A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM₁₉₇) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery

Abstract

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM₁₉₇) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM₁₉₇ and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM₁₉₇ preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM₁₉₇ and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM₁₉₇ was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM₁₉₇ antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.

Introduction

Diphtheria is a rapidly developing, acute, bacterial disease, involving both local and systemic pathology, which is caused by Corynebacterium diphtheriae. Most of the clinical symptoms of the disease are due to the release of the potent diphtheria toxin from lysogenized strains of the bacteria. Prevention may, therefore, be obtained by toxin neutralizing antibodies (antitoxin), induced through active immunization with non-toxic forms of the toxin. Current diphtheria vaccines are prepared by converting diphtheria toxin to its non-toxic, but antigenic, toxoid by formaldehyde treatment and are mostly combined with tetanus toxoid and whole-cell or acellular pertussis vaccines for infant immunization [1].

Routine immunization against diphtheria, introduced in the 1940s and 1950s, led to the almost complete eradication of this disease from developed countries by the 1970s. However, a high proportion of the European population has been shown to have diphtheria antitoxin titres below the putative protective level of 0.01 IU/ml [2]. As a result, there has been a resurgence of diphtheria in several European countries in the 1980s and 1990s [2], [3]. In most countries it is now recommended that booster doses of diphtheria vaccine be administered every 10 years. However, a limiting factor to public acceptance could be the adverse effects associated with the vaccine, due to the presence of accessory antigens in crude or partially purified toxoid preparations [4]. It would, therefore, be beneficial to develop a more refined vaccine against diphtheria.

Diphtheria toxin is composed of two disulfide-linked fragments, an enzymatically active A fragment and a B fragment, that is responsible for binding and entry of the toxin into sensitive eukaryotic cells. Protein synthesis in these cells is inhibited by inactivation of the ribosomal elongation factor 2, caused by the specific ADP ribosylating activity of fragment A, resulting in cell death [5]. Several non-toxic or partially toxic mutants of diphtheria toxin that cross-react immunologically with it and, hence, are called cross-reacting materials (CRM) have been isolated. One such mutant, CRM₁₉₇, contains a glycine to glutamic acid mutation at position 52 in the A subunit, rendering it enzymatically inactive and therefore, non-toxic [1]. Since CRM₁₉₇ is naturally non-toxic it does not require formaldehyde detoxification and can be obtained at close to 100% purity, avoiding the cross-linking to accessory antigens that occurs during formaldehyde treatment of diphtheria toxin. It has been shown to be safe in humans and is used as a carrier protein for the capsular polysaccharide of Haemophilus influenzae type b [6]. It would therefore appear to be an ideal candidate as a vaccine against diphtheria. However, studies have shown that it is less immunogenic than conventional diphtheria toxoid, but that its immunogenicity for antibody responses can be increased after stabilization with formaldehyde [7], [8].

Traditionally, vaccines such as diphtheria, tetanus and pertussis have been administered parenterally by intramuscular injection. However, mucosal immunization has the advantage over conventional parenteral immunization of stimulating both systemic and mucosal immunity. In addition, mucosal delivery of vaccines minimizes adverse effects and allows for easier administration, making vaccination in the home a possibility [9], [10]. Mucosal immunization would therefore be of particular benefit where frequent boosting is required, as is the case for diphtheria. However, soluble antigens administered mucosally tend to elicit poor immune responses and require the use of delivery vehicles, immunostimulants or adjuvants to increase their immunogenicity.

Chitosan is a cationic polysaccharide derived by deacetylation of chitin, a natural material found in the exoskeletons of various crustaceans. Chitosan derivatives have been found to have many beneficial effects including wound-healing properties [11], candidacidal [12] and anti-viral activities [13]. It has been shown that 70% deacetylated chitin could activate peritoneal macrophages [14] and natural killer cells [15] and induce various cytokines in mice [16] and in addition could confer protection against Sendai virus and E. coli infections [13]. Chitosan is non-toxic in humans and studies have shown that when administered nasally, only transient decreases in mucociliary transport rates are detected in vitro, leaving no permanent damage to this defense system [17]. Its safety, proven ability to deliver drugs across the nasal cavity and potential adjuvant activity, make it an ideal candidate for nasal delivery of vaccines. It has already been demonstrated that nasal delivery of filamentous haemagglutinin from Bordetella pertussis with chitosan can stimulate local and systemic antibody responses [18].

In this study, we have examined the T cell and antibody responses in mice and toxin neutralizing antibody production in guinea pigs following intranasal (i.n.) and parenteral immunization with CRM₁₉₇ alone or formulated with chitosan. Previous reports have shown that both native and formaldehyde-treated CRM₁₉₇ induce similar anti-DT antibody responses in mice. However, there is little information available on the effect of formaldehyde treatment on T cell responses. Here, we have examined the effects of formaldehyde treatment of CRM₁₉₇ on the induction of both T cell and antibody responses for intranasal or parenteral immunization. In addition, we assessed immune responses in mice generated by priming parenterally with DT and boosting intranasally with CRM₁₉₇ with or without chitosan. Our results provide evidence that a nasally delivered vaccine may be effective against diphtheria, especially when used for boosting individuals primed with the conventional diphtheria vaccine.