Prevention of allergy by virus‐like nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model

Abstract Background In high‐risk populations, allergen‐specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus‐like nanoparticles (VNP) expressing surface‐exposed or shielded allergens. Methods Full‐length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results Virus‐like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface‐exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1‐specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen‐specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface‐exposed but not shielded allergen induced allergen‐specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen‐protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg‐dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion Virus‐like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.

Conclusion: Virus-like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.

K E Y W O R D S
allergy prevention, mugwort pollen allergy, Treg cells, virus-like nanoparticles, prevention, lung APC

| INTRODUCTION
More than 25% of the population in Western countries is affected by allergies. Currently available options to manage allergic diseases include allergen avoidance, symptomatic pharmacotherapy, 1 or in severe cases application of biologics targeting IgE or inflammatory mediators of adaptive or innate immune cells. [2][3][4][5] while the only disease-modifying treatment available so far is allergen-specific immunotherapy with allergen extracts (AIT). 6,7 However, the poor quality of natural allergen extracts is a major limitation for the development of AIT. 8,9 Another challenge of AIT is that it can induce allergic side effects which can be potentially life-threatening. 10 To overcome the disadvantages of allergen extract-based AIT, improved forms of molecular allergy vaccines are being developed and clinically evaluated. 11

G R A P H I C A L A B S T R A C T
Allergen-expressing VNP, which are produced in HEK 293T cells with the help of MoMLV structural proteins, are unable to stimulate allergenspecific B cells or sensitized effector cells (basophils, mast cells). Instead, upon i.n. application, they are being taken up by lung-resident CD103 + DC and alveolar macrophages, which unpack their (allergic) cargo, expand Foxp3 + Treg cells and prevent from sensitization. KRATZER ET AL.

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Therefore, prevention by specific approaches has become an attractive strategy in allergy, especially in high-risk populations. 11,16,17 Today, such high-risk populations can be reliably identified by component-resolved molecular allergy diagnosis. 18,19 The presence and degree of sensitization against marker allergens early on in life (ie, at 4 years of age) serve as highly significant prediction markers for manifest disease later in life (ie, at 8 and 16 years of age) 18 and pave the way for preventive interventions such as those based on virus-like nanoparticles (VNP).
The principle of linking allergens to particles has been already suggested as possible AIT strategy. This approach can be used to reduce the allergenic activity of allergens and to enhance their immunogenicity. 20,21 Likewise, VNP may be used as allergen carriers. 22,23 They are noninfectious enveloped particles that are inducible in mammalian cells by the expression of viral structural proteins (ie, Gag) in the absence of viral nucleic acids or envelope proteins. By rational incorporation 22 of membrane-bound cytokines, [24][25][26][27] cytokine receptors, 25 growth factors, 24 fluorescent proteins, 22,25,27 MHC, costimulatory and also adhesion molecules, 28,29 VNP have been demonstrated to sustain stimulatory but also inhibitory immune responses and, as fluorescent versions, can also be used to trace, for example, subtle receptor-ligand interactions by flow cytometry. However, so far, no in-depth analysis has been performed to investigate how the mode of linking of allergens to VNP may affect their allergenic and tolerogenic properties and if such a strategy can be used to foster T cell regulation. Clinically effective AIT is associated with an increase in allergen-specific pTreg numbers in already allergic individuals. 30 In addition, we tested whether prophylactic application of allergenspecific VNP would mitigate allergic sensitization and AHR with allergen extract and we sought to identify the responsible mechanisms. Ltd., Tullagreen, Ireland), and followed by concentration using a SW41

| Statistical analyses
Groups with similar variance were compared using parametric tests (Student's t test or one-way ANOVA) followed by correction of alpha (Tukey or Holms-Sidak) using GraphPad 6.0 (GraphPad Software Inc., La Jolla, CA). Otherwise, the Mann-Whitney U test or the Kruskal-Wallis test was performed, followed by Dunn's multiple comparison testing. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Further experimental details are provided in the Materials and
Methods section in this article's Appendix S1. All antibodies used within this study are listed in Table S1. To analyze their T cell stimulatory capabilities, we incubated splenocytes of Art v 1-specific TCR/DR1 allergy mice with MA::Art v 1 VNP, Art v 1::GPI VNP, empty VNP (10 μg/mL), or rArt v 1 (0.5 μg/mL) and compared them to cultures supplemented with either medium alone or PMA plus ionomycin, respectively. Of note, both MA::Art v 1 VNP and Art v 1::GPI VNP, similar to rArt v 1, specifically stimulated T cell proliferation ( Figure 3D). In contrast, neither empty VNP nor VNP expressing the control FPs MA::Bet v 1 or Bet v 1::GPI activated Art v 1-specific T cells ( Figure 3D). Doseresponse determinations revealed a better stimulatory capacity of MA::Art v 1 VNP (EC 50 1.1 ± 0.2 μg/mL) compared to Art v 1::GPI VNP (EC 50 13.6 ± 7.8 μg/mL) ( Figure 3E and Table S2), most likely due to the higher concentration of Art v 1 in these VNP ( Figure Figure 5B-D). In contrast, some of the mice exposed to Art v 1::GPI VNP, but none of those exposed to empty VNP, developed modest, allergen-specific IgE, IgG1, and IgG2a levels ( Figure 5B-D). The fact that Art v 1::GPI VNP have the principle capacity to sensitize some of the tested mice questioned their suitability as prophylactic vaccine already at this stage. While empty VNP were recognized by the MoMLV p30Gag-specific mAb R187 in immunoblots ( Figure S5A) and ELISA ( Figure S5B), no such reactivity was detectable in mouse sera ( Figure S5C-E). In vivo uptake in lungs of fluorescently labeled VNP was dominated by alveolar macrophages and CD103 + DCs ( Figure 5E-G). Preliminary data suggest that CD103 + DC are less potent in taking up free allergen (not shown). Apart from the i.n., also the s.c. route was evaluated for prophylactic VNP application.
However, under these conditions, even MA::Art v 1 VNP turned out to be sensitizing as they induced allergen-specific IgE already after three s.c. injections. IgE levels further increased upon subsequent i.n. and intratracheal challenges with the allergen extract (not shown).
3.5 | Preventive treatment with MA::Art v 1 expressing VNP protects from subsequent allergic sensitization and ameliorates lung function As MA::Art v 1 VNP were neither immunogenic nor allergenic but clearly activated allergen-specific CD4 + T cells in vitro ( Figure 3D-E), we hypothesized that the in vivo application of MA::Art v 1 VNP might inhibit subsequent sensitization with mugwort pollen extract, akin to the mechanisms observed in peptide immunotherapy of allergy. 50 To test this, we i.n. exposed mice to MA::Art v 1 VNP, Art v 1::GPI VNP, empty VNP or PBS twice, separated by a biweekly interval followed by five challenges with aqueous mugwort pollen extract ( Figure 6A), known to induce fairly robust allergen-specific IgE titers and also allowing to monitor cytokine recall responses by lung cells. We found that, in principle, mice exposed to MA::Art v 1 VNP during preventive treatment did not produce allergen-specific IgE in response to allergen challenge (only 1 of 15 mice showed a low specific IgE titer) when compared to mice exposed to empty VNP (7 of 13 mice produced IgE, P = 0.01) or PBS, respectively ( Figure 6B). In contrast, preventive treatment with Art v 1::GPI VNP could not completely protect from the induction of specific IgE in all animals (4 of 13 mice with allergenspecific IgE) ( Figure 6B and Figure S6A and B).
Notably, restimulation of lung homogenates derived from the four groups of mice with aqueous mugwort extract revealed a clear decrease in Th2-associated cytokines in mice which had been prophylactically treated with MA::Art v 1 VNP compared to those exposed to empty VNP ( Figure 6C). In contrast, levels of IFN-γ and IL-10 were found to be equal or even significantly elevated by preventive treatment with MA::Art v 1 VNP, indicative of a Th1/Tregprone immune response ( Figure 6C). Comparable data were obtained when lung T cells were restimulated with the immunodominant Art v 1 23-35 peptide ( Figure S6C). Spurred by these observations and cognizant that Tregs are key modulators of allergen-specific immune responses, [30][31][32]51 we considered that preventive treatment with MA::Art v 1 VNP may increase the proportion of lung-resident CD3 + CD4 + Foxp3 + Tregs. Due to the different endpoints measured in these experiments, increased sensitivity could be achieved by shorter intervals between allergen exposures and a final intratracheal booster with allergen. 52 Investigation of lung homogenates of mice prophylactically treated with VNP and challenged with aqueous mugwort pollen extract as indicated ( Figure 6D) showed significantly increased CD3 + CD4 + Foxp3 + Treg numbers in mice prophylactically treated with MA::Art v 1 VNP as compared to mice treated with empty VNP (Figure 6E and Figure S7A and B). Consistently, methacholine-induced lung resistance was lowest in mice prophylactically treated with MA::Art v 1 VNP ( Figure 6F). F I G U R E 3 Uptake and immunostimulatory capacity of allergen-expressing virus-like nanoparticles (VNP). A, Binding and uptake of fluorescent MA::mCherry VNP by the indicated splenic cell types. Open bars indicate positive cells at 4°C, filled bars show positive cells at 37°C. B, Shown is flow cytometry expression of CD40, CD80, CD86, and MHC class II (HLA DR1) on bone marrow-derived dendritic cells (BMDC) after co-incubation with MA::Art v 1 VNP, Art v 1:: glycosylphosphatidylinositol (GPI) VNP, empty VNP (all at 10 μg/mL), medium alone, rArt v 1 (1 μg/mL), aqueous mugwort pollen extract (100 μg/mL), or LPS (100 ng/mL) for 24 h. C, Shown are the cytokine levels of IL-1β, IL-6, IL-10, IL-12, IL-27, and TNF-α secreted by BMDC (2 x 10 5 /well in 200 μL) upon co-incubation with VNP expressing MA::Art v 1, Art v 1:: GPI, empty particles, rArt v 1 (1 μg/mL), medium alone, aqueous mugwort extract (100 μg/mL), or LPS (100 ng/mL) for 24 h. D, Shown is proliferation of splenocytes of TCR/DR1 mice incubated with MA::Art v 1 VNP, Art v 1::GPI VNP, empty VNP (10 μg/mL), rArt v 1, (0.5 μg/mL), medium alone, or PMA/ionomycin (10 −7 M PMA, 120 ng/mL, respectively) for 72 h followed by a 16-h methyl-[3H]-thymidine pulse (1 μCi/well). E, Shown is dose-dependent proliferation of splenocytes from TCR/DR1 tg mice incubated with increasing amounts of MA::Art v 1 VNP or Art v 1::GPI VNP. Data show the summary of three independent experiments (A-E) performed in triplicates. kcpm, kilo counts per minute; Kruskal-Wallis test followed by Dunn's correction (A, D, E) or one-way ANOVA followed by Tukey's correction (B, C). ns, not significant; **P<0.01; ***P<0.001 KRATZER ET AL.  can also be induced by inhaled antigens 59 and contributes to the homeostatic, anti-inflammatory role of lung-resident CD103 + DCs. 61 Moreover, VNP are not regarded as "dangerous" by inflammatory-type DCs, as no evidence for activation ( Figure 3B and Figure S2A-C) or secretion of inflammatory cytokines ( Figure 3C and Figure S3B) was found upon coculture of allergen-expressing or empty VNP with BMDC. In marked contrast, LPS regularly upregulated all four activation markers on DC and induced the secretion of a whole collection of inflammatory cytokines. This clearly indicated that VNP preparations are free of LPS and that the VNP backbone itself does not express activating DC ligands. The latter is not entirely surprising as HEK293 cells are used as producer cells, for example, for the production of γ-retroviruses applied for (experimental) gene transfer in humans. 62,63 Our MoMLV-based VNP are clearly different from the Qβ bacteriophage-based VNP described earlier. 64,65 While Qβ-based VNP express foreign antigens/allergens exclusively as surface-exposed proteins/peptides on nonenveloped particles, the here described VNP can accommodate both surface-exposed and shielded allergens.
We here fused full-length major allergens to the MoMLV MA p15, which generates a post-translationally lipid-modified MA::allergenfusion protein, robustly targeted to the inner leaflet of the lipid bilayer envelope of budding VNP. 25 This strategy yields VNP-containing major allergens, which are efficiently shielded from the environment and prevents not only their induction of allergen-specific immunoglobulin responses but also their recognition by preformed IgE bound to FcεRI and thus basophil degranulation. This is in contrast to Art v 1::GPI VNP and Qβ particles, which express surfaceexposed allergen accessible (although at reduced levels) by FcεRIbound IgE (Schmitz et al. 20 and Figure  prophylactically treated mice, they are considered inappropriate candidates for a prophylactic vaccine formulation. Why empty VNP ameliorated lung resistance to a certain degree remains to be elucidated in future experiments. One possibility entertained in the past for the beneficial impacts on allergic symptoms of CpG-rich VNP lacking allergen was their engagement of pattern recognition receptors on both innate and adaptive immune cells. 66 So far, we did not find evidence that VNP (empty or allergen-specific) would activate DC ( Figure 3B and Figure S2A-C); however, future studies will address that question in greater detail.
What could be the advantage of applying allergen-expressing VNP over artificial liposomes containing recombinant allergens for vaccination purposes? VNP copackage complement regulatory proteins, for example, CD55 and CD59 "borrowed" from HEK-293T cells ( Figure 2G and H During VNP generation, the processes of allergen expression and envelopment are separately regulated, making the introduced allergen sequences amenable to facile manipulation (eg, display hypoallergens or altered peptide ligands 68 ) and allowing for the introduction of further bioactive molecules (eg, regulatory proteins and/or (ribo)nucleic acids 69 ).
In summary, allergen-specific VNP expressing shielded cargo are novel formulations with high potential for allergen-specific prevention of IgE sensitization and may be used as prophylactic allergy vaccines.