Development of nanoparticle based vaccine delivery systems

Abstract

Development of subunit vaccine formulation requires a careful selection of potent antigen, efficient adjuvant and route of delivery. The desirable physicochemical characteristics of the mesoporous silica nanoparticles (MSNs) such as ease of synthesis, excellent in vivo biocompatibility and good thermal and chemical stability, make them optimal nanocarriers for various biomolecules (Mody et al. Nanoscale, 2013). Freeze-drying process can be used to further improve both th e short and long-term stability of protein-loaded nanovaccine components (Mody et al. Drug Deliv. Lett., 2012). Bovine Viral Diarrhoea Virus-1 (BVDV-1) is one of the most serious pathogens, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. E2 is the structural envelope glycoprotein of BVDV-1 and is a major immunogenic determinant, making it an ideal candidate for the development of subunit vaccines. The current research project investigated range of silica nanoparticles with different physico-chemical characteristics for the development of ‘non freeze-dried’ (wet or non-FD) and ‘freeze-dried’ (FD) vaccine delivery systems using model protein Ovalbumin (OVA) and BVDV-1 Escherichia coli-expressed optimised E2 (oE2) protein. The nanoparticles repertoire included amino functionalised mesoporous silica nanoparticles (AM-41), amino functionalised hollow mesoporous silica nanoparticles (HMSAs) and novel silica vesicles (SV). The capacity of AM-41 sized 90 nm to act as self-adjuvants and nanocarriers was first investigated for model protein OVA as non-FD as well as FD [after freeze-drying with trehalose (5%) and PEG8000 (1%)] nanovaccine formulations. Administration of the non-FD and reconstituted FD OVA-41 (after storage for 2 months at ambient temperature) nanovaccine induced both humoral as well as cell-mediated immune responses after four immunisations of 10 µg OVA/150 µg AM-41 (Mody et al. Int. J Pharm, 2014). Low protein adsorption capacity of AM-41 (72 μg OVA/mg AM-41) was a major limitation of this study. Therefore, HMSAs, (particle size 120 nm, pores on the wall of entrance sized 2 nm) were investigated for developing recombinant BVDV-1 E2 nanovaccine (60-80 μg oE2 /mg HMSA). The immunogenicity of the oE2/HMSA nanovaccine before and after a freeze-drying with trehalose (5%) and glycine (1%) was evaluated in a sheep trial. The non-FD and FD oE2/HMSA generated oE2 specific antibody and cell-mediated immune responses after three subcutaneous injections with 500 µg oE2 adsorbed to 6.2 mg HMSA. Importantly, it was found that the long-term cell-mediated immune responses were detectable up to five months after immunisation (Manuscript submitted to PLoS ONE). In order to further improve the oE2 adsorption and immunogenicity of the nanovaccine as compared to conventional adjuvant Quil-A, novel silica vesicles termed SV-140 (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm3g-1) were evaluated. The SV-140 significantly improved the loading capacity (~250 μg/mg) and controlled release of oE2 protein. The in vivo functionality of the developed vaccine delivery system was validated in mice immunisation trials comparing oE2 plus Quil-A (50 μg of oE2 plus 10 μg of Quil-A) to the oE2/SV-140 (50 μg of oE2 adsorbed to 250 μg of SV-140). Compared to the oE2 plus Quil-A, which generated BVDV-1 specific antibody responses at a titre of 104, the oE2/SV-140 group induced a 10 times higher oE2 specific 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 compared to oE2 plus Quil-A (512-1369 SFU/million cells) (Mody et al. Biomaterials, 2014). The ability of oE2/SV-140 and a FD oE2/SV-140 formulation (excipients trehalose (5%) and glycine (0.1%) used to freeze-dry), to generate long-term immune response was investigated after only two subcutaneous injections in mice. The oE2 (100 µg)/SV-140 (500 µg) and FD oE2 (100 µg)/SV-140 (500 µg) nanovaccines generated oE2-specific antibody responses for up to six months post the final second immunisation in mice. Significantly, the cell-mediated responses were consistently high in all the four mice immunised with oE2/SV-140 (1500 SFU/million cells) at the six month time point. The FD oE2/SV-140 also generated strong cell-mediated responses (340-1500 SFU/million cells) at the six month time point. Histopathology studies on the site of injection and different organs of mice immunised with 500 µg SV-140 nanovaccine showed no morphological changes. This showed that the oE2/SV-140 can elicit long-term balanced immune responses for at least six months both as non-FD and FD nanoformulation with SVs acting as excellent self adjuvants and nanocarriers (Manuscript submitted to PLoS ONE). The advancement made in this project addresses key features of: reduction in vaccine dosage, adjuvants, long-term balanced immune response and elimination of cold chain storage towards vaccine delivery.