Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

Abstract

The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.

Introduction

According to a report of the World Health Organization (WHO/UNICEF, 2005), the estimated number of deaths in the world in all age groups from diseases preventable by vaccines in 2002 was 2.1 million, 600,000 deaths being due to hepatitis B. The global coverage of infants with three doses of hepatitis B vaccine in 2004 was 48%, contrasting with 3% in 1992. In the last few years, the majority of industrialized countries have introduced hepatitis B vaccination campaigns. Therefore, the above statistics of deaths due hepatitis B are representative predominantly for the less developing countries, where mass vaccination has not been implemented yet (18% of the 192 WHO member states). Its implementation is highly dependent on the development of more stable and cheaper vaccines for which the intervention of specialised human resources for the administration would not be required.

Oral administration has been appointed as the only economically feasible approach to mass vaccination. Impressive logistical advantages of orally administered vaccines were exemplified by two national vaccination days in 1996, when 121 million Indian children were vaccinated against polio at 650,000 centres (Bloom and Widdus, 1998). However, it has been shown that is very difficult to obtain a protective immune response following oral vaccination, the live-attenuated polio vaccines are being one of the few exceptions (Holmgren and Czerkinsky, 2005). For this reason, only few vaccines currently approved for human use are being administered orally.

Unfortunately, a simple oral formulation is not easily achieved for the new generation of subunit vaccines, which hold the greatest promise for disease prevention in the 21st century (Thanavala et al., 2005). Several explanations have been appointed to justify the disappointing results found for oral administration of subunit vaccines, being almost exclusively biotechnological products. One of the most important reasons is related with the adverse environment of the gastrointestinal tract (GIT), rich in acids and enzymes, which are able to destroy the antigen. Equally important is the mechanism of oral tolerance, the vital physiological role for dietary antigens in preventing hypersensitivity reactions to food or to commensal bacteria (Mowat, 2003, Nagler-Anderson, 2001). This hyporesponsivity to antigens orally administered is not yet fully understood, but it is thought that the mucosal immune system has involved a variety of mechanisms to achieve and maintain tolerance against self-antigens and against the overabundance of environmental antigens present in the microflora and food. Among them, activation-induced cell death, anergy and especially the induction of regulatory T cells are frequently reported in the literature (Holmgren and Czerkinsky, 2005). In a recent study (Gotsman et al., 2000) the induction of oral tolerance to hepatitis B virus proteins was achieved by the administration of five low oral doses of hepatitis B virus proteins, followed by two inoculations with a commercial vaccine.

In the case of the development of an oral hepatitis B vaccine, this mechanism has to be circumvented and the antigen must be protected from physical degradation and enzymatic digestion (Holmgren and Czerkinsky, 2005). For this purpose, several strategies have been described in literature. Those approaches include the encapsulation of immunogenic peptide representing residues 127–145 of the immunodominant B-cell epitope of hepatitis B surface antigen (HBsAg) in poly(d,l-lactide co-glycolide) (Rajkannan et al., 2006). Another strategies are the encapsulation of the plasmid DNA encoding hepatitis B virus protein in poly(d,l-lactide-co-glycolic acid) (PLGA) (He et al., 2005) or in Salmonella typhimurium (Gao et al., 2003, Woo et al., 2001, Zheng et al., 2001, Zheng et al., 2002) or the genetic modification of edible plants for the production and delivery of the hepatitis B vaccine, within, e.g., potato tubers (Kong et al., 2001, Thanavala et al., 2005), cherry tomatillo (Gao et al., 2003) and lettuce (Kapusta et al., 1999, Kapusta et al., 2001). A very recent clinical study (Thanavala et al., 2005) with previously vaccinated volunteers showed that the ingestion of doses of 100 g uncooked potato tubes (8.5 μg/g) induced the increase of the serum anti-HBsAg titers in about 60% of the volunteers, who ate three doses of the potatoes. Approximately 40% of the volunteers were non-responders to the HBsAg, therefore the necessity of simultaneous finding a good mucosal adjuvant in order to elicit an increase of the number of responders was emphasised by the authors of this study (Thanavala et al., 2005).

So, with this in mind the present study has as a main purpose, the evaluation of the potential of a chitosan-based nanoparticulate delivery system as a mucosal adjuvant.

Chitosan, a copolymer of d-glucosamine and N-acetyl-d-glucosamine is a derivative of chitin, one of the polysaccharides most abundant in nature. In the last few years, the properties of this biodegradable biopolymer have been intensively investigated. In particular, its ability to stimulate cells from the immune system has been shown in several studies (Babensee and Paranjpe, 2005, Borges et al., 2007, Nishimura et al., 1987, Shibata et al., 1997). For instance, the presence of chitosan in a dendritic cell culture-induced the expression levels of the co-stimulatory molecules CD86, CD40 and HLA-DQ (Babensee and Paranjpe, 2005), indicative of dendritic cell maturation. Likewise, chitosan has also shown to be able to up-regulate, in some extent, a number of macrophage functions (Nishimura et al., 1987, Shibata et al., 1997).

The polymer has also been used in the nanoparticle formulation for loading and delivering different vaccines, like meningococcal C conjugate (Baudner et al., 2002), diphtheria (van der Lubben et al., 2003) and tetanus toxoid (Jaganathan et al., 2005, Vila et al., 2004) or used without any modification, suspending the bulk powder in a solution of the meningococcal C conjugate vaccine (Baudner et al., 2005) or using a soluble chitosan derivative with the influenza vaccine (Bacon et al., 2000, Read et al., 2005) or finally using chitosan to surface-modified PLGA microspheres containing hepatitis B vaccine for intranasal immunization (Jaganathan and Vyas, 2006).

In a previous study, we have formulated and characterised alginate-coated chitosan nanoparticles (Borges et al., 2005). They consist of a chitosan core (chitosan nanoparticles) to which the hepatitis B vaccine was adsorbed and in a second step, the sodium alginate. The alginate coating is afterwards cross-linked with calcium ions. This delivery system has the particular advantage of being constructed under very mild conditions, which is a great benefit for the encapsulation of proteins, peptides and antigens. Moreover, in a very recent publication (Borges et al., 2006) we have demonstrated that these coated nanoparticles were able to be taken up by rat Peyer's patches which is one of the essential features to internalise, deliver and target the intact antigen to specialised immune cells from the gut associated lymphoid tissue (GALT) (Neutra and Kozlowski, 2006). This property makes these new nanoparticles a promising delivery system especially for oral vaccination.

Therefore, in the present study the feasibility of using the recombinant surface hepatitis B protein (HBsAg) encapsulated into the above-mentioned alginate-coated chitosan nanoparticles for the induction of local and systemic immune responses after oral vaccination was evaluated. Moreover, to improve the immune response, synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN), were also associated to the formulations, entrapped or not in the nanoparticles. CpG ODN acts as a potent adjuvant and has shown in a number of studies to induce a Th1 type immune response, not only when administered parenterally (Osorio et al., 2003, Weeratna et al., 2001) but also after mucosal vaccination (McCluskie and Davis, 2000, McCluskie et al., 2000a, McCluskie et al., 2000b). It has been shown in non-human primates, that the CpG ODNs improve the immunogenicity of the hepatitis B vaccine (Klinman et al., 2004). Regarding human clinical experience with CpG ODNs, limited information concerning the outcome of these trials has been released. However there are some few examples already reported in literature (Carpentier et al., 2006, Cooper et al., 2004, Friedberg et al., 2005). One such example is a double-blind phase I/II study with CpG 7909, as adjuvant to Engerix-B designed to evaluate the safety of CpG 7909 in healthy adults (Cooper et al., 2004). According with the authors of the same study, the most frequently reported adverse events were injection site reactions, flu-like symptoms and headache. Additionally, as reviewed somewhere else (Klinman et al., 2004), no clinically relevant changes in hematocrit or white blood cell count among immunized volunteers, nor were there any changes in liver or renal function and none of the subjects exposed to CpG ODNs developed signs or symptoms of autoimmune disease.

Therefore CpG ODN has been regarded as a promising adjuvant for the hepatitis B vaccine and in the present study the possible advantages for its encapsulation were in vivo evaluated.