Nanocapsules Comprised of Purified Protein: Construction and Applications in Vaccine Research.

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

Solid Lipid Nanoparticles Delivering a DNA Vaccine Encoding Helicobacter pylori Urease A Subunit: Immune Analyses before and after a Mouse Model of Infection.

In this study, novel solid lipid particles containing the adjuvant lipid monophosphoryl lipid A (termed 'SLN-A') were synthesised. The SLN-A particles were able to efficiently bind and form complexes with a DNA vaccine encoding the urease alpha subunit of Helicobacter pylori. The resultant nanoparticles were termed lipoplex-A. In a mouse model of H. pylori infection, the lipoplex-A nanoparticles were used to immunise mice, and the resultant immune responses were analysed. It was found that the lipoplex-A vaccine was able to induce high levels of antigen-specific antibodies and an influx of gastric CD4+ T cells in vaccinated mice. In particular, a prime with lipoplex-A and a boost with soluble UreA protein induced significantly high levels of the IgG1 antibody, whereas two doses of lipoplex-A induced high levels of the IgG2c antibody. In this study, lipoplex-A vaccination did not lead to a significant reduction in H. pylori colonisation in a challenge model; however, these results point to the utility of the system for delivering DNA vaccine-encoded antigens to induce immune responses and suggest the ability to tailor those responses.

An Evaluation of Urease A Subunit Nanocapsules as a Vaccine in a Mouse Model of Helicobacter pylori Infection.

Using removable silica templates, protein nanocapsules comprising the A subunit of Helicobacter pylori urease (UreA) were synthesised. The templates were of two sizes, with solid core mesoporous shell (SC/MS) silica templates giving rise to nanocapsules of average diameter 510 nm and mesoporous (MS) silica templates giving rise to nanocapsules of average diameter 47 nm. Both were shown to be highly monodispersed and relatively homogenous in structure. Various combinations of the nanocapsules in formulation were assessed as vaccines in a mouse model of H. pylori infection. Immune responses were evaluated and protective efficacy assessed. It was demonstrated that vaccination of mice with the larger nanocapsules combined with an adjuvant was able to significantly reduce colonisation.

Design and Synthesis of Protein-Based Nanocapsule Vaccines.

Increasing emergence of infectious diseases is driving demand for new vaccine technologies capable of improving antigen delivery and protective efficacy. Nanoparticle technology is a modern approach to antigen delivery, capable of stabilizing and increasing the amount of antigen delivered to immune cells. Protein-based nanoparticles are a biodegradable alternative to existing nanomaterials, offering a versatile and biocompatible approach to nanoparticle vaccine delivery. In this chapter, the methods for the synthesis and characterization of protein-based nanocapsule vaccines are discussed. Initially, the requirements for a suitable nanoparticle vaccine are outlined, and finally, methods for the design and synthesis of protein-based nanocapsule vaccines are explained.

Design and Preparation of Solid Lipid Nanoparticle (SLN)-Mediated DNA Vaccines.

Increasing application of nucleic acid vaccines is driving demand for new delivery systems to improve stability and efficacy of DNA vaccines. Solid lipid nanoparticles (SLN) are a particulate carrier system composed of a solid lipid core and a cationic lipid surface suitable for binding negatively charged DNA. SLN delivery systems can be used to bind DNA resulting in an SLN/DNA complex (termed "lipoplex") which can be used as a potential vaccine.In this chapter, the methodologies associated with the use of SLNs as a DNA vaccine nanocarrier are discussed. First, requirements for an effective experimental lipoplex vaccine are discussed along with current and historical examples. Then, flowcharts for design and synthesis of lipoplex vaccines are outlined, followed by detailed materials and methods for synthesis and characterization of lipoplex vaccines.

Solid Lipid Nanoparticle Carrier Platform Containing Synthetic TLR4 Agonist Mediates Non-Viral DNA Vaccine Delivery.

There is a growing demand for better delivery systems to improve the stability and efficacy of DNA vaccines. Here we report the synthesis of a non-viral DNA vaccine delivery system using a novel adjuvanted solid lipid nanoparticle (SLN-A) platform as a carrier for a DNA vaccine candidate encoding the Urease alpha (UreA) antigen from Helicobacter pylori. Cationic SLN-A particles containing monophosphoryl lipid A (adjuvant) were synthesised by a modified solvent-emulsification method and were investigated for their morphology, zeta potential and in vitro transfection capacity. Particles were found to bind plasmid DNA to form lipoplexes, which were characterised by electron microscopy, dynamic light scattering and fluorescence microscopy. Cellular uptake studies confirmed particle uptake within 3 h, and intracellular localisation within endosomal compartments. In vitro studies further confirmed the ability of SLN-A particles to stimulate expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) in human macrophage-like Tohoku Hospital Pediatrics-1 (THP-1) cells. Lipoplexes were found to be biocompatible and could be efficiently transfected in murine immune cells for expression of recombinant H. pylori antigen Urease A, demonstrating their potential as a DNA vaccine delivery system.

Protein-only nanocapsules induce cross-presentation in dendritic cells, demonstrating potential as an antigen delivery system.

Templating has been demonstrated to be an efficient method of nanocapsule preparation. However, there have been no reports of using protein-only nanocapsules as an antigen delivery system. Such a system would enable the delivery of antigen without additional polymers. This study focused on defining the structural and cellular characteristics of nanocapsules consisting of antigen (ovalbumin) alone, synthesized by the templating method using highly monodispersed solid core mesoporous shell (SC/MS) and mesoporous (MS) silica nanoparticles of 410 nm and 41 nm in diameter, respectively. The synthesized ovalbumin nanocapsules were homogeneous in structure, and cellular uptake was observed in DC2.4 murine immature dendritic cells with minimal cytotoxicity. The nanocapsules were localized intracellularly and induced antigen presentation by the cross-presentation pathway. The templating system, using SC/MS and MS silica nanoparticles, was demonstrated to be an effective nanocapsule synthesis method for a new antigen delivery system.