Peanut (Arachis hypogaea) is one of the most prevalent food allergens, affecting ~2% of people worldwide, and is the leading source of food-induced severe allergic reactions, including fatal anaphylaxis (Muraro et al. 2021). Peanut allergy, which typically manifests during childhood, has increased over the past three decades, tripling to 1.2 million children in the Unites States. According to the "hygiene hypothesis,” this marked increase is the result of cleaner living conditions that lessen exposure (“training”) of the immune system to differentiate harmful vs. harmless irritants.
Peanut allergy is an immunoglobulin E (IgE)-mediated hypersensitivity reaction induced primarily by the antigenic peanut peptide termed AraH2, as detailed elsewhere. In a sensitized individual, AraH2 cross-links IgE bound to mast cells (Figure 1), which then release histamine and a variety of cytokines and chemokines. Inflammatory cell recruitment ensues to propagate the allergic response that may lead to anaphylaxis, as reviewed by (Nguyen et al. 2021).
FIGURE 1. Depiction of antigenic peanut peptide AraH2 cross-linking two IgE antibodies (green) bound to a mast cell (orange) triggering release of histamine and other effectors (dots) of allergic response. Take from Hemmings et al. (2020) and free to use under the under the CC BY license.
When T-cells are exposed to peanut protein, they release various cytokines, such as IL-4 and IL-13, that promote the production of IgE antibodies and the activation of mast cells (Figure 1). On the other hand, T-cells can also help to suppress the immune response. In particular, regulatory T-cells (Tregs), which are a subtype of T-cells, can suppress the production of IgE antibodies and the activation of mast cells to help prevent anaphylaxis.
In people with peanut allergy, there are too many T-cells that promote the production of IgE antibodies and the activation of mast cells, and not enough Tregs to suppress the immune response and anaphylaxis. Therapeutical modulation of Tregs, which is considered (Kondĕlková et al. 2010) to be a general approach to treat allergies, has been recently demonstrated by (Xu et al. 2023) in the context of the first-ever vaccine candidate for peanut allergy. The following sections discuss how these researchers used non-allergenic fragments of peanut protein, encoded in CleanCap® nucleoside-modified mRNA from TriLink, to induce Tregs to prevent peanut allergic response and anaphylaxis in a standard mouse model-.
Design and Preparation of a mRNA Vaccine for Peanut Allergy
T-cell epitopes are antigenic peptides that can be presented to CD4+ T-cells through interaction with type II major histocompatibility (MHC-II) molecules on antigen-presenting cells (APC). Importantly, this includes epitope presentation to naïve CD4+ T- cells, which can be induced to differentiate into Tregs upon engagement of T-cell antigen receptors (TCR) with the MHC-II/epitope complex (Syed et al. 2014).
Based on this background information, Xu et al. hypothesized that mRNA-encoded T-cell epitopes from the principal peanut antigen AraH2, upon nanoparticle delivery to APCs, could serve as a tolerogenic vaccine for durable suppression of peanut-induced allergic inflammation, mast cell release, and anaphylaxis.
To test this hypothesis, the Immune Epitope Database and Analysis Resource for identifying MHC-II binding epitopes was applied to AraH2 to obtain four top-ranking 15-mer peptide T-cell epitopes with affinity binding to murine MHC-II alleles. Among these four epitopes, the synthetic 15-mer peptide termed “epitope 4” was then determined to be the most robust inducer of Tregs in mice.
To design an equivalent mRNA construct, 15-mer peptide epitope 4 was extended at each end by its two naturally occurring amino acids to give a 19-mer, which was reverse translated into its corresponding 57-mer nucleotide sequence. Because tandem-repeats of T-cell epitopes are known to enhance immunogenicity (Kjerrulf et al. 1997), a tandem-repeat of the 57-mer mRNA segment encoding epitope 4 was positioned downstream (Figure 2) of the T-cell epitope MHC-II targeting signal referred to as the “invariant Ii chain sequence” (Cloutier et al. 2021). Following the addition of requisite start (ATG) and stop codons (TAA), GenSmart™ codon optimization for mouse was applied.
FIGURE 2. Depiction of the vector for optimized IVT of mRNA encoding tandem-repeat epitope 4. Credit: Jerry Zon
Xu et al. then utilized TriLink’s custom mRNA synthesis service to prepare the mRNA epitope 4 vector to be tested as a vaccine. The mRNA was synthesized with appropriate 5’ and 3’ untranslated (UTR) sequences,120-nt poly(A) tail, ®the biologically favorable CleanCap® Cap 1 structure, andall uridines were replaced with N1-methypseudouridine.
It was desirable to formulate LNPs for targeting to liver sinusoidal epithelial cells (LSECs), which are known to act as specialized APCs to generate Tregs. To do this, Xu et al. took advantage of the demonstrated (Kim et al. 2021) ability of mannose-bearing LNPs to bind to a mannose receptor on LSECs. In the present study, this was achieved by formulating the mRNA vaccine with a mixture of lipids wherein one component was a commercially available pegylated lipid having a terminal mannose moiety (LNP-man).
mRNA Peanut Vaccine Generates Tregs in Mice
To determine whether the mRNA peanut vaccine generates Tregs in mice, four groups of animals were each intravenously (iv) injected with a different test article before spleen harvesting on day-7, preparation of splenocyte suspensions, and staining with antibodies recognizing Treg surface markers for flow cytometry. The four test articles were phosphate buffered saline (PBS), poly(A)/LNP-man, mRNA vaccine/LNP-man, and mRNA vaccine/LNP without mannose. The percent of Tregs for poly(A)/LNP-man was not statistically different compared to the PBS negative control, whereas the statistical difference for mRNA vaccine/LNP without mannose was relatively low (p < 0.01) but high for mRNA vaccine/LNP-man (p < 0.001). These results indicating greater Treg generation by mannose-mediated targeting of the mRNA vaccine were confirmed by an independent replicate experiment and an ELISpot assay for quantification of IL-10-producing Tregs.
mRNA Peanut Vaccine for Prophylactic Prevention of Anaphylaxis
The following is a brief outline of the experimental protocol used by Xu et al. to test for mRNA vaccine prophylactic prevention of anaphylaxis in mice induced by crude peanut extract (CPE). Three groups of mice received iv pre-administration (“vaccination”) of either mRNA vaccine/LNP-man or mRNA vaccine/LNP without mannose or poly(A)/LNP-man on day-0 and day-7. These animals were then sensitized by oral administration of small amounts of CPE on day-14, -21, and -28 before peritoneal challenge with a large amount of CPE on day-35.
Rectal temperatures and standard scoring of anaphylaxis scores were obtained every 15 min for 2 h. Blood, peritoneal lavage fluid, and spleen tissue were collected for further analysis. Non-sensitized mice were used as a negative control, while sensitized animals receiving CPE challenge without any co-treatment served as a positive control. Temperatures and anaphylaxis scores for all groups paralleled the results discussed above for generation of Tregs, with the most important result being nearly complete prevention of fever and anaphylaxis by prophylaxis with mRNA vaccine/LNP-man.
Apart from this prophylactic impact on physical disease manifestations, pre-administration of mRNA vaccine/LNP-man, and to a lesser extent mRNA vaccine/LNP, suppressed the rise in total serum IgE, peanut antigen-specific IgE, and murine mast cell activation. The same impact was also seen for peanut antigen-specific IgG1, which, like IgE, identifies an IL-4-mediated immunoglobulin class switching event that improves the ability of antibodies to remove the pathogenic antigen. This was also confirmed by showing mRNA vaccine/LNP-man suppression of the release of IL-4 in the peritoneal fluid. Consistent with these immunological findings, flow cytometry of spleen cells showed that mRNA vaccine/LNP-man led to the greatest increase in protective Tregs.
Finally, and perhaps the most important question addressed by Xu et al. was whether the mRNA vaccine/LNP-man could prevent anaphylaxis by administration to mice after sensitization to CPE. If so, this would suggest the possibility of using mRNA vaccine/LNP-man to prevent anaphylaxis in persons who have already developed an allergy to peanuts and accidentally ingest or come into skin contact with peanuts.
To test this important scenario in mice, the experimental protocol described in the first paragraph of this section was repeated but with reversal of the order of vaccination and sensitization with CPE. In other words, sensitization by CPE on day-0, -7, and -14 followed by administration of test articles on day-21 and day-28 before peritoneal challenge with CPE on day-35. In brief, all the measured parameters were essentially the same as with the above-mentioned original-order event, with mRNA vaccine/LNP-man preventing anaphylaxis.
The most significant finding of this study is the demonstration, for the first time, that mRNA delivery of the dominant peanut allergen epitope AraH2, as a tandem-repeat in LNP-man targeting LSECs, can induce a tolerogenic response capable of blocking anaphylaxis induced by CPE. It is also noteworthy that this outcome could be sustained for at least 60 days in mice, demonstrated by experimentally increasing the duration of time between prophylactic mRNA vaccination and of challenge with CPE.
Based on these findings, Xu et al. propose that tolerogenic allergen epitopes encoded in mRNAs and delivered in LNPs could also be useful for immunotherapy of a wide range of allergies, such as cat, house dust mite, grass pollen, and other food allergies. The estimated 2 billion persons worldwide afflicted with these and other common allergies represent a major future application for mRNA-based immunotherapy.
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