Dual role of CpG as immune modulator and physical crosslinker in ovalbumin loaded N-trimethyl chitosan (TMC) nanoparticles for nasal vaccination
Graphical abstract
Introduction
Nasal vaccination has gained much interest over the past decades as it is non-invasive and thereby expected to increase patient compliance. Additionally, vaccination via the nose has been shown to induce, besides systemic humoral (IgG mediated) and cellular responses, local as well as distal secretory immune responses (secretory IgA (sIgA) mediated) [1], [2], [3], making the mucosal linings less vulnerable to infection. Moreover, the cross reactivity of sIgA is relatively high compared to IgG antibodies [4], [5], making the induction of local immune responses a promising strategy to target highly variable pathogens, like influenza viruses [6].
Nonetheless, nasal immunization with subunit vaccines is challenging, as residence time in the nasal cavity is limited and therefore the uptake by the nasal epithelium is low. Moreover, the nasal epithelium is renowned for being a rather tolerogenic site [7], [8], making it difficult for subunit antigens to provoke an immune response. Vaccine formulation may be instrumental to successful nasal vaccination. Encapsulation of the antigen into particulate carrier systems has been explored extensively in recent years [9] and holds great promise as particles can be specifically designed to meet the challenges nasal vaccination provide [10]. Among the large variety of particles that can be found in the literature, chitosan based particles are among the most studied ones [11]. Chitosan is a cheap, biodegradable, mucoadhesive polymer. In rodents, particles prepared from chitosan have been shown to effectively induce systemic antibody responses against ovalbumin (OVA) and cholera toxin [12], Hepatitis B surface antigen [13], and Meningococcal C oligosaccharides [14]. More recently chitosan derivatives have been developed, like thiolated chitosans [15] to enhance its mucoadhesiveness and trimethylated chitosans (TMC) [16] to improve its solubility at physiological pH. Especially TMC has been shown to be a very promising nasal vaccine carrier. Nanoparticles prepared from TMC by ionic crosslinking with tripolyphosphate (TPP) increase the nasal residence time of the encapsulated antigen [17], improve the uptake of the antigen by M-cells [18] and additionally promote maturation of dendritic cells (DCs) [9], [10], [11]. Consequently, TMC particles loaded with antigens, e.g. tetanus toxoid [12], meningococcal C oligosaccharides [19] or hemagglutinin [20] induce strong systemic as well local antibody responses. Moreover, intranasally administered TMC-coated whole inactivated influenza virus resulted in protection of mice against a challenge with a lethal dose of influenza virus [21]. Nonetheless, a significant drawback of TMC is its tendency to promote a humoral (Th2 type) rather than a Th1 type immune response [20], [22]. Strong Th1 type responses are important for many vaccines that we do not have [23], such as HIV/AIDS and tuberculosis vaccines, underscoring the importance of developing vaccine carrier systems capable of inducing these responses. The bias of TMC's adjuvant effect toward a Th2 response is not restricted to the nasal administration route, as it is also observed after intradermal [24] and intramuscular administration of TMC-adjuvanted antigen (unpublished data). However, different types of immune responses have been reported after nasal vaccination [25], [26], [27], [28], depending on the adjuvant used. As TPP does not act as an adjuvant but solely services as a crosslinking agent to promote TMC nanoparticle formation, we propose it should be possible to substitute TPP with a crosslinking agent that does have an adjuvant effect. Unmethylated CpG DNA is a Toll like receptor 9 ligand and described as a Th1 response-inducing adjuvant, also after nasal administration [23]. Furthermore, phosphate groups on CpG render it negatively charged, which could make CpG a possible crosslinking agent to prepare TMC nanoparticles.
The aim of this paper was to study whether CpG can replace TPP as a crosslinker to prepare ovalbumin (OVA)-containing TMC nanoparticles and whether these new carrier systems are capable of redirecting the TMC-induced Th2 type response towards a more Th1 type response, while maintaining strong systemic and local antibody responses. The TMC/CpG/OVA nanoparticles were compared to TMC/TPP/OVA nanoparticles with respect to their physicochemical characteristics and immunogenicity after nasal administration in mice.
Section snippets
Materials
Ovalbumin (OVA) was purchased from Calbiochem (Beeston, UK) and CpG DNA (ODN 2006) as well as fluorescein isothiocyanate coupled CpG (CpG-FITC) from InvivoGen (Toulouse, France). N-trimethyl chitosan with a degree of quaternization of 15% was synthesized from 92% deacetylated (MW 120 kDa) chitosan (Primex, Avaldsnes, Norway) and characterized by NMR, as described by Bal et al. [29]. KCl, NaCl, HNa2PO4, KH2PO4 and bovine serum albumin (BSA) were purchased from Merck (Amsterdam, The Netherlands).
Nanoparticle characterization
The characteristics of the TMC/CpG/OVA nanoparticles and the TMC/TPP/OVA nanoparticles were comparable in size and zetapotential (Table 1). Both particle types showed an average hydrodynamic diameter of ca. 300 nm, were fairly monodisperse (PDI 0.1–0.2) and had a positive zetapotential of about + 20 mV. Moreover, changing the crosslinker did not alter the loading efficiency (Table 1) and the release pattern (data not shown), as both particle showed a burst release followed by no release over 48 h.
Conclusion
TMC/TPP/OVA nanoparticles have previously been shown to be very effective nasal vaccine carriers. Replacing TPP by CpG as a crosslinking agent to obtain TMC/CpG/OVA nanoparticles modulated the immune response towards a Th1 response after nasal vaccination, while maintaining the strong systemic and local antibody responses observed with TMC/TPP nanoparticles. TMC/CpG nanoparticles therefore are an interesting nasal delivery system for vaccines requiring broad humoral as well as strong Th1 type
Acknowledgements
This research was performed under the framework of TI Pharma project number D5-106 “vaccine delivery: alternatives for conventional multiple injection vaccines”. The authors thank Dr. Elly van Riet for reading the manuscript and her valuable suggestions.
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