Protection against Bordetella pertussis infection following parenteral or oral immunization with antigens entrapped in biodegradable particles: effect of formulation and route of immunization on induction of Th1 and Th2 cells
Introduction
Purified native or recombinant antigens and subunit vaccines usually require adjuvants or delivery systems and booster administrations to induce protective immune responses. One encapsulation matrix, PLG microparticles, offers real advantages as a vehicle for vaccine delivery. The dried microencapsulated antigens can be stabilized and the PLG polymer is biocompatible and has been approved for use in humans [1], [2]. Microparticles have further potential through pulsed or delayed delivery of antigens, with the possibility of a single dose of encapsulated antigen overcoming the need for repeated immunization [3], [4]. Moreover, parenterally delivered antigens entrapped in PLG particles can stimulate cell mediated immune responses in addition to circulating antibodies [5].
The most frequent portal of entry of almost all infectious agents are mucosal surfaces. Consequently, the induction of specific immune responses at mucosal surfaces is highly advantageous in vaccine strategies aimed at containing infection to the site of entry. Therefore, antibodies produced locally in the gut or respiratory tract, predominately of the IgA isotype, are important in the first line of defence [6], [7], [8]. The oral route of immunization is more attractive than systemic or nasal routes because of its ease of delivery, improved compliance and reduced possibility of side effects. Incorporating antigens into PLG microparticles protects the proteins from degradation and denaturation by proteolytic enzymes and acidic conditions in the stomach as well as enhancing uptake by M cells in the Peyer's patches [9]. The safe passage of PLG microparticles through the stomach and the ability of M cells to take up some of these PLG microparticles following oral delivery leads to a concentration of antigen at the site of mucosal stimulation [10], [11].
In order to establish if encapsulated antigens can confer protection against infection, it is necessary to test antigen(s) from an infectious pathogen in a suitable animal challenge model. Respiratory pathogens are useful to assess the hypothesis that immunization at one mucosal surface should induce immune responses at another and since a mouse model for the Bordetella pertussis infection is well established [12], we have examined the immunogenicity and protective efficacy of an acellular pertussis vaccine delivered in PLG microparticles.
B. pertussis is a gram negative bacterium which infects the human respiratory tract and causes whooping cough. Convalescence from the disease is associated with the induction of systemic and local Th1 cells, as well as circulating and secretory antibody responses [13], [14]. Immunization with the whole cell pertussis vaccine also selectively primes Th1 cells in mice and in children [15], [16]. In contrast, the new acellular vaccines induce Th2 responses in mice [15] and mixed Th1/Th2 responses in children [16], [17]. Although these conventional parenterally delivered vaccines induce potent IgG responses and confer protection against severe whooping cough in 71–85% of children, they fail to induce secretory IgA [15], [18], [19], [20].
Previous studies from this laboratory have demonstrated that systemic delivery of antigen in microparticles induces a Th1 response and when administered by a mucosal route may also induce secretory IgA [5]. There have been a number of reports suggesting that the size of the particles may affect the immunogenicity of antigens entrapped in PLG polymers [21], [22]. Most of these studies have focused on antibody responses and there has been little attempt to assess the effect of particle size on T cell responses. Moreover, it has been reported that single dose formulation with particles of different sizes and different release kinetics induce immune responses similar to the responses induced with booster doses of conventional vaccines [4]. In this study two protective antigens from B. pertussis, PTd and FHA, were encapsulated in PLG microparticles or nanoparticles and their immunogenicity and protective efficacy were evaluated in an established murine challenge model.
Section snippets
Antigens
FHA and glutaraldehyde-detoxified PT (PTd) purified from the Thoma strain of B. pertussis was purchased from Kaketsuken Laboratories, Japan. Alum adsorbed antigens were prepared by mixing 25 μg of PTd or FHA with 750 μg of alhydrogel (Superfos, Denmark) and leaving at 4°C for 24 h with constant agitation.
Particle formulations
PLG microparticles were prepared using a modification of the water-in-oil-in-water (w/o/w) solvent evaporation technique using RG504 polymer, 50:50 poly lactide: co-glycolide
Statistical analysis
Mean cytokine concentrations, antibody titres (log10 transformation) and CFU counts (log10 transformation) were compared using the Student's t-test to assess statistical significance.
Microparticle and nanoparticle analysis
Optimised formulation of antigens in PLG using the solvent evaporation and coacervation techniques produced micro- and nanoparticles respectively with characteristics summarised in Table 1. The particle size for all microparticle batches was within a fairly narrow range, with D50s from 1.5 to 4.7 μm, while those of the nanoparticles had D50s, which ranged from 200 to 600 nm. Electron microscopy confirmed visually the size ranges and that discrete smooth spherical particles were present for all
Discussion
The results of this study demonstrate that parenteral immunization with the pertussis antigens, PTd and FHA, entrapped in PLG particles induce potent T cell and antibody responses and confer a high level of protection against a B. pertussis challenge. Oral immunization also conferred protection, but 100 μg of antigen and two booster immunizations were required. In contrast a single dose of 5 μg of a combined micro- and nanoparticles formulation induced protective immune responses by parenteral
Acknowledgements
We would like to thank Geraldine Murphy, Helen Stewart and Brian O'Gorman at NUI, Maynooth, for their assistance with B. pertussis challenge experiments and Deirdra Ahern (EBR) for her assistance in the analysis of PLGA formulations. We would also like to thank Enterprise Ireland and Elan Biotechnology Research for funding.
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