Self-Replicating RNA Vaccine Delivery to Dendritic Cells

Abstract

Most current vaccines are either inactivated pathogen-derived or protein/peptide-based, although attenuated and vector vaccines have also been developed. The former induce at best moderate protection, even as multimeric antigen, due to limitations in antigen loads and therefore capacity for inducing robust immune defense. While attenuated and vector vaccines offer advantages through their replicative nature, drawbacks and risks remain with potential reversion to virulence and interference from preexisting immunity. New advances averting these problems are combining self-amplifying replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (12-15 kb) derived from viral genomes defective in at least one structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitation with RepRNA is RNase-sensitivity and inefficient uptake by dendritic cells (DCs)-absolute requirements for efficacious vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Encapsulating RepRNA into chitosan nanoparticles, as well as condensing RepRNA with polyethylenimine (PEI), cationic lipids, or chitosans, has proven effective for delivery to DCs and induction of immune responses in vivo.

Establishing Preferred Product Characterization for the Evaluation of RNA Vaccine Antigens

“…Following production, RNA transcripts are digested with DNAse I to degrade DNA templates, and then purified to remove DNA degradants, reaction components, and other contaminants. Purification method include commerical spin columns, LiCl precipitation, phenol-chloroform extraction, and chromatography [23][24][25].…”

“…Following separation by molecular mass on agarose or acrylamide gels, nucleic acids can be visualized using fluorescent dyes such as ethidium bromide, SYBR Green or SYBR Gold (Life Technologies, Carlsbad, CA, USA) [32][33][34], and then sized or quantified by densitometry using known RNA standards, as demonstrated for PhI p5, an allergen used to prevent allergic reactions to grass pollen [35]. The sharpness of the band is also sometimes considered an indicator of quality [23], as described for mRNA vaccines against influenza [36]. As with dye-based assays in solution, this method requires proper handling and disposal of potentially hazardous reagents [37], as well as capital equipment such as electrophoresis tanks, high-resolution scanners, and image-analysis software.…”

“…Although naked RNA transcripts can be physically delivered into cells by electroporation, they are, instead, often formulated with excipients that confer additional functional properties. For example, most delivery vehicles are expected or designed to protect against RNAses, and such protection is evaluated by electrophoretic mobility shift assay [23]. Transcripts formulated in particulate vehicles can also be evaluated for particle size, polydispersity, and zeta potential [23,25,48].…”

The Way Forward: Potentiating Protective Immunity to Novel and Pandemic Influenza Through Engagement of Memory CD4 T Cells

“…Critical considerations for this goal likely include administration of antigen via intranasal inoculation, inclusion of influenza proteins robustly expressed early during infection, targeting to the most efficacious lung-derived antigen-presenting cell, via engagement of specific lectin receptors on antigen presenting cell [ 75–77 ]. Also of potential value would be development of viral antigen constructs, such as self-replicating RNA, that promote antigen persistence [ 78 , 79 ], based on the studies of natural infection that suggest antigen persistence is an important parameter for effector T-cell function in the lung [ 62 , 80 ].…”

Dendritic Cell Endocytosis Essential for Viruses and Vaccines

“…Initiation of a virus infectious process may involve the viral membrane (with enveloped viruses) or virus surface proteins; thus, cytosolic translocation can be facilitated by membrane fusion and 'flipping', or through formation of ion channels and elaborating membrane pores for delivery of the RNA genome [25]. Application of this knowledge has recently been employed with synthetic biodegradable nanoparticulate vehicles for enhancing delivery of self-amplifying/replicating RepRNA vaccines to DCs [9,13,[17][18][19][20][33][34][35] (Figure 3 pathway (d)). Whilst success with this approach has been forthcoming with mRNA vaccine delivery to DCs [9,36,37], delivery of the larger replicon RepRNA molecules has required additional considerations.…”