Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.vaccine platform | SAM vaccine | respiratory syncytial virus | HIV

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem1. However, our understanding of the molecular interaction of WNV (and related flaviviruses) with mammalian host cells is limited1. WNV encodes only 10 proteins, implying that the virus may use many cellular proteins for infection1. WNV enters the cytoplasm through pHdependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway1 -3. RNAinterference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions4 -6. Here we report the identification of 305 host proteins impacting WNV infection,

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

The Japanese encephalitis virus (JE) structural glycoprotein (E) and two nonstructural glycoproteins (NS1 and NS1') were processed differently by JE-infected vertebrate and invertebrate cell lines. All three proteins were released slowly (t1/2 greater than 6 hr) from JE-infected monkey cells (Vero cells). Mosquito cell lines released E at a similar rate (t1/2 greater than 8 hr), while NS1 and NS1' were retained in an undegraded form in the cell layer. The proteolytic processing of the three proteins appeared identical in both cell types, but some differences in N-linked glycosylation were observed. E, NS1, and NS1' found within the infected cells of both types contained high-mannose oligosaccharide groups for more than 8 hr after synthesis. Additional sugar residues were added to the single E protein oligosaccharide group prior to release from Vero cells, while sugar residues were trimmed from the E protein oligosaccharide group prior to release from mosquito cells. The forms of NS1 and NS1' found in the culture fluid of infected Vero cells contained one complex and one high-mannose oligosaccharide. All three glycoproteins released from JE-infected Vero cells were associated with extracellular particles, the virion in the case of E and a low density particle in the case of and NS1' exhibited amphipathic properties in Triton X-114 extraction experiments. Taken together, these results suggest that both the structural (E) and nonstructural (NS1 and NS1') glycoproteins were pathway of the infected Vero cells, assembled into particles, and then released into the extracellular fluid.