Research PaperOral hepatitis B vaccine: chitosan or glucan based delivery systems for efficient HBsAg immunization following subcutaneous priming
Graphical abstract
Introduction
The World Health Organization (WHO) estimates that, in 2015, there were 887 000 hepatitis B virus (HBV) related deaths (WHO, 2017) mainly representative of developing countries. The limited used of current licensed injectable HBV vaccines in those countries could be overcome by the development of stable oral formulations. Moreover, oral vaccines are seen as an economically feasible approach to mass vaccination, regarding easier and safer administration and, theoretically, would have better stability, facilitating shipping and storage (Giudice and Campbell, 2006, Kochhar et al., 2013). Other than logistic advantages, mucosal vaccination induce a varied immune response. In addition to serum immunoglobulin G (IgG), the generation of a strong secretory IgA (sIgA) response (resistant to protease and nuclease activity) will allow a prolonged protection to viral infection at mucosal surfaces (Wang et al., 2015), which is relevant for sexually transmitted HBV, the major route of dissemination in low endemic areas. Although no oral peptide vaccines are currently approved, mainly due to the phenomenon of oral tolerance or poor antigen immunogenicity and degradation in gastrointestinal tract (Poonam, 2007), antigen encapsulation in polymeric particles is a vital tool to exceed all of these hurdles, as they may improve antigen stability/bioavailability and act as adjuvant (Koppolu and Zaharoff, 2013, Wang et al., 2015). Indeed, some promising results for HBV oral immunization have already been published using polymeric delivery systems (Borges et al., 2007, Dinda et al., 2016, Gupta et al., 2007, Mishra et al., 2011). Chitosan is a widely used hydrophilic polymer for particles preparation, which allows the antigen encapsulation in an aqueous environment. Moreover, it is a biodegradable polycationic biopolymer with interesting immunostimulatory and mucoadhesive properties, directing both cellular and humoral responses (Bernkop-Schnurch and Dunnhaupt, 2012, Xia et al., 2015) and thus it is seen as potential adjuvant for mucosal vaccines (Wang et al., 2015). Chitosan particles mediated oral delivery is limited due to its solubility at acidic pH, (gastric lumen) leading, most probably, to the antigen release in the stomach. To provide a greater stability to the particles, a process of particles coating with sodium alginate, an acid insoluble polymer, was developed (Borges et al., 2006, Li et al., 2008). The oral vaccination with hepatitis B surface antigen (HBsAg) loaded alginate coated chitosan particles, led to significant induction of the anti-HBsAg sIgA in the gut and systemic specific antibodies in only less than 20% of the mice. In the same study, performed in our laboratory, when CpG oligodeoxynucleotide (ODN) loaded particles were added to prior formulation and administered orally to a second group, a percentage of 40 % responders was observed (Borges et al., 2007). In fact, combined use of protein antigens and CpG emerged as one of the innovative approaches for adjuvant development. Synthetic ODNs containing unmethylated CpG motifs trigger cells that express Toll-like receptor 9 (TLR9) to mount an innate immune response characterized by the activation and maturation of dendritic cells (DCs) and B cells differentiation into antibody-secreting plasma cells (Vollmer and Krieg, 2009). Thus, the first aim of the present work is to improve our previous developed formulation, co-entrapping both, the HBsAg and the CpG in the same particle, as their temporal and physical proximity is an important requirement for an effective adjuvant effect (Bode et al., 2011), in an attempt to increase the low seroconversion rate observed. The second aim is to prepare and evaluate another delivery system for HBsAg oral immunization: glucan particles. The intention is to compare the efficacy of both formulations for oral vaccination. The potential of glucan particles (GPs) as an oral vaccine delivery system/adjuvant for HBsAg has never been evaluated. However, the literature suggests GPs as an optimal oral vaccine platform (Baert et al., 2015; De Smet et al., 2014; De Smet et al., 2013, Shah et al., 2016), protecting the encapsulated agent from gastrointestinal conditions (De Smet et al., 2013, Shah et al., 2016, Zhang et al., 2014) and eliciting a robust humoral response in the intestine comprising the production of antigen specific IgA, sIgA and secretory component antibodies (De Smet et al., 2013). However, systemic immune responses were only elicited following parenteral administrations (Berner et al., 2015, Huang et al., 2010, Huang et al., 2013, Specht et al., 2015). GPs, porous micrometer-sized shells derived from baker’s yeast (Saccharomyces cereviseae) cell walls (Soto and Ostroff, 2007), are an excellent choice for targeting antigen to antigen presenting cells (APC), as their β-1,3-d-glucan surface composition selectively binds to Dectin-1 and TLR2 receptors, highly expressed on innate immune cell surface (Taylor et al., 2002, Tesz et al., 2011, Zhang et al., 2014). Prior immunization studies, to evaluate the elicited systemic and mucosal immune responses, both developed delivery systems should be able to interact with APCs and to be internalized by the Peyer’s Patches (PPs), which play a key role in the development of mucosal immune responses toward vaccine antigens. We also evaluated the ability to activate mast cells as the possible adjuvant mechanism of the delivery systems, as they act as sentinels of infection strategically located close to host-environment interface, such as mucosal tissues, directing immune modulation (Wernersson and Pejler, 2014).
Section snippets
Material
Chitosan (ChitoClear™ - 95 % DD and 8 cP viscosity measured in 1 % solution in 1 % acetic acid) was purchased from Primex BioChemicals AS (Avaldsnes, Norway) and purified as previously described (Jesus et al., 2013). A low molecular weight (18 kDa) pharmaceutical grade sodium alginate (MANUCOL LB® - 61 % mannuronic acid and 39 % guluronic acid) was kindly donated by ISP Technologies Inc. (Surrey, UK). The CpG ODN 1826 solution was obtained from InvivoGen (California, USA) and the HBsAg
Particle characterization
Particles characterization is an important task in particulate-based adjuvant vaccine formulation as their physicochemical properties might dictate their adjuvant activity. Size is a perfect example, and it was recently reviewed as an intrinsic characteristic that affects the generation of long last adaptive immune responses, namely through the impact of particle size on the polymer half-life in vivo and the influence of that in the duration of the innate and adaptive immune responses elicited (
Conclusion
Vaccination is the most effective intervention in modern medicine. The development of new mucosal formulations for underutilized vaccines could be the way towards vaccine-preventable diseases eradication. Antigen encapsulation in polymer-based particles is a primordial tool for effective vaccine delivery to mucosal sites. The appellative features of chitosan and glucan based particles as promising delivery systems for an oral immunization as well as interesting immunostimulatory properties made
Conflicts of interest
None.
Acknowledgements
This work was supported by FEDER European funds through the Programa Operacional Fatores de Competividade —COMPETE 2020 and by Portuguese funds through FCT—Portuguese Foundation for Science and Technology in the framework of the projects PTDC/SAU-FAR/115044/2009, PEst-C/SAU/LA0001/2011 and UID/NEU/04539/2013 and fellowship SFRH/BD/96167/2013. We also wish to thank Dr. Butterfield for his generous gift of the human mast cell line HMC1.1, Luisa Cortes from the MICC Imaging facility of CNC, Isabel
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