Donna Mahony

Mesoporous silica nanoparticles as antigen carriers and adjuvants for vaccine delivery

Vaccines have been at the forefront of improving human health for over two centuries. The challenges faced in developing effective vaccines flow from complexities associated with the immune system and requirement of an efficient and safe adjuvant to induce a strong adaptive immune response. Development of an efficient vaccine formulation requires careful selection of a potent antigen, efficient adjuvant and route of delivery. Adjuvants are immunological agents that activate the antigen presenting cells (APCs) and elicit a strong immune response. In the past decade, the use of mesoporous silica nanoparticles (MSNs) has gained significant attention as potential delivery vehicles for various biomolecules. In this review, we aim to highlight the potential of MSNs as vaccine delivery vehicles and their ability to act as adjuvants. We have provided an overview on the latest progress on synthesis, adsorption and release kinetics and biocompatibility of MSNs as next generation antigen carriers and adjuvants. A comprehensive summary on the ability of MSNs to deliver antigens and elicit both humoral and cellular immune responses is provided. Finally, we give insight on fundamental challenges and some future prospects of these nanoparticles as adjuvants.

Structure of Vps26B and Mapping of its Interaction with the Retromer Protein Complex

Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single‐binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G‐protein‐coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure‐based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C‐terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.

Mesoporous silica nanoparticles act as a self-adjuvant for ovalbumin model antigen in mice

Immunization to the model protein antigen ovalbumin (OVA) is investigated using MCM-41 mesoporous silica nanoparticles as a novel vaccine delivery vehicle and adjuvant system in mice. The effects of amino surface functionalization and adsorption time on OVA adsorption to nanoparticles are assessed. Amino-functionalized MCM-41 (AM-41) shows an effect on the amount of OVA binding, with 2.5-fold increase in binding capacity (72 mg OVA/g AM-41) compared to nonfunctionalized MCM-41 (29 mg OVA/g MCM-41). Immunization studies in mice with a 10 μg dose of OVA adsorbed to AM-41 elicits both antibody and cell-mediated immune responses following three subcutaneous injections. Immunizations at a lower 2 μg dose of OVA adsorbed to AM-41 particles results in an antibody response but not cell-mediated immunity. The level of antibody responses following immunization with nanoformulations containing either 2 μg or 10 μg of OVA are only slightly lower than that in mice which receive 50 μg OVA adjuvanted with QuilA, a crude mixture of saponins extracted from the bark of the Quillaja saponaria Molina tree. This is a significant result, since it demonstrates that AM-41 nanoparticles are self-adjuvanting and elicit immune responses at reduced antigen doses in vivo compared to a conventional delivery system. Importantly, there are no local or systemic negative effects in animals injected with AM-41. Histopathological studies of a range of tissue organs show no changes in histopathology of the animals receiving nanoparticles over a six week period. These results establish the biocompatible MCM-41 silica nanoparticles as a new method for vaccine delivery which incorporates a self-adjuvant effect.

Towards defining the nuclear proteome

BackgroundThe nucleus is a complex cellular organelle and accurately defining its protein content is essential before any systematic characterization can be considered.ResultsWe report direct evidence for 2,568 mammalian proteins within the nuclear proteome: the nuclear subcellular localization of 1,529 proteins based on a high-throughput subcellular localization protocol of full-length proteins and an additional 1,039 proteins for which clear experimental evidence is documented in published literature. This is direct evidence that the nuclear proteome consists of at least 14% of the entire proteome. This dataset was used to evaluate computational approaches designed to identify additional nuclear proteins.ConclusionThis represents direct experimental evidence that the nuclear proteome consists of at least 14% of the entire proteome. This high-quality nuclear proteome dataset was used to evaluate computational approaches designed to identify additional nuclear proteins. Based on this analysis, researchers can determine the stringency and types of lines of evidence they consider to infer the size and complement of the nuclear proteome.

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene.

Conventional kinesin is a microtubule‐based molecular motor involved in the transport of membranous and non‐membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre‐mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3′ of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3′ untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.

Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates

BackgroundProtein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB).ResultsThe expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies.ConclusionWe have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (~6 nm), large cavity (~40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (∼ 250 μg/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 μg of oE2 plus 10 μg of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 μg of oE2 adsorbed to 250 μg of SV-140) or oE2/SV-140 together with 10 μg of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery.

Identifying novel peroxisomal proteins.

Peroxisomes are small subcellular compartments responsible for a range of essential metabolic processes. Efforts in predicting peroxisomal protein import are challenged by species variation and sparse sequence data sets with experimentally confirmed localization.

Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein

Bovine Viral Diarrhoea Virus (BVDV) is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV) based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2) antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm3g-1) have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD) as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 μg)/SV-140 (500 μg) and FD oE2 (100 μg)/SV-140 (500 μg) to induce long-term immunity was compared to immunisation with oE2 (100 μg) together with the conventional adjuvant Quil-A from the Quillaja saponira (10 μg) in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells) at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 μg SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications.

Shaping Nanoparticles with Hydrophilic Compositions and Hydrophobic Properties as Nanocarriers for Antibiotic Delivery.

Inspired by the lotus effect in nature, surface roughness engineering has led to novel materials and applications in many fields. Despite the rapid progress in superhydrophobic and superoleophobic materials, this concept of Mother Nature’s choice is yet to be applied in the design of advanced nanocarriers for drug delivery. Pioneering work has emerged in the development of nanoparticles with rough surfaces for gene delivery; however, the preparation of nanoparticles with hydrophilic compositions but with enhanced hydrophobic property at the nanoscale level employing surface topology engineering remains a challenge. Herein we report for the first time the unique properties of mesoporous hollow silica (MHS) nanospheres with controlled surface roughness. Compared to MHS with a smooth surface, rough mesoporous hollow silica (RMHS) nanoparticles with the same hydrophilic composition show unusual hydrophobicity, leading to higher adsorption of a range of hydrophobic molecules and controlled release of hydrophilic molecules. RMHS loaded with vancomycin exhibits an enhanced antibacterial effect. Our strategy provides a new pathway in the design of novel nanocarriers for diverse bioapplications.