Oral Administration of Lactococcus lactis Expressing Mite and Cockroach Major Allergens Alleviates Progression of Atopic March in a Mouse Model

Abstract

Purpose

Atopic march is defined as the development of atopic dermatitis in early childhood. We recently developed an atopic march mouse model through skin sensitization with aeroallergens from house dust mites and cockroaches. Using this model, this study aimed to evaluate the oral immunotherapy efficacy of Lactococcus lactis harboring specific antigens on the progression of atopic march.

Methods

Dust mite major allergen Der p 2 and cockroach Per a 2-372 were expressed in L. lactis as a fusion recombinant clone (D2P2). L. lactis-D2P2 was administered intragastrically to Aeroallergen patch-sensitized mice once a day for a total of 35 times. The immunological variables in sera, scratching behavior, airway hyperresponsiveness (AHR), and pathology of lungs and skin were evaluated.

Results

Our data showed that L. lactis-D2P2 significantly lowered total immunoglobulin E levels, decreased scratch bouts, and relieved AHR compared with the control mice. Histological analysis of the skin and lung tissue demonstrated the therapeutic effects of L. lactis-D2P2 to modulate immune responses via decreased eosinophil infiltration and reduced expression of key cytokines, interleukin (IL)-31 and IL-13, respectively.

Conclusions

The results imply that mucosal allergen-specific immunotherapy of L. lactis-D2P2 is a more cost-effective alternative to conventional subcutaneous allergen-specific immunotherapy. This study provides a promising platform for the development of novel oral protein-based vaccines in the early prevention of allergies.

INTRODUCTION

The term atopic march is used to describe the clinical features of patients who developed atopic dermatitis (AD) in early childhood, followed by immunoglobulin E (IgE)-mediated food allergies and allergic airway disorders.¹,² AD is one of the most common chronic inflammatory skin diseases, and it has a prevalence of 15%–30% in children and 2%–10% in adults worldwide.³,⁴,⁵,⁶,⁷ Various factors are involved in the pathogenesis of AD, including susceptibility genes, environmental factors, and immunological mediators.⁶ Stepwise treatment to optimize control of AD symptoms includes topical emollients to restore skin hydration, minimizing exposure to environmental triggers, and pharmacologic therapy, such as anti-inflammatory and biological agents blocking specific cytokines.⁵,⁸,⁹,¹⁰

Cutaneous exposure to proteolytic aeroallergens through activation of protease-activated receptor-2 (PAR2) and IgE binding can also initiate AD and promote allergy development. The most common aeroallergens responsible for AD are house dust mites and cockroaches.¹¹ House dust mites are one of the most important perennial allergen sources for airway allergy worldwide.¹²,¹³ Up to 80% of mite-allergic subjects have an IgE response to the 14 kDa allergen Der p 2, derived from Dermatophagoides pteronyssinus.¹⁴,¹⁵ Oral administration Der p 2 in Lactococcus lactis has been shown to protect mice from asthma¹⁶,¹⁷; however, the efficacy of this approach concerning AD is unknown. American cockroach, as another common aeroallergen in Taiwan,¹⁸,¹⁹,²⁰ is a potent sensitization inducer with 10- to 100-fold lower levels than other allergens to induce an IgE response.²¹ Among the identified 12 groups of cockroach allergens, sensitization to Per a 2 protein, which is derived from body and feces, correlated with more severe airway allergies.¹⁹,²⁰

Allergen-specific immunotherapy (AIT) or allergy vaccine is one of the therapeutic options for allergic diseases. Using recombinant allergens for vaccination has been shown to be more effective in inducing immunogenicity than using allergen extract, which contains both allergic and non-allergic components.²² In addition, since environmental allergens include many different substances, allergen extract vaccination may be challenging for patients affected by multiple allergens.¹⁷ To overcome the disadvantage, the fusion of several allergenic molecules for AIT may be a better choice to induce an immune response for each component. Thus, in this study, we constructed a L. lactis fusion clone D2P2 producing the major mite allergen Der p 2 and cockroach hypoallergen Per a 2-372 The therapeutic effects of this L. lactis-D2P2 on AD and the progression of atopic march were examined using an atopic march mouse model sensitized with Per a 2 or Der p 2.²³

MATERIALS AND METHODS

Construction of pNZ8149-Der p 2-Per a 2-372 (D2P2) fusion clone in L. lactis NZ3900 strain

The 773-bp fusion gene D2P2 comprised genes of Der p 2 from D. pteronyssinus (accession number: AM263560.1) and Per a 2-372 from Periplaneta Americana (accession number: GU188391.1) was cloned into the NcoI/XbaI sites of the pNZ8149 vector in-frame.²⁴,²⁵ The constructed plasmids were extracted, purified, and transformed into L. lactis strain NZ3900.

L. lactis-D2P2 fusion proteins analysis by Western blot

Cell lysates of 0.5-mL L. lactis-D2P2 culture were separated on 4-12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 2% bovine serum albumin (BSA) and then probed with rabbit anti-rPer a 2-372 or anti-rDer p 2 antibodies at 1μg/mL, followed by peroxidase-labeled goat anti-rabbit IgG antibody (10,000-fold dilution; Millipore, Burlington, MA, USA). The membrane was developed using an enhanced chemiluminescent solution (ECL) (Millipore), and the signals were recorded by exposure to G:BOX Chemi XX9 gel imaging system (Syngene, Cambridge, UK). The density of the protein bands was quantified using Gel-Pro image analysis software version 3.1 (Media Cybernetics, Rockville, MD, USA).

Preparation of L. lactis-D2P2 for oral administration

The L. lactis-D2P2 was cultured in a bench-top fermentor (Firstek, New Taipei, Taiwan).¹⁷ According to pre-experiments, the L. lactis-D2P2 fusion clone grown until an OD₆₀₀ of 0.2 was reached and induced by nisin (40 ng/mL; Sigma, St. Louis, MO, USA) for 5 hours. The harvested cells were monitored for protein expression by immune-detection with either lab-made rabbit anti-rPer a 2-372 or anti-rDer p 2 polyclonal antibodies for double confirmations.¹⁷,²⁴ The harvested cells for oral gavage were washed twice with sterile phosphate-buffered saline (PBS), aliquoted into 200 μL, and then stored at −80°C.

Preparation of Per a 2 and Der p 2 recombinant proteins for sensitization

Escherichia coli-expressed Per a 2 and Der p 2 recombinant proteins (E-rPer a 2, E-rDer p 2) were purified by rapid affinity column chromatography (Novagen, Darmstadt, Germany) as described previously.¹⁷,²³

Experimental design of mice sensitization and oral treatment with L. lactis-D2P2

Six-week-old female BALB/c mice from the National Laboratory Animal Center, Taiwan, were used for the experiments. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Taichung Veterans General Hospital (approval number: La-1101826). The sensitization and treatment schedule are summarized in Fig. 1. Briefly, back fur was removed with a depilatory cream. Then, 1 cm² gauze pads containing recombinant allergens Per a 2 (Experiment I) or Der p 2 (Experiment II) in 100 μL of PBS were applied to the shaved skins and patched with a transparent dressing (3M HealthCare, Bracknell, UK). The patches were placed for 1 week and then removed. Two weeks later, an identical patch was reapplied onto the same skin site. Each mouse had a total of three 1-week exposures at 2-week intervals. Between days 8–55, mice from the 2 groups, Per a 2-D2P2 (Experiment I) and Der p 2-D2P2 (Experiment II), received intragastric (IG) administration of 200 μL of L. lactis-D2P2 once a day on weekdays for a total of 35 times. Mice in the Per a 2-DEX group were subcutaneously (SC) injected with dexamethasone (2.5 mg/mL) twice a week for 3 weeks as the therapeutic positive control. On days 50, 53, and 55, all of the mice were challenged both intradermally (ID) and intratracheally (IT) with allergens E-rPer a 2 (Experiment I) or E-rDer p 2 (Experiment II), respectively. On days 55 and 56 after final IT and ID challenges, airway hyperresponsiveness (AHR) and skin scratching behavior were assessed, respectively. Control mice (Ck-PBS group) received PBS at the time of patching and challenges. Serum samples were collected from the submandibular vein bi-weekly and stored at −20°C until analysis. All mice were sacrificed on day 56, and skin, lung, and spleen were removed for further study.

Fig. 1. Experimental design of allergen-induced atopic march in a murine model. Mice were sensitized via skin with E. coli-expressed Per a 2 (Experiment I) and Der p 2 (Experiment II) in 100 μL PBS to a sterile patch. The patches were placed for a 1-week period and then removed. Each mouse had a total of three 1-week sensitizations at 2-week intervals. From days 8–55, mice of groups Per a 2-D2P2 and Der p 2-D2P2 received IG administration of L. lactis-D2P2 once a day on weekdays for 7 weeks. Mice of Per a 2-DEX group were SC injected with dexamethasone twice a week for 3 weeks as a therapeutic positive control. On days 50, 53, and 55, the mice were challenged both ID and IT, and sacrificed on day 56.

PBS, phosphate-buffered saline; IG, intragastric; SC, subcutaneously; ID, intradermally; IT, intratracheally.

Measurement of total IgE

Total IgE levels were measured using an IgE mouse ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Ninety-six-well plates (Nunc) were coated with 5 μg/mL monoclonal antibody overnight at 4°C. After washing twice with PBS/0.05% Tween 20, the plates were blocked with PBS plus 1% BSA for 2 hours and then incubated for 2 hours with 1:10 diluted sera or 2-fold serial dilutions of mouse IgE standards. After washing, the detection antibody diluted at 1:250 was added and incubated for an additional 2 hours. The plates were washed, and streptavidin-horseradish peroxidase conjugate (1:400) was subsequently added for 30 minutes. After final washing, tetramethylbenzidine (TMB) was added for 10 minutes, and the reaction was stopped with 1 M phosphoric acid. The relative concentration of total IgE was measured at OD₄₅₀ for serum samples and control from the standard curve.

Scratching behavior

The scratching behaviors were videotaped for 1 hour after the final allergen by ID challenge. Counts of scratching around the ID sites were recorded using video playback as described previously.²⁶

Histological analysis of skin lesions

The skin lesions from sacrificed mice were removed immediately. The excised skin specimens were fixed in 10% neutral formalin and embedded in paraffin. Five μm-thick sections were stained with hematoxylin and eosin (H&E) to analyze the inflammatory infiltrate. All slides were examined under a Hamamatsu NanoZoomer 2.0 HT slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) at 400-fold magnification.

Measurement of AHR

The airway resistance of mice was measured using a whole-body Buxco mouse plethysmograph (Buxco Electronics, Troy, NY, USA) on day 55 after the last IT challenge. Mice were placed in the main chamber and challenged with aerosolized methacholine at 0 and 50 mg/mL concentrations generated by a nebulizer (Buxco aerosol distribution system). The degree of bronchoconstriction was measured and averaged for 3 minutes after each nebulization. Data were expressed as enhanced pause (Penh) by the following equation: Penh = Pause × (PEP/PIP). Pause, PEP, and PIP refer to the expiration time, peak expiratory pressure, and peak inspiratory pressure, respectively.

Quantitative real-time polymerase chain reaction (PCR)

Total RNA was extracted from the skin lesions and lungs using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s recommendation. cDNA synthesis was prepared with a SuperScript III kit (Invitrogen) using isolated RNAs and an oligo (dT) primer. Quantitative PCR was performed on a StepOnePlus system (Applied Biosystems, Foster City, CA, USA) to measure the expression of cytokines, using the primer sequences listed in Table. Data were imported into an Excel database and expressed as 2^−ΔCt after normalizing by the housekeeping gene β-actin.

Table. The sequences of gene-specific primers for mice used in real-time PCR.

Gene (mouse)		Sequences	Product size (bp)
IL-13			123
	F	5′ AGA CCA GAC TCC CCT GTG CA 3′
	R	5′ TGG GTC CTG TAG ATG GCA TTG 3′
IFN-γ			118
	F	5′ GGC CAT CAG CAA CAA CAT AAG CGT 3′
	R	5′ TGG GTT GTT GAC CTC AAA CTT GGC 3′
IL-31			121
	F	5′ CAG CTG TTT CAA CCC ACT G 3′
	R	5′ CAG TTC TGC CAT GCA GTT TG 3′
β-actin			251
	F	5′ GGC CAA CCG TGA AAA GAT GA 3′
	R	5′ CAC GCT CGG TCA GGA TCT TC 3′

PCR, polymerase chain reaction; IL, interleukin; IFN, interferon.

Statistical analysis

Statistical analysis was performed with SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA) using the Bonferroni multiple range test. P-values less than 0.05 were considered to be significant.

RESULTS

Expression and quantification of the D2P2 fusion proteins in recombinant L. lactis

The food-grade expression vector pNZ8149 was chosen to design the constitutive expression of fusion protein D2P2 in L. lactis. The constructed plasmid was electrotransformed into L. lactis NZ3900, which was then plated onto Elliker plates. A total of 10 lactose-positive colonies were selected and confirmed by restriction enzyme digestion, as shown in Supplementary Fig. S1. The isolated pNZ8149-D2P2 clone was confirmed by DNA sequencing using an automated DNA analyzer (ABI Prism 3700) (Supplementary Fig. S2). The successful expression of the 28.4-KDa fusion protein via nisin induction in the engineered L. lactis NZ3900 was evaluated by Western blot using the anti-Der p 2 (Fig. 2A) and anti-Per a 2-372 (Fig. 2B) polyclonal antibodies, respectively. We used the extract of L. lactis NZ3900 as a negative control, and no band was observed in Fig. 2. The maximal protein expression level of recombinant D2P2 clone was about 225 ng/mL observed at 40 ng/mL of inducer nisin for 5 hours.

Fig. 2. Immunoblot analyses of protein expression of recombinant pNZ8149-D2P2 L. lactis. After 40 ng/mL of nisin induction for 5 hours, the fusion protein of D2P2 was immune-detected with lab-made (A) rabbit anti-Der p 2 or (B) anti-Per a 2-372 polyclonal antibodies. The D2P2 protein was indicated by the arrow. Lane NZ3900 was cell lysate from L. lactis NZ3900 with pNZ8149 as a negative control. E. coli-expressed recombinant Der p 2 (E-rDer p 2) and E-rPer a 2-372 were used as semi-quantified standards for fusion protein D2P2.

The effects of oral L. lactis-D2P2 on serum total IgE and scratching behavior in sensitized mice

One of the key immunological features of allergy is a high level of serum total IgE. Fig. 3A revealed that the total IgE concentrations of Per a 2 or Der p 2-sensitized groups (Ck-Per a 2 and Ck-Der p 2) on week 8 were elevated significantly compared with the control groups (Ck-PBS or Ck-sentinel). Administration of engineered L. lactis-D2P2 resulted in a significant decrease in the total IgE in both therapeutic groups, which had similar IgE levels compared to Per a 2-DEX mice treated with dexamethasone injection.

Fig. 3. Effects of oral L. lactis-D2P2 on serum total IgE and scratching in sensitized mice. (A) Changes in serum levels of total IgE and (B) Scratching bouts of mice from Experiments I and II, respectively. The scratching counts were recorded for 20 minutes after induction by intradermal injection of PBS, rPer a 2 (Experiment I), and rDer p 2 (Experiment II). Data are mean ± standard error of the mean of 6 mice.

IgE, immunoglobulin E; PBS, phosphate-buffered saline.

Denoted ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by one-way analysis of variance with the Bonferroni multiple range test.

AD is a serious skin disorder that is characterized clinically by intense itching and scratching, leading to eczematous lesions. Fig. 3B reveals that both the L. lactis-D2P2 and Per a 2-DEX groups had significantly decreased scratch bouts at the challenge sites compared with the control mice.

The effects of oral L. lactis-D2P2 on Per a 2/Der p 2-induced AD-like skin lesions

AD skin lesions are characterized by histological analysis for dermal infiltration of inflammatory cells. H&E staining showed epithermal hyperplasia and accumulation of inflammatory cells in the dermis of the Per a 2/Der p 2-patched groups compared to the PBS group (Fig. 4A and B, left panel). We found that repeated oral feeding of L. lactis-D2P2 significantly decreased eosinophil and total cell counts in comparison with the sensitized groups, respectively (Fig. 4A and B, right panel), and showed equal efficacy compared to the steroid group of Per a 2-DEX.

Fig. 4. H&E staining and the cell counts of infiltrating inflammatory cells in skin lesions from Experiments I (A) and II (B). (C) Expression levels of IL-31 mRNA in skin lesions from both experiments by real-time PCR. Data are mean ± standard error of the mean of 6 mice.

H&E, hematoxylin and eosin; IL, interleukin; PCR, polymerase chain reaction.

Denoted ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by one-way analysis of variance with the Bonferroni multiple range test.

Interleukin (IL)-31 overexpressed in the skin might contribute to the development of AD-induced skin inflammation and pruritus.²⁷ Fig. 4C shows that mice treated with steroid or L. lactis-D2P2 exhibited a marked decrease in mRNA expression of IL-31 in skin lesions compared with controls-Per a 2/Der p 2, respectively.

The effects of oral L. lactis-D2P2 on Per a 2/Der p 2-induced AHR and inflammation of lungs

Dexamethasone is the currently recommended systemic steroid for moderate to severe asthma exacerbations. We aimed to compare the therapeutic efficacy of oral L. lactis-D2P2 with steroid injection against murine asthma induced by cutaneous exposure to cockroaches or mite allergens. After the final IT challenge, mice were treated with methacholine aerosol, and Penh was calculated. As shown in Fig. 5, mice from groups of Ck-Per a 2 and Ck-Der p 2 showed significantly increased Penh upon methacholine exposure. Mice from the L. lactis-D2P2-fed groups showed a significant reduction of Penh, which was as effective as a dexamethasone injection.

Fig. 5. Effects of oral L. lactis-D2P2 on AHR in mice. Mean enhanced pause (Penh) values were evaluated at 50 mg/mL of methacholine in mice from both experiments. Data are expressed as the mean ± standard error of the mean of 5 mice from each group.

AHR, airway hyperresponsiveness; PBS, phosphate-buffered saline.

^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by one-way analysis of variance with the Bonferroni multiple range test.

Following the measurement of airway resistance, lungs were fixed, sliced, and stained by H&E to reveal histological changes in the mice. Both the Ck-Per a 2 and Ck-Der p 2 groups showed marked inflammatory infiltration into the lungs, as shown in Fig. 6, respectively. However, the L. lactis-D2P2-fed groups showed a significant decrease in eosinophils and total cell counts. IL-13 is a key driver of Th2 allergic inflammation and IgE production. Significant down-regulation of IL-13 mRNA expression was detected in the L. lactis-D2P2-fed groups (Fig. 7A). In contrast, Th1 cytokine interferon-γ showed no difference in all of the groups of mice (Fig. 7B). Taken together, oral administration of L. lactis-D2P2 appeared to significantly alleviate lung inflammation in terms of both Per a 2 and Der p 2-sensitization in skin patch murine models.

Fig. 6. H&E staining and the cell counts of infiltrating inflammatory cells in lungs from Experiments I (A) and II (B). Data are expressed as the mean ± standard error of the mean of 5 mice from each group.

H&E, hematoxylin and eosin; PBS, phosphate-buffered saline.

^**P < 0.01, ^***P < 0.001 by one-way analysis of variance with the Bonferroni multiple range test.

Fig. 7. Expression levels of mRNA for cytokines IL-13 and IFN-γ in lungs of mice from Experiments I (A) and II (B) by real-time PCR. Data are expressed as the mean ± standard error of the mean of 5 mice from each group.

IL, interleukin; IFN, interferon; PCR, polymerase chain reaction; ns, not statistically significant.

^*P < 0.05, ^**P < 0.01 by one-way analysis of variance with the Bonferroni multiple range test.

DISCUSSION

In this study, we successfully demonstrated the expression of a recombinant D2P2 fusion protein of the major allergens of European dust mite (Der p 2) and American cockroach (Per a 2) in L. lactis. Our data showed that oral administration with L. lactis-D2P2 significantly lowered total IgE levels and scratching behavior, and reduced AHR in mite- and cockroach-sensitized mice. Histological analysis of the skin and lung tissue samples demonstrated the ability of L. lactis-D2P2 to decrease eosinophil and total infiltrated cell counts. This immune modulation was also evidenced by reducing expression levels of key cytokines, such as IL-31 and IL-13. Notably, the observed effects were similar to those achieved with dexamethasone, a glucocorticoid commonly employed for treating various allergic inflammation. We found evidence suggesting that the therapeutic effects of L. lactis-D2P2 were induced by the corresponding recombinant D2P2 proteins that were engineered, but not by L. lactis alone, as demonstrated in our previous study.¹⁷ These results imply that L. lactis-D2P2 has considerable potential as a therapeutic agent for allergy-related conditions.

The therapeutic potential of oral probiotics on allergic animal models has been studied previously.²⁸,²⁹,³⁰ However, this is the first study to use a skin sensitization model that mimics the atopic march to investigate the effects of food-grade engineered Lactococcus containing corresponding allergens. The successful expression of the 28.5-KDa fusion protein D2P2 in recombinant L. lactis, as confirmed by Western blot, highlights the feasibility of using food-grade expression vectors, such as pNZ8149, for constitutive expression of recombinant proteins. Zhai et al. recently reported the use of L. lactis to deliver recombinant proteins containing PDCoV-S1 and PDCoV-S1-Co1, the antigens of porcine delta-coronavirus (PDCoV), and demonstrated its potential as a vaccination option for PDCoV in piglets.³¹ Furthermore, Zhu et al. successfully used engineered L. lactis secreting Flt3L and OX40 ligand as a vaccine for in situ cancer immunotherapy.³² These approaches offer a promising platform for the development of novel oral protein-based vaccines.

To simulate the conditions that occur in human disease, our animal model was first sensitized by a skin patch with allergens to imitate early childhood exposure through a disrupted skin barrier, followed by administering D2P2 to prevent asthma. The results of this study provide us with an alternative approach to the early prevention of atopic dermatitis, which might stop the subsequent development of respiratory allergies. However, the ability to prevent both allergic diseases by administering D2P2 during early childhood is not fully verified, given the uncertainty in predicting the exact timing of sensitization due to the amount and duration of allergen exposure and other immune regulatory factors in humans.

Our previous study provided evidence supporting that epicutaneous sensitization to aeroallergens also led to nasal and airway symptoms comparable to atopic march as described in humans.²³ In this study, allergen exposure through a disrupted skin barrier provided the initial sensitization in the animal model, followed by allergen-specific oral immunotherapy. It is effective in inducing tolerance in systemic sera (total IgE), skin (scratch bouts, eosinophils, anti-inflammation), and airways (AHR, lung). However, as a study limitation, we did not test the mechanism of this allergen-specific oral immunotherapy. A unique feature of the oral route is the large surface of contact of the gut mucosa, and it lodges immune elements. Further studies will focus on exploring the mechanism by analyzing RNA sequencing of intestinal Peyer patches to identify possible therapeutic targets.

Despite the promising results, several limitations should be acknowledged in this study. First, the long-term effectiveness of the L. lactis-D2P2 was not evaluated in this study. It would be advantageous to assess the long-term safety and efficacy of L. lactis-D2P2, in addition to conducting an in-depth exploration of its mechanism of action, especially concerning mucosal immunology. Gaining a precise understanding of the immunological basis of the observed effects could inform efforts to optimize this therapeutic approach and it may provide insights into the development of other allergy-related treatments. Secondly, it would be valuable to investigate the efficacy of L. lactis-D2P2 in other pre-clinical models, such as larger animals, to determine its translational potential. Thirdly, further studies could evaluate the optimal dosing regimen for achieving maximal therapeutic effects. Finally, clinical trials in human subjects will be essential to confirm the safety and efficacy of L. lactis-D2P2 in treating allergy-related conditions. These trials could also explore the potential for individual variations in response to treatment and may inform strategies for tailoring the therapy to specific patient populations.

In conclusion, our study presents compelling evidence supporting the potential of L. lactis-D2P2 as a novel therapeutic agent for allergy-related conditions in an atopic march mouse model. Our study also demonstrated that a single oral food-grade Lactococcus vaccine could alleviate subsequent airway and skin allergic inflammation. These findings warrant further investigation into the long-term safety, efficacy, and mechanism of action of this promising treatment, intending to improve the quality of life for individuals suffering from allergies and related disorders.

SUPPLEMENTARY MATERIALS

Supplementary Fig. S1

Agarose gel electrophoresis of pNZ8149-D2P2 recombinant plasmids double-digested with NcoI and XbaI. The numbers at left indicate the size of standard DNA fragments in kilobase pairs (kb). Lanes 1-10, clones 1-10.

Supplementary Fig. S2

(A) The modified nucleotide and (B) deduced amino acid sequences of a novel D2P2 clone. The DNA sequences of D2P2 included 389-bp of Der p 2, 372-bp of Per a 2, and an extra 12-bp cloning sites. The theoretical protein of pNZ8149-D2P2 in L. lactis NZ3900 consists of 265 amino acids, with pI and MW of 6.29 and 28.42 kDa, respectively.