Research paper
Oral Helicobacter pylori vaccine-encapsulated acid-resistant HP55/PLGA nanoparticles promote immune protection

https://doi.org/10.1016/j.ejpb.2016.11.007Get rights and content

Abstract

Oral vaccination, is notoriously weak or nonimmunogenic. One of the major reasons is the inefficient antigen uptake caused by enzymolysis and hydrolysis in the gastrointestinal tract. In this study, acid-resistant HP55/PLGA nanoparticle was developed as an oral delivery system to protect H. pylori recombinant antigen CCF against the complex gastrointestinal environment. These ∼200 nm particles controlled the release of antigen in the acidic environment (pH  5.5). Immunized mice with HP55/PLGA-CCF nanoparticles induced high levels of urease-specific antibodies and memory T cell responses. A month after H. pylori challenge, 43% of mice were completely protected. The protection was highly associated with the Th1/Th17-bias immune response, which had been recognized as an optimal immunity against H. pylori infection. In addition, a mass of T-cells were observed in the lamina propria of mice immunized with CCF, especially in the HP55/PLGA-CCF nanoparticles administered recipients, and contributed to the development of postimmunization gastritis. These results indicate that oral immunization with acid-resistant HP55/PLGA nanoparticles encapsulating vaccine antigens represent a promising strategy for antigen protection, slow-release and targeting, and thus prevented gastrointestinal infection.

Introduction

The gastric Gram-negative pathogen Helicobacter pylori (H. pylori) is a highly successful bacteria that colonizes 50% of worldwide population [1]. The virulence factors harboring by H. pylori can trigger host immunopathology and further cause chronic gastritis, which is asymptomatic in the majority of carriers but a potential risk factor for the development of gastric ulcers, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma [2], [3]. As common dogma, the human stomach is hard to live with bacteria, H. pylori not only survives but tends to be numerically dominant [4]. Although the infection can be efficiently eradicated by antibiotic therapy, the risks of reinfection, antibiotic resistance and associated adverse effects remain unneglectable in the areas where H. pylori is endemic. Vaccination is considered as a cost-effective alternative to control the prevalence of H. pylori [5], [6]. Mounting evidences indicate that inducing a mucosal immune response is indispensable to prevent or treat H. pylori infection, thus administering vaccine directly via the gut mucosa by oral immunization possesses immunologic and practical advantages [7]. Thereby, a protective strategy in an oral vaccine formulation preparation is desperately needed to overcome the degradation and degeneration induced by gastrointestinal protease and extremely acidic pH [8].

d,l-lactide-co-glycolic acid (PLGA) nanoparticles (NPs) are extensively applied in vaccine delivery [9]. Further, PLGA is a FDA-approved biocompatible and biodegradable polymer and has the potential to be qualified as the competent vaccine delivery device and drug controlled release system. Delivering antigen with PLGA NPs significantly improved the vaccine-induced immune responses [10], [11]. Liu et al. reported that oral administration with pH-responsive PLGA NPs could target to dendritic cells, induce lymphocyte activation and facilitate a memory T cell response [12]. In addition, Hydroxypropyl methylcellulose phthalate (HPMCP), an enteric coating agent, is used to protect drugs from degradation by gastric acid and prevent side effects caused by gastric release, which is admitted into the U.S. National Formulary, European pharmacopeia and Japanese Pharmacopeia [13], [14]. Sharma et al. have designed enteric submicron particulates using pH-sensitive polymers of HPMCP to avoid gastric inactivation of papain [15]. HP-55, a special type of HPMCP, is distinguished by its solubility at pH 5.5 and high resistance to simulated gastric fluid. The polymer PLGA NPs could be physically modified by HP-55, then possessed the characteristic of excellent stability in acid environment, resulted in the acid-resistant nanoparticles (HP55/PLGA NPs), which could protect antigen from gastric acid degradation, improve antigen uptake and control the release of antigen [16]. In our previous study, we constructed a dual-antigen epitope and dual-adjuvant vaccine called CCF [17]. A novel chimeric flagellum (CF) was constructed by replacing the central variable region of H. pylori flagellin FliC with the central variable region of Salmonella typhimurium (S. typhimurium) FlaA. Then, the CF was linked to the C-terminus of CTB-UE. CTB-UE was composed of the cholera toxin B (CTB) subunit as well as tandem copies of the Th and B cell epitopes from H. pylori urease [18]. Although we demonstrated that oral co-administration CCF with an aluminum adjuvant could activate TLR5 and benefit to the elimination of H. pylori, it still needed a formulation to enhance the immunogenicity and overcome the extreme gastrointestinal environment.

Here we synthesized acid-resistant HP55/PLGA nanoparticles, and evaluated its oral preventive effect in mice model. Increased levels of antigen-specific antibodies, switched IgG2a/IgG1 ratio and proinflammatory cytokines were detected in the mice vaccinated with CCF-encapsulated HP55/PLGA NPs. Furthermore, a strong Th1/Th17-type immune response was observed and 43% of mice received HP55/PLGA-CCF NPs acquired complete protection. Together, these results indicate that an acid-resistant nanoparticles formulation represents a promising strategy for oral vaccine delivery and facilitates the development of anti-H. pylori vaccines for human use.

Section snippets

Reagents

PLGA (An inherent viscosity of 0.17 dL/g, a lactic/glycolic molar ratio of 75:25) was obtained from Shandong Academy of Pharmaceutical Sciences (Shandong, China). The HPMCP (HP55, Nominal Phthalyl content 31%, Labeled viscosity 40 cSt) was purchased from Shin-Etsu Chemical Co., Ltd (Tokyo, Japan). Poloxamers 188 (Pluronic F68, av. mol wt 8400) was provided by BASF (Shanghai, China). Acetone and dichloromethane (DCM) was purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Native

Preparation and characterization of HP55/PLGA NPs and PLGA NPs

HP55/PLGA NPs and PLGA NPs were developed by the modified membrane emulsification technology and double-emulsion solvent evaporation technique (Fig. 1a). The blend polymers of HP55 and PLGA were dissolved in the resultant solvent mixture of acetone and DCM to prepare multiple emulsions. The low temperature environment and transitory sonication were used to protect the CCF from degradation. After sonicate, CCF were distributed into the matrix of HP55 and PLGA via the carboxyl-amide interaction.

Discussion

Here we prepared an acid-resistant HP55/PLGA NPs formulation using a membrane emulsification and double-emulsion solvent evaporation technology, and H. pylori vaccine CCF was used as a model protective antigen. The HP55/PLGA NPs showed pH-sensitive profiles and had comparative antigen encapsulation and release efficiency compared with normal PLGA NPs. Mice orally immunized with HP55/PLGA-CCF NPs developed antigen-specific antibodies, Th1/Th17-type cell-medicated immunity and significant

Acknowledgements

This work was supported by the National Major Special Program of New Drug Research and Development (No. 2012ZX09103301–008), Natural Science Foundation of Jiangsu Province (No. BK20130647), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and Qing Lan Project, National Natural Science Foundation of China (No. 81502970), and the Fundamental Research Funds for the Central Universities (No. 2015ZD004).

References (58)

  • M. Shakweh et al.

    Poly (lactide-co-glycolide) particles of different physicochemical properties and their uptake by peyer’s patches in mice

    Eur. J. Pharm. Biopharm.: Off. J. Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV

    (2005)
  • P. Brandtzaeg

    Induction of secretory immunity and memory at mucosal surfaces

    Vaccine

    (2007)
  • A. Azizi et al.

    Mucosal HIV vaccines: a holy grail or a dud?

    Vaccine

    (2010)
  • X. Song et al.

    PLGA nanoparticles simultaneously loaded with vincristine sulfate and verapamil hydrochloride: systematic study of particle size and drug entrapment efficiency

    Int. J. Pharm.

    (2008)
  • K. Chen et al.

    Th17 cells mediate clade-specific, serotype-independent mucosal immunity

    Immunity

    (2011)
  • Z. Wang et al.

    Regulatory T cells promote a protective Th17-associated immune response to intestinal bacterial infection with C. rodentium

    Mucosal Immunol.

    (2014)
  • J.F. Mann et al.

    Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection

    Vaccine

    (2009)
  • Z. Zhang et al.

    Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery

    Biomaterials

    (2011)
  • T. Jung et al.

    Tetanus toxoid microspheres consisting of biodegradable poly(lactide-co-glycolide)- and ABA-triblock-copolymers: immune response in mice

    Int. J. Pharm.

    (2002)
  • L. Shi et al.

    Pharmaceutical and immunological evaluation of a single-shot hepatitis B vaccine formulated with PLGA microspheres

    J. Pharm. Sci.

    (2002)
  • C.S. Chong et al.

    Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery

    J. Control. Release: Off. J. Control. Release Soc.

    (2005)
  • D. Mucida et al.

    Regulation of TH17 cells in the mucosal surfaces

    J. Allergy Clin. Immunol.

    (2009)
  • A. Sokic-Milutinovic et al.

    Role of Helicobacter pylori infection in gastric carcinogenesis: current knowledge and future directions

    World J. Gastroenterol.

    (2015)
  • G. Ayala et al.

    Exploring alternative treatments for Helicobacter pylori infection

    World J. Gastroenterol.

    (2014)
  • S.F. Moss et al.

    Helicobacter pylori. Current opinion in infectious diseases

    (2003)
  • A. O’Connor et al.

    Treatment of Helicobacter pylori infection 2014

    Helicobacter

    (2014)
  • V. Papastergiou et al.

    Treatment of Helicobacter pylori infection: past, present and future

    World J. Gastr. Pathophysiol.

    (2014)
  • H. Sijun et al.

    Helicobacter pylori vaccine: mucosal adjuvant & delivery systems

    Indian J. Med. Res.

    (2009)
  • S. Hamdy et al.

    Activation of antigen-specific T cell-responses by mannan-decorated PLGA nanoparticles

    Pharm. Res.

    (2011)
  • Cited by (61)

    • Development of an oral nanovaccine for dogs against Echinococcus granulosus

      2023, European Journal of Pharmaceutics and Biopharmaceutics
    • RNA nanotechnology: A new chapter in targeted therapy

      2023, Colloids and Surfaces B: Biointerfaces
    • PLGA-based nanoparticles for treatment of infectious diseases

      2023, Poly(lactic-co-glycolic acid) (PLGA) Nanoparticles for Drug Delivery
    • Oral organic nanovaccines against bacterial and viral diseases

      2022, Microbial Pathogenesis
      Citation Excerpt :

      Mice orally and I.P. immunized with 100 μg of CCF (four times at one-week intervals) were I.P. challenged with H. pylori SS1 (0.4 × 109 CFU) two weeks post-last immunization. Strong specific IgG antibody was produced and, moreover, oral immunization induced higher protection than the I.P. immunization by reducing the number of H. pylori colonies (4 x 102 vs 7 × 103 CFU) in gastric tissue [120]. This recent study highlights the need to promote the use of PLGA more and more for developing effective vaccines against infectious human diseases.

    • A new poly(I:C)-decorated PLGA-PEG nanoparticle promotes Mycobacterium tuberculosis fusion protein to induce comprehensive immune responses in mice intranasally

      2022, Microbial Pathogenesis
      Citation Excerpt :

      In another study, PLGA loaded with OVA antigen activated higher levels of antigen-specific CD8+ T cells in mice [26]. Furthermore, the efficacy of PLGA nanoparticles as adjuvants was also demonstrated in pathogen models such as hepatitis B virus, Corynebacterium diphtheriae and Helicobacter pylori [27,28]. However, PLGA material is highly hydrophobic, the stability of nanoparticles and the encapsulation effect is poor [29].

    View all citing articles on Scopus
    View full text