Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery.

Biodegradable polymer and particulate carriers have been shown to be of considerable potential for the delivery of peptides, proteins and DNA in animal models. In the context of vaccine delivery to the upper and lower respiratory tracts, the use of mucoadhesive agents offers a strategy for the facilitation of increased residence time and increased vaccine efficacy. Additional concerns addressed here include the potential of uptake of vaccine formulations by the primary olfactory nerves in the nasal cavity, effective delivery to the lung, strategies to maximise the immunopotentiation of candidate vaccine formulations, as well as the evaluation of animal models and interpretation of engendered immune responses in terms of antigen-specific antibody production. Experimental data are presented that demonstrate the potential of muco- and bioadhesive agents in combination with liposomes for intranasal (i.n.) delivery of tetanus toxoid in mice. A delivery system utilising chitosan for the formulation of microspheres by the spray-drying method is described and assessed for intranasal vaccine delivery, and porous particles with potential for pulmonary administration are also outlined.

Adjuvant synergy: the effects of nasal coadministration of adjuvants.

Modern peptide and protein subunit vaccines suffer from poor immunogenicity and require the use of adjuvants. However, none of the currently licensed adjuvants can elicit cell-mediated immunity or are suitable for mucosal immunization. In this study we explored the immunological effect of nasal co-administration of adjuvants with distinct functions: cholera toxin subunit B, a potent mucosal adjuvant that induces strong humoral responses, muramy di-peptide (MDP), an adjuvant known to elicit cell mediated immunity but rarely used nasally, and chitosan, an adjuvant that achieves specific physiological effects on mucosal membranes that improve antigen uptake. Groups of five female BALB/c mice received on days 1 and 56 nasal instillations of the recombinant Helicobacter pylori antigen urease admixed to single or multiple adjuvant combinations. Serum IgG kinetics were followed over 24 weeks. At the conclusion of the experiment, local antibody responses were determined and antigen-specific recall responses in splenocyte cultures were assayed for proliferation and cytokine production. The combination of adjuvants was shown to further contribute to the increased antigenicity of recombinant H. pylori urease. The data presented here outline and support facilitation of increased immunomodulation by an adjuvant previously defined as an effective mucosal adjuvant (chitosan) for another adjuvant (MDP) that is not normally effective via this route.

Stimulation of spleen cells in vitro by nanospheric particles containing antigen.

Activation of cells, in primary culture, by nanospheres containing antigen has been investigated. Single cell suspensions of spleen cells from primed and nai;ve animals were cocultured with escalating quantities of soluble tetanus toxoid (TT) or TT encapsulated within nanospheres fabricated from poly(lactide-co-glycolide) (PLGA). Concomitantly, spleen cells were also cultured in the presence of 'empty' PLGA nanospheres that contained no TT. Nanospheres loaded with antigen were found to elicit increased proliferation of splenocytes from preimmunised mice in comparison to free antigen during coculture at equivalent doses of immunogen (at low and intermediate doses). Interestingly, cellular proliferation was abolished if B-cells were removed from the splenocyte cultures. Production of IFN-gamma and IL-6 was increased, for formulated as compared to free antigen, in microcultures from both nai;ve and pre-immunised animals. Secretion of IFN-gamma or IL-6 was not observed when primed or nai;ve spleen cells were stimulated with 'empty' polymeric spheres. Some unspecific cytotoxicity was detected if cells were cocultured with high concentrations of PLGA particles, although toxic effects were not seen at concentrations where maximum levels of cytokine secretion and cellular proliferation were recorded. These cell culture data indicate that, at least in this in vitro model, nanoparticulate TT is able to elicit cytokine production that is probably consistent with increased stimulation. This mechanism is likely to be distinct from non-specific effects caused by components of the delivery vehicle itself.

Adjuvant action of melittin following intranasal immunisation with tetanus and diphtheria toxoids.

Melittin, a 26-amino acid peptide and the major active component of the venom of the honey bee--Apis mellifera--has recently been shown to have absorption enhancing properties in Caco-2 cells at levels well below the level required for the generation of cytotoxicity. Given the potential of absorption enhancing agents to act as adjuvants when administered nasally [Alpar, H.O., Eyles, J.E., Williamson, E.D. and Somavarapu, S. (2001) "Intranasal vaccination against plague, tetanus and diphtheria", Adv. Drug Delivery Rev. 51, 173-201] we hypothesized that melittin may have potential as a mucosal adjuvant. Following our initial studies reported here, it was found that the co-administration of 4 microg of melittin in conjunction with tetanus toxoid in BALB/c mice was effective in eliciting markedly enhanced antibody titres in comparison to control groups and groups receiving free antigen administered intranasally. Lower concentrations of melittin were less effective and mice receiving 4 microg of melittin plus antigen exhibited antibody titres significantly higher (i.e. P<0.05) than any of the other groups tested. The observed IgG2a titres were shown to be dependent upon the dose of melittin co-administered with the immunising antigen in a similar fashion to the observed total IgG responses. In summary, melittin has been shown here to have potential as a novel mucosal adjuvant for antigens administered via the nasal route.

Oral plasmid DNA delivery systems for genetic immunisation.

The use and optimisation of plasmid DNA delivery systems for the purposes of eliciting transgene specific immune responses to orally administered DNA encoded antigen represents a significant challenge. Here, we have outlined a multicomponent polymer modified liposomal delivery system that offers potential for oral administration of plasmid DNA. It is shown that the polymer/liposome formulated DNA is able to elicit markedly enhanced transgene specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen. This is discussed with reference to recent publications and the potential of plasmid DNA delivery systems for the purposes of genetic immunisation, as reported in selected literature, is assessed.

Microsphere translocation and immunopotentiation in systemic tissues following intranasal administration.

With a view to developing improved mucosal immunisation strategies, we have quantitatively investigated the uptake of fluorescent polystyrene carboxylate microspheres (1.1 microm diameter), using histology and fluorescence-activated cell sorting, following intranasal delivery to BALB/c mice. To qualify these biodistribution data, antigen specific memory and effector responses in the spleens of mice immunised nasally with Yersinia pestis V antigen loaded poly(lactide) (PLA) microspheres (1.5 microm diameter) were assessed at 4, 7 and 11 days. Irrespective of administration vehicle volume (10 or 50 microl), appreciable numbers of fluorescent microspheres were detected within nasal associated lymphoid tissues (NALT) and draining cervical lymph nodes. Nasal administration of the particles suspended in 50 microl volumes of phosphate-buffered saline (PBS) served to deposit the fluorescent microspheres throughout the respiratory tract (P<0.05). In these animals, appreciable particle uptake into the mediastinal lymph node was noted (P<0.05). Also, spleens removed from mice 10 days after fluorescent particle application contained significantly more microspheres if the suspension had been nasally instilled using a 50 microl volume (P<0.05). Appreciable memory (and effector from day 7) responses were detected in mediastinal lymph nodes removed from mice immunised nasally with 50 microl volumes of microparticulated or soluble V antigen. Immunological responses in splenic tissue removed 7 days after intranasal immunisation corroborated the thesis that the spleen can act as an inductive site following bronchopulmonary deposition of particulated antigen: upon exposure to V in vitro, splenic T-cells from mice nasally immunised with 50 microl volumes of microspheres incorporated statistically greater (P<0.05) quantities of [3H]thymidine into newly synthesised DNA than did T-cells from cohorts nasally immunised with 50 microl volumes of V in solution. Similarly, significant numbers of anti-V IgG secreting cells were only detected in spleens from mice immunised intramuscularly or nasally with microparticles. These immunological and biodistribution data support the tenet that, following an appropriate method of mucosal delivery, microparticles can translocate to tissues in the systemic compartment of the immune system and thence provoke immunological reactions therein.