Vincent W. Bramwell

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.

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.

Liposome/DNA complexes coated with biodegradable PLA improve immune responses to plasmid encoding hepatitis B surface antigen.

We hypothesized that the addition of polymer to the surface of liposome/DNA complexes may potentially enhance in vivo delivery of plasmid DNA to antigen‐presenting cells and thereby facilitate enhanced immune responses to encoded protein. BALB/c mice were immunized subcutaneously or intramuscularly three times with a total of 50 µg of the plasmid pRc/CMV‐HBs(S) (ayw subtype) encoding for the hepatitis B surface antigen. We measured transgene‐specific total immunoglobulin G (IgG), IgG2a, IgG2b and IgG1 antibody responses as well as splenocyte and T‐cell proliferation and cytokine production upon re‐stimulation following immunization. Modification of lipid/DNA complexes by the polymer precipitation method used here for the addition of poly(d,l‐lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal DNA or naked DNA in eliciting transgene‐specific immune responses to plasmid‐encoded antigen when administered by the subcutaneous route. In addition, the polymer‐modified formulations delivered by this route were more effective than naked DNA delivered by the intramuscular route in inducing antibody responses (n=5, P<0·03). Our observations provide ‘proof of principle’ for the use of these multicomponent formulations, which offer potential for manipulation and increased transfection efficiency in vivo for the purposes of genetic immunization.

Strategies for DNA vaccine delivery

Strategies for gene delivery comprise a diverse range of live and synthetic approaches; DNA delivery for the purposes of immunisation in turn comprises a large part of this research. This review mainly discusses synthetic systems for application in the delivery of plasmid DNA vaccines, outlining polylactide-co-glycolide, liposome, chitosan and complex combination delivery systems. Areas of promise for DNA vaccine candidates include immune modulation of allergic responses and veterinarian application. The potential for realistic consideration of DNA vaccines as an alternative to existing approaches is dependent on the development of efficient DNA vaccine vectors and improved systems for DNA vaccine delivery. DNA vaccine technology may yet prove to be an important asset in an environment where there is a critical need for therapeutic and prophylactic strategies to combat a wide range of disease states.

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.

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 μg 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 μg 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.