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Title: Investigating alternative delivery systems for self-amplifying RNA vaccines
Author: Anderluzzi, Giulia
ISNI:       0000 0004 8509 3591
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2019
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The rabies virus is an enveloped, single stranded, negative-sense RNA virus of the Lyssavirus genus, zoonotic pathogens within the family Rhabdoviridae. Although extensive effort has been made in the last decades to develop efficacious vaccines to prevent rabies spread, the virus is still responsible for the mortality of about 24,000 to 90,000 people per year especially in developing countries and it has been classified as one of the major causes of death from infectious diseases in humans. Commercially available rabies vaccines for humans are considered effective, however the production costs are very high and multiple injections are required to achieve protection. Therefore, the development of new vaccines to reduce the toll of rabies disease in the developing world would be highly desirable. Within this project a nucleic acid based vaccine strategy - in particular self-amplifying RNA vaccine (SAM) - has been investigated since this platform was previously reported to elicit protective immune responses, particularly in the case of cell-mediated responses in a safe manner and for a variety of virus disease. To enhance biological stability and cell internalisation, SAM was combined with four cationic delivery systems. Oil-in-water cationic nano emulsions (CNE), polymeric nanoparticles (NPs), lipid nanoparticles (SLNs) and liposomes were formulated in the absence of or in combination with a specific SAM vaccine. Despite the differences in formulation composition, all samples contained the same concentration of cationic lipid - 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), or dimethyldioctadecylammonium (DDA) - known as immunostimulants. In the preliminary studies, two different manufacturing processes such as Microfluidics and Microfluidisation were applied. As a proof of concept, anionic liposomes and solid lipid nanoparticles were formulated and ovalbumin was encapsulated within the delivery systems as model protein antigen. Resulting carriers were compared in terms of their physico-chemical properties. The purpose was to obtain homogeneous formulations with a diameter in the nanometres range with a given manufacturing method. Furthermore, dialysis, tangential flow filtration (TFF) and size exclusion chromatography (SEC) have been tested as purification methods and compared in terms of the ability to remove both residual organic solvent and unloaded protein from samples without altering physico-chemical attributes. These process parameters and purification method optimisations were then applied to produce cationic CNE, NPs, SLNs and liposomes in combination with a specific SAM vaccine. In the preliminary studies and during formulations development optimisation, SAM encoding for green fluorescent protein (SAM-GFP) was used as a model SAM with a reporter function,given the ease of detection in in-vitro cell cultures. However, SAM encoding for rabies glycoprotein (SAM-Rabies) represented the actual antigen of interest, employed in this project for further in vivo analysis. Cationic SLNs, NPs and liposomes were produced using microfluidics, since this method required smaller volumes compared to the Microfluidisation, thus avoiding waste of reagents. However, the Microfluidizer was used to reduce CNE size,due to incompatibility between CNE component and microfluidics chip. Moreover, particles were formulated with SAM encoding the antigen of interest and loaded into or adsorbed onto cationic carriers. All delivery systems were evaluated according to their physico-chemical properties: hydrodynamic radius, sample homogeneity (polydispersity index - PDI) and surface charge. Furthermore, in vitro activity was investigated using three different cell lines:bone marrow derived macrophages (BMDM), bone marrow derived dendritic cells (BMDC) and baby hamster kidney cells (BHK). SAM uptake and antigen expression from each formulation in each cell line were used to discriminate and down-select formulations for invivo studies. In the in vivo studies, biodistribution of carriers alone or in combination with SAM were performed. Briefly the selected SAM-carriers were administered intramuscularly (i.m.) to BALB/c mice and their movement in the animal body was tracked using a radiolabelling technique thereby allowing measurement of formulations at chosen time-points and in specific organs. The aim of the study was to understand the pharmacokinetic profile of formulations in a mouse model and assess whether biodistribution might correlate with subsequent immunogenicity studies. The initial attempt of these studies was to (i) find the antigen dose to induce high antibody and cellular responses in vivo and (ii) to compare the adjuvant properties of selected cationic candidates (i.e. SAM encapsulating DOTAP NPs, DOTAP liposomes and DDA liposomes) after i.m. injection. Formulations were selected according to the potency of inducing antigen expression in vitro. The commercial vaccine Rabipur, which is an inactivated virus rabies vaccine, was used as comparator. The aim was to find a valid and more cost-effective alternative formulation which induced an immune response comparable or superior to the commercial vaccine. Data showed that DOTAP NPs were the most potent in triggering IgG titers among candidates and the antibody levels were equivalent to the ones induced by the commercial vaccine after a single dose. Interestingly, the GMT was well above the protective threshold despite the antigen dose used, thus meaning that elicited antibodies were functional against rabies glycoprotein G. In terms of cellular response all candidates were able to activate both CD4+ and CD8+ T cells in a comparable manner to the vaccine on the market. Moreover, to evaluate if changing the route of administration might affect carriers' potency,SAM encapsulating candidates were also administered intradermally (i.d.) and intranasally (i.n.), and formulations immunogenicity was evaluated according to IgG titres and cellular response. To do so, DOTAP NPs and DOTAP SLNs were selected; NPs were tested considering the promising outcome from the first in vivo study, whereas SLNs were introduced although poor in vitro antigen expression. The aim was to understand the power of in vitro models to predict in vivo antigen immunogenicity. Results highlighted that SLNs injected i.m.showed increased immunogenicity compared to both NPs and the licenced vaccine after a single dose. Moreover, the potency of SLNs was also seen after intradermal administration,where SLNs were as potent as Rabipur to elicit IgG titer in mice after two vaccinations, inducing comparable innate and adaptive immunity to the vaccine on the market. Herein it was also reported that two doses of SAM SLNs injected i.n. induced a humoral immunity which was higher than the one elicited by Rabipur. Interestingly, intranasal administration of SLNs led to a higher percentage of IL-2 producing antigen specific CD4+ T cells compared to the licenced in both spleens and lungs. Although a significant difference was observed among formulations in the ability to enhance antigen-specific IgG titres, immunogenicity did not directly correlate with biodistribution, where carriers' pharmacokinetics were indeed similar. All together, these findings are encouraging and demonstrate that coformulation of SAM vaccine and solid lipid nanoparticles might be a valid and more advantageous alternative to produce rabies vaccines, with augmented patient' safety and compliance.
Supervisor: Baudner, Barbara C. ; Perrie, Yvonne Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral