Investigating alternative delivery systems for self-Amplifying RNA vaccines

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.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

Investigating the impact of delivery system design on the efficacy of self-amplifying RNA vaccines

Messenger RNA (mRNA)-based vaccines combine the positive attributes of both live-attenuated and subunit vaccines. In order for these to be applied for clinical use, they require to be formulated with delivery systems. However, there are limited in vivo studies which compare different delivery platforms. Therefore, we have compared four different cationic platforms: (1) liposomes, (2) solid lipid nanoparticles (SLNs), (3) polymeric nanoparticles (NPs) and (4) emulsions, to deliver a self-amplifying mRNA (SAM) vaccine. All formulations contained either the non-ionizable cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDA) and they were characterized in terms of physico-chemical attributes, in vitro transfection efficiency and in vivo vaccine potency. Our results showed that SAM encapsulating DOTAP polymeric nanoparticles, DOTAP liposomes and DDA liposomes induced the highest antigen expression in vitro and, from these, DOTAP polymeric nanoparticles were the most potent in triggering humoral and cellular immunity among candidates in vivo

Microfluidics based manufacture of liposomes simultaneously entrapping hydrophilic and lipophilic drugs

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300 nm. In terms of drug loading, microfluidics production promoted high loading within ∼100 nm vesicles for both the water soluble drug (20–25% of initial amount added) and the bilayer embedded drug (40–42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug

Rational design of adjuvants for subunit vaccines : the format of cationic adjuvants affects the induction of antigen-specific antibody responses

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity

Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP : a case study using protein-loaded liposomes

Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product

Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens

Microfluidic manufacture of solid lipid nanoparticles : a case study on tristearin-based systems

Solid lipid nanoparticles are lipid-based carriers and that can be used for a range of drugs and biomolecules. However, most manufacturing methods currently used do not offer easy translation from laboratory to scale-independent production. Within this study, we have investigated the use of microfluidics to produce solid lipid nanoparticles and investigated their protein loading capability. In the development of the process we have investigated and identified the critical process parameters that impact on the production of the SLNs. Solid lipid nanoparticles based on Tristearin and 1,2-Distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjugate-2000 were formulated using on the Nanoasemblr® Benchtop system from Precision Nanosystems and the flow rate ratio and total flow rate were investigated as process parameters and the particle size, PDI, zeta potential, drug loading and drug release was quantified. Our results demonstrate the suitability of microfluidics as a valid method for solid lipid nanoparticles containing protein production. In terms of key process parameters to consider, both the solvent/aqueous ratio (FRR) and total flow rate were shown to have a notable impact on particle size. However, protein loading capacity was similar across all flow rates tested. Within this study we outline a rapid and easy to adopt protocol for the scale-independent production of solid lipid nanoparticles and this process can support the rapid translation of production methods from bench to clinic

Neuropilin-1 and Integrins as Receptors for Chromogranin A-Derived Peptides

Human chromogranin A (CgA), a 439 residue-long member of the &ldquo;granin&rdquo; secretory protein family, is the precursor of several peptides and polypeptides involved in the regulation of the innate immunity, cardiovascular system, metabolism, angiogenesis, tissue repair, and tumor growth. Despite the many biological activities observed in experimental and preclinical models for CgA and its most investigated fragments (vasostatin-I and catestatin), limited information is available on the receptor mechanisms underlying these effects. The interaction of vasostatin-1 with membrane phospholipids and the binding of catestatin to nicotinic and b2-adrenergic receptors have been proposed as important mechanisms for some of their effects on the cardiovascular and sympathoadrenal systems. Recent studies have shown that neuropilin-1 and certain integrins may also work as high-affinity receptors for CgA, vasostatin-1 and other fragments. In this case, we review the results of these studies and discuss the structural requirements for the interactions of CgA-related peptides with neuropilin-1 and integrins, their biological effects, their mechanisms, and the potential exploitation of compounds that target these ligand-receptor systems for cancer diagnosis and therapy. The results obtained so far suggest that integrins (particularly the integrin avb6) and neuropilin-1 are important receptors that mediate relevant pathophysiological functions of CgA and CgA fragments in angiogenesis, wound healing, and tumor growth, and that these interactions may represent important targets for cancer imaging and therapy