Generation of Toxoplasma gondii GRA1 protein and DNA vaccine loaded chitosan particles: preparation, characterization, and preliminary in vivo studies

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Abstract

Chitosan microparticles as carriers for GRA-1 protein vaccine were prepared and characterized with respect to loading efficiency and GRA-1 stability after short-term storage. Chitosan nanoparticles as carriers for GRA-1 pDNA vaccine were prepared and characterized with respect to size, zeta potential, and protection of the pDNA vaccine against degradation by DNase I. Both protein and pDNA vaccine preparations were tested with regard to their potential to elicit GRA-1-specific immune response after intragastric administration using different prime/boost regimen. The immune response was measured by determination of IgG2a and IgG1 antibody titers. It was shown that priming with GRA1 protein vaccine loaded chitosan particles and boosting with GRA1 pDNA vaccine resulted in high anti-GRA1 antibodies, characterized by a mixed IgG2a/IgG1 ratio. These results showed that oral delivery of vaccines using chitosan as a carrier material appears to be beneficial for inducing an immune response against Toxoplasma gondii. The type of immune response, however, will largely depend on the prime/boost regimen and the type of vaccine used.

Introduction

Toxoplasma gondii is an obligate intracellular parasite which—after oral ingestion by the host—generally induces a mild asymptomatic infection, both in humans and animals. Control of the infection in the host occurs through the induction of strong and persistent cell mediated immunity, characterized by production of gamma-interferon (IFN-γ), immunoglobulin (Ig) IgG2a, and cytotoxic T cells (Denkers, 1999, Yap and Sher, 1999). In humans, a primary T. gondii infection and subsequent transplacental transmission during pregnancy can result in miscarriage or in severe disease in the infant (Holliman, 1995). On the other hand, this infection may reactivate under conditions of immunosuppression, resulting in toxoplasma encephalitis and other complications (Denkers and Gazzinelli, 1998). These pathological consequences associated with congenital toxoplasmosis not only represent a threat to humans but are also a cause of economic losses due to abortions in farm animals (Dubey and Beattie, 1988).

Reports on vaccination with the T. gondii excreted/secreted dense granule protein 1 (GRA1) have been accumulating and shown to induce protective immune responses against experimental T. gondii infection in different animal models (Duquesne et al., 1991, Supply et al., 1999, Vercammen et al., 2000, Scorza et al., 2003). Protection against infection with T. gondii, obtained after intramuscular (i.m.) GRA1 DNA vaccination, was shown to be associated with Th1 type responses, characterized by the production of IgG2a, IFN-γ, and T. gondii-specific cytolytic CD8+ T cells (Vercammen et al., 2000, Scorza et al., 2003). However, as T. gondii infects the host through the gut, local immune responses may be more appropriate to reduce the risk of infection. Whereas i.m. vaccination with naked plasmid DNA can induce systemic humoral and cellular immune responses, oral delivery systems may be used to induce mucosal immune responses in the gut.

The mucosal route of vaccine administration is attractive due to the increased patient compliance and ease of application (i.e. no need of trained personnel). Furthermore, vaccination at mucosal surfaces may result in humoral and cellular responses, both systemic and local; the latter not only at the site of vaccination, but also at distant mucosal epithelia. The rationale of the work presented here was to orally immunize mice using chitosan nanoparticles as a non-viral delivery system for GRA1 encoding plasmid DNA (pDNA), and chitosan microparticles as carriers for the recombinant GRA1 protein vaccine and to compare the immune responses elicited by both systems.

Particulate mucosal delivery systems that encapsulate protein or pDNA encoding antigens have been widely explored for their ability to induce an immune response. Examples of materials used for this purpose are poly(lactide glycolide acid) (PLGA; O’Hagan, 1998, Raghuvanshi et al., 2002), starch (Wikingsson and Sjoholm, 2002), and different cationic polymers among them chitosan (McNeela et al., 2000, Illum et al., 2001, van der Lubben et al., 2001c). Chitosan is the deacetylated form of chitin that has many properties suitable for vaccine delivery. It is positively charged in acidic solutions, biodegradable, biocompatible, and is very cheap for being a waste product of the seafood industry. Chitosan is a mucoadhesive polymer that is able to open tight junctions and allow the paracellular transport of molecules across mucosal epithelium, therefore is suitable for the mucosal delivery of vaccines (Artursson et al., 1994, Luessen et al., 1996, van der Lubben et al., 2001b). Previously, properties of chitosan microparticles were explored in our lab and exhibited suitable in vitro and in vivo characteristics for oral vaccination (van der Lubben et al., 2001a, van der Lubben et al., 2001c). Microparticles loaded with diphtheria toxoid (DT) strongly enhanced local (IgA) and systemic (IgG) immune responses against DT after oral administration in mice (van der Lubben et al., 2003). When used for DNA vaccination, oral administration of chitosan nanoparticles loaded with DNA plasmid that encoded a peanut allergen gene, protected AKR mice from food allergen-induced hypersensitivity (Roy et al., 1999). Recently, intranasal immunization with chitosan nanoparticles loaded with pDNA encoding respiratory syncytial virus (RSV) proteins, was reported to induce protective Th1 type immune responses in BALB/c mice (Kumar et al., 2002).

In the present study, both GRA1 pDNA and recombinant GRA1 protein loaded chitosan formulations were generated and characterized with regard to their physico-chemical parameters such as loading efficiency, size, and zeta potential. In addition, their stability was evaluated with respect to DNA and protein degradation. The immunogenicity of these chitosan-based delivery systems was addressed in a preliminary in vivo study.

Section snippets

Materials

Chitosan was purchased from Primex (Karmsund, Norway) and had a viscosity of 12 mPa s and a degree of deacetylation of 93.2%, as measured by the manufacturer. PicoGreen dsDNA quantitation kit was from Molecular Probes (Leiden, The Netherlands). Tween-80, Tween-20, lysozyme, imidazol, 4-chloro-napthol substrate tablets, 3,3′,5,5′-tetramethylbenzidine (TMB) and peroxidase-conjugated rat anti-mouse immunoglobulin (IgG) were obtained from Sigma–Aldrich (Zwijndrecht, The Netherlands). Bio-Rad protein

Expression and purification of recombinant GRA1

After the cloning of GRA1 into pQE-81L, clones harboring pQE81-GRA1 were grown for the production of recombinant GRA1. Following protein induction and purification, the recombinant 23 kDa GRA1 was analyzed on SDS–PAGE gels, revealing that the preparation was devoid of contaminating proteins (Fig. 1, panel A). An average yield of approximately 9 mg recombinant GRA1 per liter bacterial culture was obtained. In order to confirm that the antigenic properties of the recombinant GRA1 were conserved,

Conclusions

In this study we evaluated the possibility of loading chitosan particles with T. gondii GRA1 protein and GRA1 encoding pDNA. These chitosan formulations were easy to prepare, shown to be stable and therefore appropriate for mucosal delivery. GRA1 protein loading efficiency was high, GRA1 protein was protected from degradation over short-term storage by loading onto chitosan microparticles. Efficiency of nanoparticle formation from chitosan and GRA1 pDNA was also very high. Nanoparticles

Acknowledgements

We would like to thank Mariken van der Lubben for her guidance during the chitosan microparticles preparation. Raphaël Zwier from the Fine Mechanical Department at LACDR is acknowledged for preparing the feeding needles for the mice. This work was in part supported by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen, grant GO40598. We are indebted to R. Zaugg (Vical, San Diego, CA, USA) for allowing us to work with the VR1020 plasmid.

References (28)

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    Several antigens have been evaluated as vaccine candidates of dense granule organelles including GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8,GRA14, GRA15, GRA24 and GRA41. Most of studies were focused on GRA7 (11 studies) (Fatoohi et al., 2002; Hiszczyńska-Sawicka et al., 2010; Hiszczyńska-Sawicka et al., 2011; Jongert et al., 2008; Liu et al., 2014; Min et al., 2012; Quan et al., 2012; Rosenberg et al., 2009; Vazini et al., 2018; Vercammen et al., 2000; Yin et al., 2015), GRA4 (9 studies) (Desolme et al., 2000; Hiszczyńska-Sawicka et al., 2011; Li et al., 2007; Martin et al., 2004; Meng et al., 2013; Mévélec et al., 2005; Picchio et al., 2018; Yácono et al., 2012; Zhang et al., 2007), GRA2 (8 studies) (Allahyari et al., 2016; Babaie et al., 2018; Ching et al., 2016; Golkar et al., 2007; Golkar et al., 2005; Xue et al., 2008; Zhou et al., 2007; Zhou et al., 2012) and GRA1 (7 studies) (Bivas-Benita et al., 2003; Fatoohi et al., 2002; Hiszczyńska-Sawicka et al., 2011; Jongert et al., 2008; Scorza et al., 2003; Supply et al., 1999; Vercammen et al., 2000). These antigens were tested either alone or in combination (18 as single and 21 as cocktail) for evaluation of potential effects against T. gondii.

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