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

Pneumococcal Pili Are Composed of Protofilaments Exposing Adhesive Clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2–3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host

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

Safety and immunogenicity in animals of subunit influenza vaccine given intranasally with mutants of Escherichia coli heat-labile enterotoxin (LT)

Mucosal delivery of inactivated vaccine against respiratory diseases offers several advantages over the traditional intramuscular injection. This requires the use of a potent adjuvant and/or of delivery systems to obtain strong local and systemic immune responses. Site-directed mutagenesis has allowed the generation of mutants such as LTK63 (Ser to Lys substitution at position 63 in the A subunit) and LTR72 (Ala to Arg at position 72) with abolished or strongly reduced toxicity while still retaining strong mucosal adjuvanticity. Both of them have been used to formulate a subunit influenza vaccine, administered intranasally together with nanoparticles and were shown to be able to potentiate its immunogenicity. These vaccine formulations were totally safe in different animal models. © 2004, Elsevier B.V

The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency

The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID-19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNA-vaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy

Dose Dependent Binding of RrgA to Selected Extracellular Matrix (ECM) Components.

<p>Shown are the results of binding increasing concentrations of purified T4 pilus proteins HisTag-RrgA, -RrgB and -RrgC (A) and HMW pilus preparations (B) to fibronectin, collagen I and laminin. BSA and delta pilus mock preparation served as negative controls. Binding was quantified by ELISA at an absorbance of 405 nm. Points represent the means (error bars, standard errors of the means) of measurements made in triplicate.</p

Triple Immunoelectron-Microscopy (IEM) Analysis of the Pilus Subunits of <i>Streptococcus pneumoniae</i>.

<p>Isolated pili material (A) was incubated with antisera raised against His-tagged RrgA, RrgB and RrgC and conjugated respectively to 15 nm, 5 nm and 10 nm gold particles. The image shows the pilus backbone stained with gold-labelled antibodies raised against the main pneumococcal pilus component (RrgB). Clusters of RrgA ancillary proteins (open arrows) are present along the entire pilus. Single copies of the ancillary protein RrgC (arrows) were found alone or co-localized with the RrgA clusters. Scale bar, 100 nm. The same protocol for triple immunogold EM has been applied to bacteria preparation <i>of Streptococcus pneumoniae</i> T4 (B), showing a similar pattern of gold distribution (scale bar, 100 nm).</p

Micrograph of Negative Stained Whole Cell <i>Streptococcus pneumoniae</i> TIGR4.

<p>Sample stained with 1% buffered phosphotungstic acid (PTA). Open arrows indicate an individual single pilus; arrow indicates bundles of individual pili. Scale bar, 200 nm. (Philips TEM CM200 FEG microscope at 50000× magnification, working at low-dose conditions).</p