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. 2020 Apr 23;15(4):e0232042. doi: 10.1371/journal.pone.0232042

Therapeutic DNA vaccine attenuates itching and allergic inflammation in mice with established biting midge allergy

Mey Fann Lee 1,#, Yi-Hsing Chen 2,3,4,*,#, Pei-Pong Song 1, Tzu-Mei Lin 2
Editor: Junji Xing5
PMCID: PMC7179863  PMID: 32324792

Abstract

Forcipomyia taiwana is a tiny hematophagous midge that attacks en masse. It is responsible for the most prevalent biting insect allergy in Taiwan. For t 2 is its major allergen. The intense itchy reactions can prevent allergic individuals from performing their regular daily outdoor activities. This study aimed to investigate whether the For t 2 DNA vaccine was effective in treating mice with established biting midge allergy. Mice were sensitized with recombinant For t 2 proteins or whole midge extracts. Two to four consecutive shots of various concentrations of For t 2 DNA vaccine, with or without CpG adjuvants, were then administered to midge-sensitized mice. Mice that received two shots of 50–100 μg For t 2 DNA vaccine showed a significant reduction in allergen-induced bouts of scratching, For t 2-specific IgE, specific IgG1/IgG2a ratio in sera, skin eosinophil infiltration, and IL-31 production, as well as IL-4 and IL-13 production by splenocytes. Two doses of For t 2 DNA vaccine one week apart was sufficient to treat mice with established biting midge allergy. The treatment resulted in clinical, immunological, and histopathological improvements. We recommend that this low-cost, convenient treatment strategy be developed for use in humans who are allergic to biting midges.

Introduction

The biting midge, Forcipomyia taiwana, is a very small (1–1.5 mm) blood-sucking insect, which is widely distributed throughout Taiwan and southern China [1]. It is the most prevalent biting insect allergy in Taiwan, and as many as 60% of exposed individuals develop intense itchy reactions to the bites; 58–64% of individuals are sensitized to its major allergen, For t 2 [2–4]. The hematophagous genus of Forcipomyia has been reported around the world, including Singapore, Poland, Italy, North India and Mexico [5–9]. Unlike mosquitos or other midges, F. taiwana do not transmit infectious diseases to human subjects through biting and blood sucking [10], however they can cause annoying, intense, itchy allergic reactions. People with allergic reactions to insects frequently avoid normal outdoor activities, which may negatively impact their quality of life [11–13]. Allergic reactions to the bites of members of the midge family are not isolated to humans, they also occur in other animals, such as horses [14].

The major allergen of F. taiwana is For t 2; it has a 65–77% overlap with the eukaryotic translation initiation factor 3 subunit of many insects. The For t 2 protein not only binds with serum IgE in patients allergic to midges, it also induces the production of key inflammatory chemokines from skin fibroblasts in a concentration-dependent manner [3].

A previous study by the authors showed that a DNA vaccine encoding the For t 2 midge allergen was able to prevent the development of allergic skin inflammation induced by the biting midge allergen in a mouse model [15,16]. However, as biting midge allergy is so prevalent in Taiwan, a large scale preventive vaccination does not seem to be practical in the real world. A vaccine that is able to treat patients with an established midge allergy is more clinically relevant.

The present study aimed to investigate whether the For t 2 DNA vaccine was effective in treating mice with an established biting midge allergy.

Materials and methods

Expression and purification of the recombinant For t 2 from E. coli for sensitization

Plasmid pQE30 containing For t 2 coding sequences was transformed into Escherichia coli strain M15, the protein expression was performed as previously described [3]. After a His-tag affinity column, the E.coli-expressed For t 2 recombinant protein (E-rFor t 2) was further purified using Endotoxin Detoxi-Gel (Pierce, Rockford, IL, USA) and passed through a 0.22 μm syringe filter (Millipore, Billerica, MA, USA) as previously described [15]. The endotoxin content was determined using an E-TOXATE kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The lowest detection limit of the test was 0.05 endotoxin units per ml.

Preparation of pCI-For t 2 plasmid for vaccination

The eukaryotic expression plasmid pCI-For t 2 was propagated in E. coli DH5α and large-scale purification was performed with the EasyPrep EndoFree Maxi Plasmid Extraction kit according to the manufacturer’s instructions (Tools, New Taipei City, Taiwan). The expression level of pCI-For t 2 was confirmed in mammalian cells by transfecting the human epidermal keratinocyte cell line HaCaT (ATCC, Manassas, VA, USA) with 2.5 μg of the plasmid using Lipofectamine 2000® (Invitrogen: Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were cultured for 24 h and then the supernatants from the transfected cells were examined by western blot analysis using rabbit anti-E-rFor t 2 polyclonal antibodies.

Experimental design of therapeutic DNA vaccination

Female 6-week-old BALB/c mice were purchased from the National Laboratory Animal Center, Taiwan and raised under specific pathogen-free conditions. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Taichung Veterans General Hospital (approval no. LA1051380).

A total of 30 mice were sensitized with 2 intraperitoneal injections (IP) of 10 μg E-rFor t 2 absorbed by 2 mg alum adjuvant, with a 1-week interval between each injection. On days 14 and 21 the three groups of vaccinated mice (V50, V100 and V200) received intra-muscular (IM) injections of the For t 2 DNA vaccine at doses of 50, 100 and 200 μg, respectively. The not-vaccinated and vector only (VO) groups received IM injections of PBS and 100 μg pCI-neo plasmid DNA as the controls. All groups of mice were challenged intra-dermally (ID) with 3 doses of 1 μg E-rFor t 2 for 3 consecutive days between days 59–61. Blood samples were collected bi-weekly from the retro-orbital venous plexus. The scratching behaviors of the mice were video recorded on days 0 and 61, and the mice were sacrificed on day 63.

Same protocol was performed for the second set of experiments using mice sensitized with crude midge extracts as well as experiments testing effects of CpG adjuvants.

ELISA for the measurement of For t 2-specific antibodies in the sera

The titer of anti-For t 2 IgE, IgG1 and IgG2a antibodies in the mice sera was detected using ELISA. 96-well ELISA plates (NUNC MaxiSorp, Thermo Fisher Scientific, Inc.) were coated with 0.5 μg E-rFor t 2/well in a coating buffer and ELISA was performed as previously described [15].

Evaluation of mouse scratching behavior immediately following an intra-dermal challenge

On days 0 and 61, the scratching behaviors of the mice were video recorded for 1 h starting immediately after the intra-dermal challenge with E-rFor t 2. Counts of scratching were made using video playback. The observation of scratching behavior was blinding measured and performed as previously described [15,16].

Histological examination of delayed reactions

On day 63 the mice were sacrificed and the abdominal skin from the challenge sites was removed and fixed in 10% formalin solution. Briefly, the tissues were embedded in paraffin, cut into 5-μm-thick sections and then stained with either hematoxylin and eosin (H&E) to examine cell infiltration, or immunostained with rabbit anti-mouse CD4 (800-fold dilution; Bioss, MA, USA) or IL-31 (100-fold dilution; Abcam, Cambridge, UK) polyclonal antibodies for the analysis of T cells. Endogenous peroxidases were blocked using peroxide for 30 min at room temperature. To detect positive cells the sections were incubated with peroxidase-conjugated goat anti-rabbit IgG and visualized using diaminobenzidine solution using the Bond automatic system (Leica, Newcastle, UK). Inflammatory cell infiltrates were examined by light microscopy and corresponding images were captured using an Olympus BX51 microscopic/DP71 Digital Camera System (Nagano, Japan).

Measurement of in vitro cytokine production by For t 2-treated splenocytes

Splenocytes from the experimental mice were harvested on day 61 (48 h after the final challenge) and processed to form a single-cell suspension. Cells were cultured in 24-well flat-bottomed plates at a concentration of 1×106/ml and stimulated with 1 μg/ml E-rFor t 2 at 37°C for 3–5 days. The culture supernatants were collected at each time interval and stored at -20°C until required for the cytokine assay. The levels of IL-4, IL-10, IL-13 and interferon (IFN)-γ in the culture supernatants were measured using mouse ELISA kits (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.

RNA preparation and quantitative reverse transcription (qRT)-PCR of For t 2-treated splenocytes

Predesigned primer sequences [16] were used to measure the levels of cytokine mRNA expression. Briefly, on day 3 the total RNA from E-rFor t 2-stimulated splenocytes was purified using the Invitrogen TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc.). cDNA was obtained from 1 μg total RNA using a SuperScript III kit (Life Technologies). The reaction mixtures were amplified with SYBR Green PCR master mix (Life Technologies) in the presence of 0.2 μM of each of the specific primer sets. PCR amplification was conducted with an initial 10-min step at 95°C followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min using the StepOnePlus™ system (Applied Biosystems, CA, USA). The fluorescence signal was detected immediately after the extension step of each cycle, and the cycle at which the product was first detectable was recorded as the cycle threshold. Data were imported into an Excel database and analyzed using the comparative cycle threshold method with normalization of the raw data to β-actin.

Statistical analysis

Data were presented as the mean ± standard error of the mean, and statistical analyses were performed using IBM SPSS Statistics software version 22 (IBM Corporation, Armonk, NY, USA) with appropriate methods. P-values <0.05 were considered to indicate a statistically significant difference.

Results

Mouse model and in vitro expression of the For t 2 DNA vaccine

Therapeutic schedule of For t 2 DNA vaccination on E-rFor t 2-sensitized mice is shown in Fig 1A. The endotoxin content of E-rFor t 2 after removing gel was under the detection limit (0.05 U/ml) of the Limulus Amebocyte Lysate test.

Fig 1. Experimental procedure.

Fig 1

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(A) Therapeutic regimen of For t 2 DNA vaccination in E-rFor t 2-sensitized mice. BALB/c mice received 2 intra-peritoneal injections of E-rFor t 2/alum on days 0 and 7. Subsequently, the sensitized mice were treated intra-muscularly with For t 2 DNA vaccine on days 14 and 21. From days 56 to 61 the mice were challenged intra-dermally (ID) with 3 doses of E-rFor t 2 on 3 consecutive days. Blood samples were collected bi-weekly from the retro-orbital venous plexus. The scratching behaviors of the mice were videotaped at days 0 and 61. All experimental mice were sacrificed on day 63. (B) Western blot analysis of culture supernatant from HaCaT cells transfected with empty vector or pCI-For t 2 using rabbit anti-E-rFor t 2 antibodies. E. coli-expressed recombinant protein was seen at 36 kDa as detected in the positive control.

The full-length cDNA of For t 2 was cloned in the pCI-neo vector and the expression level of the construct was confirmed in HaCaT cells (a keratinocyte cell line from adult human skin). Culture supernatants from the transfected cells after 24 h were collected and analyzed via western blotting using a E-rFor t 2-specific antibody. The purity of E-rFor t 2 and the specificity of lab-made rabbit anti-E-rFor t 2 antibody were demonstrated in S1 Fig and described previously [3,15]. The expression of the 36 kDa For t 2 protein was detected and is shown in Fig 1B. No For t 2 protein was seen in the control sample transfected with the empty vector.

Down-regulation of For t 2-specific IgE production and the ratio of IgG1/IgG2a after therapeutic For t 2 DNA vaccination

In the dose-response experiments, two consecutive shots of For t 2 DNA vaccine were given to E-rFor t 2-sensitized mice. ELISA was used to determine the effects of the DNA vaccine on the allergen specific-IgE, -IgG1 and -IgG2a levels in the sera on day 63. E-rFor t 2-induced specific IgE antibodies reduced by 40–56% in the V50, V100 and V200 groups compared with either the not-vaccinated group or the VO group (Fig 2A). However, only the change in the V50 group was statistically significant when compared with the VO group. For t 2-specific IgG2a, a surrogate maker of successful allergen-specific immunotherapy in mice, was elevated in all treatment groups in compared with not-vaccinated and VO groups, but no significant difference (Fig 2B). However, a significant decrease in the ratios of IgG1/IgG2a was observed in all For t 2 DNA-vaccinated groups compared with the VO control group (Fig 2C).

Fig 2. Serum levels of For t 2-specific antibodies on week 9 as determined by ELISA.

Fig 2

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(A) IgE, (B) IgG2a and (C) the ratio of IgG1/IgG2a. The statistics show comparisons between the nonvaccinated or vector only groups and the DNA vaccine groups by one-way analysis of variance with Bonferroni multiple range test. *p<0.05, ***p<0.001; NS, not statistically significant.

Therapeutic For t 2 DNA vaccine ameliorates scratching bouts induced by midge allergen challenge

Post-sensitization vaccination was administered to all groups of mice and comprised 3 ID challenges with E-rFor t 2 protein during days 56–61. After the final challenge, the scratching behaviors (the surrogate marker of an itch) were immediately videotaped for 1 h. In the not-vaccinated group and the VO group, the mean number of scratching bouts was >300 in the initial 20 min (Fig 3). All For t 2 DNA vaccine-treated groups showed significantly fewer scratching bouts on the challenge sites during the first 20 min compared with both the not-vaccinated group and the VO group. Treatment of For t 2-sensitized mice with the For t 2 DNA vaccine significantly inhibited the incidence of skin itching, which is the most annoying problem for human allergy patients.

Fig 3. The number of scratching bouts induced in mice following administration of E-rFor t 2 by intradermal injection.

Fig 3

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The statistics show comparisons between the not-vaccinated and VO groups with the DNA vaccine groups using Dunnett’s t-test. ***p<0.001; NS, not statistically significant.

Effect of the For t 2 therapeutic DNA vaccine on cytokine gene expression and the production of rFor t 2-stimulated splenocytes

The spleen plays an important role in humoral and cellular immune responses. It contains a variety of immune cells which provide the organ with a cytokine-rich environment [17]. Splenocytes were harvested from all groups of mice and stimulated with rFor t 2 allergen to determine whether Th1 or Th2 cytokines were affected by the For t 2 DNA vaccination. The cytokine mRNA expression on day 3 and the protein levels on day 5 in response to rFor t 2 were determined using qRT-PCR and ELISA, respectively. Significant down regulation of IL-13 mRNA expression and protein secretion was only detected only in the V50 group compared with the not-vaccinated group (Fig 4A). There was a marked decrease in IL-4 mRNA expression in the V50 and V100 groups compared with the nonvaccinated group. The protein levels of IL-4 were >500 pg/ml in the not-vaccinated group. All DNA vaccine-treated groups produced extremely low amounts of IL-4 protein, which were below the detection limits (Fig 4B). The mRNA level of IL-10 was only significantly increased in the V50 group compared with the nonvaccinated group, and there were no significant differences in IL-10 protein secretion between all groups (Fig 4C). The levels of IFN-γ mRNA and protein expression showed no significant changes between all groups (Fig 4D).

Fig 4. Cytokine protein expression and the mRNA levels of splenocytes from each group of mice as determined by ELISA and qRT-PCR, respectively.

Fig 4

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(A) IL-13, (B) IL-4, (C) IL-10 and (D) IFN-γ. Cytokine release data are shown as the mean ± SEM from 3 independent experiments. qRT-PCR data are expressed as the mean fold increase ± SEM from 3 independent experiments. The statistical significance of differences among the groups was assessed by one-way analysis of variance with Dunnett’s t-test. *p<0.05; **p<0.01; NS, not statistically significant; SEM, standard error of the mean.

Effect of the For t 2 therapeutic DNA vaccine on delayed skin reactions

Skin tissue from the challenged sites was harvested 48 h after the final ID challenge with the E-rFor t 2 allergen, and paraffin sections were prepared for histopathology. Fig 5A and 5B shows that the total number of infiltrate cells, mainly the eosinophils, decreased significantly in the V50, V100 and V200 groups compared with the nonvaccinated and VO groups. IL-31 is known to be one of the key cytokines that induces itching and promotes scratching in mouse models of atopic dermatitis [18]. As shown at Fig 5C, expression of intradermal IL-31 was significantly decreased in all of the DNA vaccinated groups, including the VO group, compared with the nonvaccinated group. However, the reduction of IL-31 is more significant in the V50, V100 and V200 groups compared with the VO group.

Fig 5. Skin histopathology of mice from different groups after challenge with rFor t 2 allergen.

Fig 5

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(A) Representative sections of the abdominal skin was obtained 48 h after the intradermal challenge test and stained with H&E or with rabbit anti-mouse CD4 and IL-31 antibodies. The sections were observed under a 400x light microscope. (B) The infiltrating inflammatory cells were quantified in the H&E-stained skin. The results are expressed as the mean ± SEM of the cell number per square mm of skin specimen. (C) The intensity levels of IL-31 in the epidermis from (A) as determined by Gel-Pro image software. The statistical significance of differences among the groups was assessed by one-way analysis of variance with Dunnett’s t-test. *p<0.05, **p<0.01, ***p<0.001; NS, not statistically significant.

Therapeutic effects of the For t 2 DNA vaccine in crude midge extract-sensitized mice

In a second set of experiments (n = 6 per group), the mice were sensitized with crude midge extracts (containing all midge allergens) and then treated with the For t 2 DNA vaccine (Fig 6A). Administration of the For t 2 DNA vaccine at 100 or 200 μg ameliorated the midge allergen-induced scratch bouts (Fig 6B), as well as midge allergen-induced IL-13 production from splenocytes (Fig 6C), and inflammatory cell infiltration in the lesions 48 h after ID challenge.

Fig 6. Therapeutic effects of For t 2 DNA vaccines on crude midge extract-sensitized mice.

Fig 6

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(A) Experimental groups of therapeutic For t 2 DNA vaccinations on crude midge extract-sensitized mice. (B) Scratching bouts induced by intradermal challenge of crude midge extract. (C) IL-13 protein levels of midge extract stimulated-splenocytes from each group of mice, as determined by ELISA. The statistical significance of differences among the groups was assessed by one-way analysis of variance with Dunnett’s t-test. *p<0.05, ***p<0.001.

Effects of the booster doses and CpG adjuvant (TLR-9 ligand) on the For t 2 therapeutic DNA vaccine

An additional experiment was designed to investigate whether additional booster doses (increasing the number of injections from 2 doses to 4 doses), or adding CpG adjuvant (TLR-9 ligand) had beneficial therapeutic effects on the For t 2 DNA vaccine. It was found that after vaccination, decreasing levels of For t 2-specific IgE and increasing levels of specific IgG2a were only observed in group V50-2 (2 doses) but not the CPG groups or the V50-4 groups (four doses). The number of scratching bouts in mice induced by ID challenge of rFor t 2 allergen only decreased in the V50-2 group but not in the CGP groups or the V50-4 groups. These results suggest that 2 additional doses and the CpG adjuvant do not provide additional benefits to 2 doses of the For t 2 therapeutic DNA vaccine. Taken together, these findings suggest that two shots of 50–100 μg DNA is sufficient to improve biting midge-induced allergic symptoms and inflammation.

Discussion

In the present study, the therapeutic effect of the For t 2 DNA vaccine was investigated in mice with an established biting midge allergy. It was found that two doses of For t 2 DNA vaccine against the major allergen of biting midge F. taiwana, attenuated itching and allergic inflammation in mice with an established biting midge allergy. After treatment with two doses of For t 2 DNA vaccine, the mice that were already sensitized to biting midges demonstrated a significant decrease in the number of scratching bouts as well as the secretion of type II cytokines, such as IL-13 and IL-4; skin eosinophil infiltration and intra-dermal IL-31 production was also significantly reduced in response to ID midge-allergen challenge. To the best of our knowledge, this is the first report on a therapeutic DNA vaccine treating established biting insect allergies.

IL-31 is a cytokine principally produced by activated Th2 cells. IL-31 receptors are constitutively expressed on epithelial cells and keratinocytes [18–20]. There is mounting evidence showing that IL-31 is correlated with the itchy pathophysiology of atopic dermatitis, cutaneous T-cell lymphoma, uremic pruritus, allergic contact dermatitis and chronic urticaria [21,22]. In the current study, For t 2-specific IgE, the For t 2 specific IgG1/IgG2a ratio, IL-4, IL-13 and skin inflammation were all reduced following administration of the DNA vaccine, there was also an increase in For t 2-specific IgG2a, as one would expect to see in a successful allergen specific immunotherapy [23,24]. In addition to this, allergen-induced intra-dermal IL-31 production was significantly reduced following the administration of DNA vaccine therapy. This change in IL-31 supported the significant reduction in the number of itchy scratching bouts observed in the mice after therapy in the present study.

Interestingly, the optimal number of doses for this therapeutic For t 2 DNA vaccine was two consecutive doses one week apart, which is the same as previously recommended for the prevention of the development of biting midge allergies [15]. Increasing the number of injections to four doses or adding CPG adjuvants seemed to have no benefits. Our data also showed that increasing the dose of vaccine from 50 to 100 or 200 μg did not seem to influence the therapeutic effects. Fifty μg was capable of achieving the same clinical effects as those achieved by 100 and 200 μg. As our previous study [15] showed that 25 μg had less effect than 50 μg in preventing biting midge allergy, it does not seem to be the case that any dose is effective. A possible explanation may be the capacity limitation of cells at injection sites to express the target protein.

Although there are at least 11 allergens in the biting midge F. taiwana [2], a DNA vaccine encoding the single major allergen For t 2, seems to be adequate to alleviate the itchy scratching bouts, as well as allergen-induced IL-13 production in mice sensitized with whole crude midge allergens. These results imply that it may not be necessary to use multiple DNA sequences of midge allergens in the future when applying this DNA vaccine to human midge allergy subjects.

There are limitations to the current study. First, whether the therapeutic effects of the DNA vaccine as observed in mice could be generalized to humans remains unknown. It is known that DNA-based vaccines tend to have weaker immunogenicity in primates and humans compared with rodents, particularly when administered by conventional injection. Further studies on primates may be necessary prior to human trials. Second, conventional intra-muscular injection was used in the current study. Using alternative routes of administration, such as electroporation, have been reported to enhance the effect of a DNA vaccine encoding the major house dust allergen Der p 2 [25]. Third is the issue of adjuvants. Using the CpG motif as an adjuvant in the present study provided no additional benefits to the vaccine. However, other adjuvants were not tested. For example poly-L-lysine (PLL), which has been reported to improve the prophylactic and therapeutic effect of the peanut allergen Ara h 2 DNA vaccine in peanut-allergic mice [26]. Furthermore, whether the For t 2 DNA vaccine can last long enough to cover the whole midge season requires further investigation.

In conclusion, two doses of allergen specific-immunotherapy administered one week apart, using DNA encoding the major midge allergen For t 2, is effective at treating mice with established biting midge allergies. The treatment effects are evident by clinical, immunological and histopathological improvements. With its convenience and low cost the For t 2 DNA vaccine could be a favorable strategy for the treatment of this problem in human patients.

Supporting information

S1 Fig. The purity of E-rFor t 2 and the specificity of lab-made rabbit IgG against recombinant and natural For t 2 proteins.

(A) Coomassie Blue-stained SDS-PAGE of purified E.coli-expressed For t 2 recombinant protein. (B) Immunoblotting of E-rFor t 2 or (C) midge extracts with rabbit anti-E-rFor t 2 polyclonal antibodies (lane 3). Lane 1, midge extract probed with non-immunized rabbit serum; lane 2, midge extract probed with pre-immunized rabbit serum.

(TIF)

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S1 Raw images

(PDF)

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Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by grants from the Taiwan Ministry of Science and Technology (grant nos. MOST-105-2321-B-010-017 and MOST-106-2321-B-010-013). The URL of the funder is www.most.gov.tw. The funder did not participate the study design, data collection and analysis, decision to publish and preparation of the manuscript.

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Decision Letter 0

Junji Xing

21 Feb 2020

PONE-D-20-00004

Therapeutic DNA vaccine attenuates itching and allergic inflammation in mice with established biting midge allergy

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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5. Review Comments to the Author

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Reviewer #1: This manuscript by Chen et al examines the potential therapeutic benefit of a DNA vaccine against the biting midge-Forcipomyia taiwana. This is of importance regionally as this is the most prevalent biting insect allergy in Taiwan, with t2 being the major allergen component. Using a mouse model, they aimed to show if a DNA vaccine agains For t2 allergens could prevent or attenuate various readouts for the allergen response, namely, T2 cytokines and the physical readout of scratching. Overall, they discover there appears to be some benefit to this vaccine when delivered in the proper dosage to mice. This has implications that further studies could be useful for humans as treatment-also noted that this would be reasonably affordable option for treatment as well. These studies, while of importance regionally, would be less relevant to those outside of Taiwan.

The abstract needs editing for better grammar.

Figure legends are scattered throughout the methods and results sections making it awkward to read. These should be placed in the appropriate locations as directed by the journal.

A discussion of potential reasons contributing to why increasing dosing of vaccine had less activity against the midge allergy should be discussed.

When analyzing scratching behavior from video tapes, were the persons assessing blinded to the groups?

Reviewer #2: Lee et al., present a manuscript seeking to assess the impact of therapeutic vaccination with a midge protein (For t 2) to protect from allergic reaction in mice. The data, as presented, are inconclusive and somewhat confounding. Some of the terminology in the text needs to be corrected for accuracy. A number of methods are incomplete, antigens employed not completely described and key antisera is not sufficiently defined. Resolution of major concerns will require additional experiments focused on demonstrating that For t 2 in crude midge extracts and in recombinant form are relevant target of vaccination.

Materials and Methods section:

“sterilized with a 0.22 um syringe filter” is not the correct terminology when material is passed through a 0.22 um filter owing to the filterability of most viruses through this pore size. “passed through” would be appropriate.

The detection limit of the endotoxin test should be stated where the results of the test are described, which should be at the beginning of the Results section. Currently, no description is included.

The description of Figure 1 should be moved to the Results section. The time post transfection when cells were collected and lysed for panel B should be more completely described. Also, E-rFor t 2 should be defined as the “E. coli-expressed recombinant protein”. In addition to the immunoblot, a gel stained for total protein should be shown to demonstrate the purity of the E-rFor t 2. Given that “rabbit anti-rFor t 2 polyclonal antibodies” are employed to show that the 36 kDa protein in the gel is antigenically related to For t 2, the source and reference for the antibody must be better documented. Evidence that the material produced in bacteria or following DNA transfection of HaCaT cells are related to natural antigen is crucial here, particularly if the polyclonal sera used was raised by immunizing with these same recombinant sources of protein.

Results section:

Figure 1 should start this section. Where “rFor t 2” is written, “E-rFor t 2” should be used to be consistent, Both Figure 1A and Figure 2 currently indicate “rFor t 2”. The term “not-vaccinated” should be corrected to “nonvaccinated”.

If the source of “rFor t 2 used in ELISA assays is the same as used for sensitization of the animals, more need to be included to demonstrate that the bacterially produced antigen is pure.

It would be appropriate to use a different source of recombinant protein for the ELISA to avoid cross reactivity due to undisclosed impurities.

Figure 3 attempts to show that post-sensitization vaccination can reduce inching, but the description “Five weeks after For t 2 DNA vaccination, all groups of mice received 3 ID challenges with rFor t 2 protein during days 56-61” does not state that animals were first sensitized by IP inoculation as shown in Figure 1A. This must be the case for the controls to have shown high levels of scratching, but the text description must be unambiguous. This data suggests that any dose of therapeutic vaccine (V50, V100 or V200) tested was sufficient to reduce scratching to a similar degree.

Authors do not provide a rationale or evidence that the spleen is the appropriate organ to assess for cytokines in this model. Results observed are confounding, likely due to an incomplete experimental strategy.

Figure 4 shows that, in splenocytes, cytokines IL-13, IL-4 and IL-10 showed consistent patterns by ELISA at any dose of vaccine tested, although there is a surprising absence of IL-4 in a control group as well, making this set of observations impossible to interpret. IFNg or IFN mRNA did not show any differences between groups. IL-13 mRNA was only significantly reduced in the V50 group; whereas IL-4 mRNA was reduced most in the V100 group and IL-10 mRNA was only elevated in the V50 group. The number of times this experiment was performed is not stated, but none of these peculiarities seems likely. The patterns observed seem arbitrary.

Figure 5 shows that eosinophils are higher in controls than any of the For t 2 vaccinated groups. in situ immunostaining in skin sections for CD4+ cells and intradermal IL-31. Patterns are referred to a “significantly” changed, without any possibility of statistical significance in this type of experiment. Furthermore, intensity of IL-31 staining is quantified in Fig 5C, with statistics applied inappropriately, but showing high significance. Text and statistics must be better aligned with the type of experiment shown.

Figure 6 shows an experiment where groups of mice are sensitized with crude midge extract and then vaccinated before challenge with crude midge extract. Vaccination is shown to reduce scratching and IL-13 levels.

In order to have a more rigorous series of animal experiments, two additional experiments are needed, where E-rFor t 2 is used to sensitize and crude midge extract is used to challenge, either with or without vaccination, and also where crude midge extract is used to sensitize and E-For t 2 is used to challenge. These variations will help address some of the concerns about antigens that have been applied here.

**********

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Reviewer #2: No

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PLoS One. 2020 Apr 23;15(4):e0232042. doi: 10.1371/journal.pone.0232042.r002

Author response to Decision Letter 0

22 Mar 2020

Reviewer #1: This manuscript by Chen et al examines the potential therapeutic benefit of a DNA vaccine against the biting midge-Forcipomyia taiwana. This is of importance regionally as this is the most prevalent biting insect allergy in Taiwan, with t2 being the major allergen component. Using a mouse model, they aimed to show if a DNA vaccine against For t2 allergens could prevent or attenuate various readouts for the allergen response, namely, T2 cytokines and the physical readout of scratching. Overall, they discover there appears to be some benefit to this vaccine when delivered in the proper dosage to mice. This has implications that further studies could be useful for humans as treatment-also noted that this would be reasonably affordable option for treatment as well. These studies, while of importance regionally, would be less relevant to those outside of Taiwan.

Question 1. The abstract needs editing for better grammar.

Reply: The abstract has been sent for English editing by a professional English editor as per the reviewer’s suggestion.

Question 2. Figure legends are scattered throughout the methods and results sections making it awkward to read. These should be placed in the appropriate locations as directed by the journal.

Reply: All figure legends have been moved to the last part of the manuscript, from page 23 to 26.

Question 3. A discussion of potential reasons contributing to why increasing dosing of vaccine had less activity against the midge allergy should be discussed.

Reply: We have added the following to the discussion on page 16 “…. Our data also showed that increasing the dose of vaccine from 50 to 100 or 200 �g did not seem to influence the therapeutic effects. Fifty �g was capable of achieving the same clinical effects as those achieved by 100 and 200 �g. As our previous study [15] showed that 25��g had less effect than 50��g in preventing biting midge allergy, it does not seem to be the case that any dose is effective. A possible explanation may be the capacity limitation of cells at the injection site to express the target protein.”

Question 4. When analyzing scratching behavior from video tapes, were the persons assessing blinded to the groups?

Reply: Yes. This has been added to the revised manuscript. Please see page 7, line 16. “The observation of scratching behavior was blinding measured and performed as previously described [15,16].”

Reviewer #2: Lee et al., present a manuscript seeking to assess the impact of therapeutic vaccination with a midge protein (For t 2) to protect from allergic reaction in mice. The data, as presented, are inconclusive and somewhat confounding. Some of the terminology in the text needs to be corrected for accuracy. A number of methods are incomplete, antigens employed not completely described and key antisera is not sufficiently defined. Resolution of major concerns will require additional experiments focused on demonstrating that For t 2 in crude midge extracts and in recombinant form are relevant target of vaccination.

Materials and Methods section:

Question 1. “sterilized with a 0.22 um syringe filter” is not the correct terminology when material is passed through a 0.22 um filter owing to the filterability of most viruses through this pore size. “passed through” would be appropriate.

Reply: The term on page 5 line 8 has been changed to “passed through” as per the suggestion of the reviewer.

Question 2. The detection limit of the endotoxin test should be stated where the results of the test are described, which should be at the beginning of the Results section. Currently, no description is included.

Reply: A description of the detection limit of the endotoxin has been added to Page 5, Lines 9-11: “The endotoxin content was determined using an E-TOXATE kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The lowest detection limit of the test was 0.05 endotoxin units per ml.” The following was also added to Page 10, Lines 10-11: “The endotoxin content of E-rFor t 2 after removing gel was under the detection limit (0.05 U/ml) of the Limulus Amebocyte Lysate test.”

Question 3. 1) The description of Figure 1 should be moved to the Results section. The time post transfection when cells were collected and lysed for panel B should be more completely described. 2) Also, E-rFor t 2 should be defined as the “E. coli-expressed recombinant protein”. 3) In addition to the immunoblot, a gel stained for total protein should be shown to demonstrate the purity of the E-rFor t 2. Given that “rabbit anti-rFor t 2 polyclonal antibodies” are employed to show that the 36 kDa protein in the gel is antigenically related to For t 2, the source and reference for the antibody must be better documented. Evidence that the material produced in bacteria or following DNA transfection of HaCaT cells are related to natural antigen is crucial here, particularly if the polyclonal sera used was raised by immunizing with these same recombinant sources of protein.

Reply: 1) The description of Figure 1 has been moved to the first part of the Results section, Page 10, Lines 9-11. 2) All descriptions of E-coli-expressed For t 2 protein in this manuscript have been changed to “E-rFor t 2”. 3) A supplemental figure, Fig S1, has been added to demonstrate the purity of E-rFor t 2 (Lane A and B) as well as the specificity of rabbit anti-rFor t 2 polyclonal antibodies to 36 kDa natural For t 2 protein from whole midge extract (Lane C).

Fig S1. The purity of E-rFor t 2 and the specificity of lab-made rabbit IgG against recombinant and natural For t 2 proteins. (A) Coomassie Blue-stained SDS-PAGE of purified E.coli-expressed For t 2 recombinant protein. (B) Immunoblotting of E-rFor t 2, or (C) midge extracts with rabbit anti-E-rFor t 2 polyclonal antibodies (lane 3). Lane 1, midge extract probed with non-immunized rabbit serum; lane 2, midge extract probed with pre-immunized rabbit serum.

Results section:

Question 4. 1) Figure 1 should start this section. 2) Where “rFor t 2” is written, “E-rFor t 2” should be used to be consistent, Both Figure 1A and Figure 2 currently indicate “rFor t 2”. 3) The term “not-vaccinated” should be corrected to “nonvaccinated”.

Reply: 1) The description of Figure 1 has been moved to the first part of the Results section, Page 10 Lines 9-10. 2) All descriptions of E-coli-expressed For t 2 protein in this manuscript have been changed to “E-rFor t 2”. 3) The term “not-vaccinated” has been changed to “nonvaccinated”, as shown in figure 1A, and in all related parts of the manuscript.

Question 5. If the source of “rFor t 2 used in ELISA assays is the same as used for sensitization of the animals, more need to be included to demonstrate that the bacterially produced antigen is pure. It would be appropriate to use a different source of recombinant protein for the ELISA to avoid cross reactivity due to undisclosed impurities.

Reply: A supplemental figure, Fig S1, has been added to demonstrate the purity of E-rFor t 2 (panel A and B).

Question 6. Figure 3 attempts to show that post-sensitization vaccination can reduce inching, but the description “Five weeks after For t 2 DNA vaccination, all groups of mice received 3 ID challenges with rFor t 2 protein during days 56-61” does not state that animals were first sensitized by IP inoculation as shown in Figure 1A. This must be the case for the controls to have shown high levels of scratching, but the text description must be unambiguous. This data suggests that any dose of therapeutic vaccine (V50, V100 or V200) tested was sufficient to reduce scratching to a similar degree.

Reply: We apology for the confusion. For greater clarity, the description has been rephrased as follows: “Post-sensitization vaccination was administered to all groups of mice and comprised 3 ID challenges with E-rFor t 2 protein during days 56-61.” Please see Page 11, Lines 17-18.

As our previous study (MF Lee, et al. Allergy 71 (2016) 522–531) showed that 25��g had less effect than 50��g in preventing biting midge allergy, it does not seem to be the case that any dose is effective. The following sentences have been added to the Discussion section, Page 16 and 17: “Our data also showed that increasing the dose of vaccine from 50 to 100 or 200 �g did not seem to influence the therapeutic effects. Fifty �g was capable of achieving the same clinical effects as those achieved by 100 and 200 �g. As our previous study [15] showed that 25��g had less effect than 50��g in preventing biting midge allergy, it does not seem to be the case that any dose is effective. A possible explanation may be the capacity limitation of cells at injection sites to express the target protein.”

Question 7. Authors do not provide a rationale or evidence that the spleen is the appropriate organ to assess for cytokines in this model. Results observed are confounding, likely due to an incomplete experimental strategy.

Reply: In order to obtain sufficient cells for experiments, especially for those requiring further culture and stimulation with allergen, we used splenocytes from mice as a surrogate for peripheral mononuclear cells in humans. We have add a description and a reference (Mebius RE, Kraal G. Nat Rev Immunol. 2005; 5: 606-616. ) on Page 12 to explain the rationale for using splenocytes, as follows: “The spleen plays an important role in humoral and cellular immune responses. It contains a variety of immune cells, which provide the organ with a cytokine-rich environment.”.

Question 8. Figure 4 shows that, in splenocytes, cytokines IL-13, IL-4 and IL-10 showed consistent patterns by ELISA at any dose of vaccine tested, although there is a surprising absence of IL-4 in a control group as well, making this set of observations impossible to interpret. IFNg or IFN mRNA did not show any differences between groups. IL-13 mRNA was only significantly reduced in the V50 group; whereas IL-4 mRNA was reduced most in the V100 group and IL-10 mRNA was only elevated in the V50 group. The number of times this experiment was performed is not stated, but none of these peculiarities seems likely. The patterns observed seem arbitrary.

Reply: We believe it is reasonable to suppose that a reduction in scratching bouts after challenge (equivalent to a reduction in itching sensation in humans after midge bites) would indicate that the DNA vaccine had a demonstrable therapeutic effect. The immunologic change induced by an allergen-specific DNA vaccine may not be the same as that of a protein-based allergen-specific vaccine. We have added a number of experiments in the legend of figure 4, as follows: Fig 4. Cytokine protein expression and the mRNA levels of splenocytes from each group of mice as determined by ELISA and qRT-PCR, respectively. (A) IL-13, (B) IL-4, (C) IL-10 and (D) IFN-γ. Cytokine release data are shown as the mean ± SEM from 3 independent experiments. qRT-PCR data are expressed as the mean fold increase ± SEM from 3 independent experiments. The statistical significance of differences among the groups was assessed by one-way analysis of variance with Dunnett’s t-test. *p<0.05; **p<0.01; NS, not statistically significant; SEM, standard error of the mean.”

Question 9. Figure 5 shows that eosinophils are higher in controls than any of the For t 2 vaccinated groups. in situ immunostaining in skin sections for CD4+ cells and intradermal IL-31. Patterns are referred to a “significantly” changed, without any possibility of statistical significance in this type of experiment. Furthermore, intensity of IL-31 staining is quantified in Fig 5C, with statistics applied inappropriately, but showing high significance. Text and statistics must be better aligned with the type of experiment shown.

Reply: To avoid confusion, we have rephrased the description of figure 5 on Page 13 as follows: “Fig 5A and 5B show that the total number of infiltrate cells, mainly the eosinophils, decreased significantly in the V50, V100, and V200 groups compared with the nonvaccinated and VO groups. IL-31 is known to be one of the key cytokines that induces itching and promotes scratching in mouse models of atopic dermatitis [18]. As shown in Fig 5C, expression of intradermal IL-31 was significantly decreased in all of the DNA vaccinated groups, including the VO group, compared with the nonvaccinated group. However, the reduction of IL-31 was more significant in the V50, V100, and V200 groups compared with the VO group.”

Question 10. Figure 6 shows an experiment where groups of mice are sensitized with crude midge extract and then vaccinated before challenge with crude midge extract. Vaccination is shown to reduce scratching and IL-13 levels.

In order to have a more rigorous series of animal experiments, two additional experiments are needed, where E-rFor t 2 is used to sensitize and crude midge extract is used to challenge, either with or without vaccination, and also where crude midge extract is used to sensitize and E-For t 2 is used to challenge. These variations will help address some of the concerns about antigens that have been applied here.

Reply: The experiments shown in figure 6 are an attempt to reproduce a real-world scenario involving a human being bitten by midges (i.e., being exposed to all midge allergens after being bitten in the field). It is very unlikely that a human would be exposed to For t 2 protein only after being bitten by midges. We believe that a protocol involving a crude midge extract-challenge in which mice are sensitized to crude-extract is sufficient to represent a human response to midge allergy.

Thank you again for reviewing our manuscript and for the reviewers’ valuable comments. We welcome your suggestions and advice. I look forward to hearing from you soon.

Sincerely,

Yi-Hsing Chen, MD, PhD.

Associate Professor of Medicine

Division of Allergy, Immunology and Rheumatology

Taichung Veterans General Hospital

Faculty of Medicine, National Yang-Ming University,

1650 Taiwan Boulevard, Section 4, Taichung 40705, Taiwan

Tel. 886-4-23592525 ext. 3330

Fax 886-4-23592705

ysanne@vghtc.gov.tw

yhchen8@ym.edu.tw

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Decision Letter 1

Junji Xing

7 Apr 2020

Therapeutic DNA vaccine attenuates itching and allergic inflammation in mice with established biting midge allergy

PONE-D-20-00004R1

Dear Dr. Chen,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Junji Xing, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Authors have responded to all concerns and comments and have introduced better explanations of phenomena

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Acceptance letter

Junji Xing

10 Apr 2020

PONE-D-20-00004R1

Therapeutic DNA vaccine attenuates itching and allergic inflammation in mice with established biting midge allergy

Dear Dr. Chen:

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    Supplementary Materials

    S1 Fig. The purity of E-rFor t 2 and the specificity of lab-made rabbit IgG against recombinant and natural For t 2 proteins.

    (A) Coomassie Blue-stained SDS-PAGE of purified E.coli-expressed For t 2 recombinant protein. (B) Immunoblotting of E-rFor t 2 or (C) midge extracts with rabbit anti-E-rFor t 2 polyclonal antibodies (lane 3). Lane 1, midge extract probed with non-immunized rabbit serum; lane 2, midge extract probed with pre-immunized rabbit serum.

    (TIF)

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    Data Availability Statement

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