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Cited by 9Advanced Drug Delivery SystemsAllergic Rhinitis and SensitizationFood Allergy and Anaphylaxis Research

Characterization of Vibrio cholerae neuraminidase as an immunomodulator for novel formulation of oral allergy immunotherapy

Open access
Diesner, Susanne C., Bergmayr, Cornelia, Wang, Xue-Yan, Heiden, Denise, Exenberger, Sarah, Roth-Walter, Franziska, Starkl, Philipp, Ret, Davide +3 more·2018·Clinical Immunology
DOI 10.1016/j.clim.2018.03.017PMC PMC6002774
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Abstract

To improve current mucosal allergen immunotherapy Vibrio cholerae neuraminidase (NA) was evaluated as a novel epithelial targeting molecule for functionalization of allergen-loaded, poly(D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) and compared to the previously described epithelial targeting lectins wheat germ agglutinin (WGA) and Aleuria aurantia lectin (AAL). All targeters revealed binding to Caco-2 cells, but only NA had high binding specificity to α-L fucose and monosialoganglioside-1. An increased transepithelial uptake was found for NA-MPs in a M-cell co-culture model. NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2. Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response. In conclusion, NA has enhanced M-cell specificity compared to the other targeters. NA functionalized MPs induce a Th1 and T-regulatory driven immune response and avoid allergy effector cell activation. Therefore, it is a promising novel, orally applied formula for allergy therapy.

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Crossref · 10.1016/j.clim.2018.03.017

Cites

39 of 39 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 45 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    NA-coated microparticles showed increased transepithelial uptake in an M-cell co-culture model.

    “An increased transepithelial uptake was found for NA-MPs in a M-cell co-culture model.”
    Quote in the paperSubstantial claim
  • Findingown data

    Only NA among the tested targeters had high binding specificity to α-L fucose and monosialoganglioside-1 (GM1).

    “All targeters revealed binding to Caco-2 cells, but only NA had high binding specificity to α-L fucose and monosialoganglioside-1.”
    Quote in the paperSubstantial claim
  • Findingown data

    NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes.

    “NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2.”
    Quote in the paperSubstantial claim
  • Findingown data

    NA and NA-MPs induced CCL20 expression in Caco-2 cells.

    “NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2.”
    Quote in the paperSubstantial claim
  • Findingown data

    Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response.

    “Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response.”
    Quote in the paperSubstantial claim
  • May combine two claims. 24
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 16

  • May combine two claims.

    Findingown data

    NA, WGA, and AAL all bound to Caco-2 cells.

    “All targeters revealed binding to Caco-2 cells, but only NA had high binding specificity to α-L fucose and monosialoganglioside-1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In intestinal lavage, OVA-specific IgA titers were significantly lower in the AAL-MPs group than in naive animals, while other groups were substantially but not significantly lower.

    “Screening intestinal lavage after oral provocation for antibody levels revealed significantly lower OVA-specific IgA titers in the group fed AAL-MPs compared to naïve animals, while all other groups were only substantially, but not significantly lower (Fig. 6C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    OVA-specific IgE was not induced; titers were significantly lower in NA-MPs, WGA-MPs, and Plain-MPs groups than in naive animals.

    “No OVA-specific IgE was induced as measured titers were significantly lower in the groups fed NA-MPs, WGA-MPs and Plain-MPs than in naïve animals, the latter representing background IgE levels (Fig. 6A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NA stimulation substantially induced IL8 in Caco-2 cells, but its levels did not differ significantly from those with AAL or WGA.

    “IL8 was substantially induced after NA stimulation but levels were not significantly different from AAL and WGA stimulation (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    α-L fucose inhibited NA attachment to Caco-2 cells in a concentration-dependent way, reducing binding by up to 40%, with mean inhibition at 318 μM.

    “Binding of NA to Caco-2 cells was inhibited in a concentration dependent manner (Fig. 1C) by the addition of α-L fucose (reduced binding up to 40%; mean inhibition with 318 μM α-l fucose)”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    In systemically sensitized OVA-allergic mice given six feeds of OVA-loaded functionalized MPs, IgE slightly decreased in NA-MP-fed mice, without statistical significance.

    “After MP gavages, there was a slight decrease of IgE in NA-MP fed mice (Fig. 7A)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    PLGA particles are fully biocompatible, eliminated via the Krebs cycle, and cause no polymer-specific immune response.

    “and are fully biocompatible being eliminated via the Krebs cycle with no polymer-specific immune response [2,4,5].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [2] source

      Not checked in the free run.

    • cites [4] source

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    • cites [5] source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The main advantage of oral immunotherapy over subcutaneous injections is painless application resulting in higher patient compliance.

    “Its main advantage over subcutaneous injections is the painless application resulting in a higher patient compliance [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    For oral application, drugs must be digestion stable and have sufficient bioavailability by overcoming first-pass metabolism.

    “For oral application, drugs have to fulfill several criteria, such as digestion stability and sufficient bioavailability by overcoming the first pass metabolism [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Targeting intestinal M-cells by particle surface modification can enhance the efficacy of PLGA carriers, as demonstrated for AAL.

    “The efficacy of PLGA carriers can be enhanced targeting intestinal microfold cells (M-cells) by particle surface modification, as demonstrated for AAL [7].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Unlike CT, NA cannot stimulate cAMP overproduction, as shown by the lack of pro-allergenic effects in the study's results.

    “However, due to its functionality NA is not able to stimulate cAMP overproduction like CT, which was confirmed by the absence of pro-allergenic effects in our results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Statistics were calculated from 6 wells per condition.

    “Statistics were calculated from 6 wells per condition.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Since M-cells highly express α-L fucose and GM1, NA may be a promising tool to prolong residence on intestinal epithelial cells and directly target M-cells, enhancing transepithelial uptake of allergen-loaded microparticles.

    “As α-L fucose and GM1 are both highly expressed on M-cells [34,35], NA might represent a promising tool not only to enhance the residence time on intestinal epithelial cells but also to target M-cells directly leading to an enhanced transepithelial uptake of allergen loaded”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [34] source

      Not checked in the free run.

    • cites [35] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Significant outliers were calculated and excluded from statistical analysis.

    “Significant outliers were calculated and excluded from statistical analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    NA is highly stable under gastric and intestinal proteolytic conditions, making it an ideal targeting molecule for oral delivery.

    “In addition, NA was revealed to be extremely stable under gastric and intestinal proteolytic conditions and, thus, represents an ideal targeting molecule for the oral route [19,36].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [19] source

      Not checked in the free run.

    • cites [36] source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The Vibrio cholerae genome contains NA within a pathogenicity island.

    “NA is encoded within a pathogenicity island of the Vibrio cholerae genome [30].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In vitro data support that NA binds via α-L fucose and also via GM1, as AAL does.

    “Our in vitro data support the assumption that, similarly to the lectin AAL, NA binds through α-L fucose and additionally through GM1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    NA increases its own binding partner on enterocytes and M-cells and may prolong intestinal residence.

    “Thereby, NA enhances its own binding partner on enterocytes and M-cells and might increase the intestinal residence time.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Animals were treated according to European Union rules of animal care and with approval by the local ethics committee and the Austrian Federal Ministry of Science and Research.

    “Animals were treated according to European Union rules of animal care and with approval by the local ethics committee and the Austrian Federal Ministry of Science and Research (permission numbers GZ BMWF-66.009/0051-II/10b/2008 and BMWF-66.009/0070-II/3b/2012).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    PCR reactions were performed in technical triplicates for each of four independent experiments.

    “PCR reactions were performed in technical triplicates for each of the four independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    Sample size calculation was based on previous own data with a power calculation based on two-sided, two-sample t-test.

    “Sample size calculation was based on previous own data [19] with a power calculation based on two-sided, two-sample t-test.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    NA has been reported to enhance cholera toxin binding to GM1, possibly contributing to CT's pro-allergenic effect via cAMP overproduction.

    “NA was even reported to promote cholera toxin (CT) binding to GM1 [32], which might contribute to the pro-allergenic effect of CT induced by cAMP over-production [33].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] source

      Not checked in the free run.

    • cites [33]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductnone given

    Three independent sets of inhibition experiments were performed.

    “Three independent sets of experiments were performed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    LPS levels of targeters and functionalized microparticles were determined to rule out strong endotoxin contamination.

    “To rule out a strong endotoxin contamination, lipopolysaccharide (LPS) levels of the targeters and the functionalized microparticles were determined using Endosafe–PTS cartridges (Charles River Laboratories, Wilmington, MA, USA) according to manufacturer's instructions.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Caco-2/Tc7 cells have a small intestinal phenotype.

    “with a small intestinal phenotype [21]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Animals were treated in random order within each group.

    “Experimental procedures were started after a 2-weeks acclimation period in a separate animal experimentation room by treating animals in random order within each group.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The authors do not expect NA to be a strong pro-inflammatory trigger at the intestinal epithelial level, since no significant effects appeared on tight junction integrity or molecules such as ZO-1, ZO-2, claudin-1, and claudin-4.

    “However, we do not anticipate that NA is a strong pro-inflammatory trigger on intestinal epithelial level, as we observed no significant effects on tight junction integrity and molecules such as ZO-1, ZO-2 and claudin-1 and claudin-4.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    CCL20 is considered crucial for M-cell differentiation and regulates lymphocyte recruitment via CCL20-CCR6.

    “Additionally, CCL20 is regarded as a crucial chemokine for M-cell differentiation and regulates lymphocyte recruitment in a CCL20-CCR6 dependent manner [38].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    NA and NA-functionalized MPs can induce a strong Th1 and T-regulatory environment, which in allergy may counteract an ongoing Th2 response.

    “NA and NA-functionalized MPs are able to induce a profound Th1 and T-regulatory environment, which, in the case of allergy, might counteract an ongoing Th2 response.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Literature associates CCL20 and IL8 with a pro-inflammatory response when Caco-2 cells are incubated with entero-invasive bacteria such as Salmonella and Shigella.

    “In literature, CCL20 and IL8 were associated with a pro-inflammatory response upon incubation of Caco-2 cells with entero-invasive bacteria, such as Salmonella and Shigella [37].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [37] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    CCL20 triggers dendritic cell chemotaxis, which together with Th1 and T-regulatory induction may benefit an ongoing allergic response.

    “CCL20 triggers the chemotaxis of dendritic cells, which in combination with an induction of Th1 and T-regulatory immunity might be beneficial in cases of an ongoing allergic response.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Single and repeated long-term oral dosing of NA-functionalized MPs was safe, with no adverse effects in naive and allergic mice regarding allergen-specific IgE formation or activation of allergy effector cells such as mast cells.

    “Single and long-term, repeated oral application of NA-functionalized MPs were safe as no adverse effects were observed in naïve and allergic mice with regards to the formation of allergen-specific IgE or the activation of allergy effector cells, such as mast cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: NA is part of the mucinase complex and acts as an important virulence factor that enhances Vibrio cholerae pathogenicity.

  • May combine two claims.

    Interpretationown data

    Only NA induces CCL20, which recruits DCs and lymphocytes to the subepithelial layer and starts MALT formation.

    “Additionally, only NA induces CCL20, which recruits DCs and lymphocytes to the subepithelial layer and initiates MALT formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The presented results show NA is suitable for oral use because of its digestion stability, and it may have adjuvant, immunomodulatory effects on epithelial and lymphoid cells.

    “Based on the here presented results, NA was proven to be suitable for oral application due to its digestion stability and additionally might have adjuvant, immunomodulatory properties on epithelial and lymphoid cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Coating MPs with NA increases intestinal uptake, protects antigens from degradation, and ensures intact antigens are presented to immune cells at mucosal induction sites.

    “Coating of MPs with NA increases intestinal uptake, protects the antigens against degradation and ensures presentation of intact antigens to immune cells at mucosal induction sites.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The particulate nature of microparticles ensures uptake via Peyer's patches and enables release and presentation of intact allergen to immune cells beneath M-cells.
35 of 35

  • The paper relies on this without support.

    The Caco-2/Raji-B co-culture model's M-cell-like cells and transepithelial transport measurements are representative of M-cell uptake in the human intestine.

    “co-cultured with Raji-B cells (1 × 106 Raji cells/filter; ATCC Raji (ATCC® CCL-86)) for the last 4 days of incubation period on the basolateral side of the epithelial layer as described [23].”

  • The paper relies on this without support.

    Mouse splenocyte cytokine responses and oral gavage outcomes in mice predict human immune responses and clinical allergy therapy.

    “NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2.”
    “Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response.”

  • The paper relies on this without support.

    OVA is a valid model allergen for evaluating allergy immunotherapy.

    “Aqueous solution of OVA (400 μl, 500 mg/ml, Grade V, lyophilized powder > 98%, Sigma-Aldrich, Lot SLBD2312V and 076 K7045) was emulsified with 400 mg PLGA in ethyl acetate by sonication for 2 min.”

  • The paper relies on this without support.

    Mouse mast cell protease-1 levels and rectal temperature adequately capture allergy effector cell activation and anaphylaxis.

    “For analysis of immediate mast cell activation, mice were gavaged once with the respective MPs preparations and sera taken 1 h after gavage were screened for mouse mast cell protease-1 (mMCP-1) using the mouse MCPT-1 Ready-set-go ELISA Kit (eBioscience, San Diego, CA) according to manufacturer's instructions.”
    “Rectal temperature as indicator of anaphylaxis was measured before and 15 and 30 min after OC.”

  • The paper relies on this without support.

    NA is part of the mucinase complex and acts as an important virulence factor that enhances Vibrio cholerae pathogenicity.

    “NA is part of the mucinase complex, and as such, is an important virulence factor, which enhances the pathogenicity of Vibrio cholerae.”

  • The paper relies on this without support.

    NA degrades the gastrointestinal mucin layer, facilitating bacterial penetration.

    “By degrading the mucin layer of the gastrointestinal tract, it facilitates bacterial penetration.”

  • The paper relies on this without support.

    The particulate nature of microparticles ensures uptake via Peyer's patches and enables release and presentation of intact allergen to immune cells beneath M-cells.

    “The particulate nature of MPs ensures uptake via the Peyer's patches and enables the release and presentation of intact allergen to immune cells residing underneath the M-cells.”
7 of 7

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Authors
Diesner, S. C., Bergmayr, C., Wang, X.-Y., Heiden, D., Exenberger, S., Roth-Walter, F., Starkl, P., Ret, D., Pali-Schöll, I., Gabor, F., & Untersmayr, E.
Affiliations
  • Medical University of Vienna (AT)
  • TU Wien (AT)
  • University of Vienna (AT)
Corresponding author
Untersmayr, Eva
ORCID
Venue
Clinical Immunology
Publisher
Elsevier BV
Published
2018-07
Volume
192
Pages
30-39
ISSN
1521-6616 (ISSN-L)
DOI
10.1016/j.clim.2018.03.017
PMC
PMC6002774
OpenAlex
W2794841308
Cited by
9
References
39
Citations by year
2020: 6 · 2019: 3
FWCI
1.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Clinical Immunology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion, cc-by)
  • PubMed Central (acceptedVersion, cc-by)
Funders
  • Austrian Science fund (P21884, KLI284)
  • FWF (F4606-B28, W1205-B09)
  • FRW and FG (EP1638531A1)
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advanced Drug Delivery Systems
  • Health Sciences › Medicine › Immunology and Allergy › Allergic Rhinitis and Sensitization
  • Health Sciences › Medicine › Immunology and Allergy › Food Allergy and Anaphylaxis Research
Keywords
Vibrio cholerae neuraminidase, PLGA microspheres, M cells, Caco-2 cells, alpha-L-fucose, wheat germ agglutinin, Aleuria aurantia lectin, Th1/Th2 balance, regulatory T cells, CCL20, allergen-specific immune response, immunomodulation
MeSH terms
Allergens, Animals, Bacterial Proteins, Desensitization, Immunologic, Humans, Hypersensitivity, Immunologic Factors, Mice, Inbred BALB C, Microspheres, Mouth Diseases, Neuraminidase, Protein Binding, Vibrio cholerae, Coculture Techniques, Caco-2 Cells, Cell Line, Tumor

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