mice and was well tolerated by all animals throughout the treatment. This indicates that\nany potential leakage of the expressed allergen from transfected cells was insufficient to trigger allergic\nreactions, which would have manifested as reduced activity or a drop in body temperature. Notably,\nmRNA-LNP formulations have also shown a favorable safety profile in sensitized models in previous\nstudies (Xu et al., 2023).\n3. The IgE inhibition studies are nice, but this doesn't exactly predict the efficacy of IgG in a therapeutic\ncontext. The authors should use cell based assays (human basophil activation tests, or RBL cell lines with\nhuman IgE) to better define whether the cns-LTP IgG can interfere with IgE function.\nResponse: We thank the reviewer for this suggestion, which has significantly strengthened our\nmanuscript. We included functional assays using hRBL-2H3 cells sensitized with human IgE. Our results\ndemonstrate that antibodies induced by cnsLTP-1 immunization effectively block allergen-induced\ndegranulation, highlighting the functional relevance of IgG responses beyond IgE binding inhibition.\nThese findings are summarized in Figure 3 and the discussion section.\n4. I would strongly recommend including T cell readouts. Does the cnsLTP alter T cell cytokine responses\nin an allergic (or even naive) mouse.\nResponse: We analyzed T cell populations (Th1 and Th2) in the spleen and draining lymph nodes of\nimmunized mice (Supplementary figure 5). While we observed a modest increase in Th2 cells in the\nspleens of intramuscularly mRNA-immunized mice, there were no significant differences in the lymph\nnodes, nor in the subcutaneous injections, and the results were inconclusive in this non-allergenic\ncontext. This highlights the need for further studies in sensitized models to assess T cell responses more\ncomprehensively. This has been emphasized in the discussion in lines 554-556.\n5. The sample sizes for the mouse studies are very low (n=3 per treatment group). More mice should be\nincluded for reproducibility and rigor. Similarly, for Figures 3 and 4, there appears to be trends in\ndifferences between mRNA and Protein-treated mice, however, the sample sizes are too small to detect\nthese differences.\nResponse: We have increased the number of mice in our immunization experiments (n=5 per group) and\nconducted additional experiments using both intramuscular and subcutaneous routes with different\nadjuvants. This improved the statistical power of our analyses and allowed for more robust comparisons\nbetween protein and mRNA formulations. These updates are reflected in Figures 2 and Supplementary\nfigure 4.\nMinor Concerns:\nLine 83 - Xolair (Omalizumab) is now also approved for treating food allergies.\nResponse: Updated in the introduction in lines 66-67\nLine 367 - Figure 4C is referenced but did not appear in the submitted version of the manuscript.\nResponse: We have reorganised the figures for clarity.\nFigure 1A - why is the cnsLTP-1 a mixture of proteins? Shouldn't it be a single band like the other LTPs\nshown in the SDS-PAGE gels?\nResponse: The multiple bands observed for cnsLTP-1 in SDS-PAGE (Supplementary figure S2) can be\nexplained by the presence of multiple isoforms with shuffled disulfide bonds which might undergo\nvariable reduction in the assayed conditions. This phenomenon has been observed with other nsLTPs\ndue to their extensive disulfide bonding. We have clarified this point in the manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and input and hope that our manuscript served as a\ngood training ground."}]

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