*3.3. RNA-Based Delivery*

Similar to recombinant alphavirus particle delivery, RNA replicon administration has proven efficient in vaccine development [34]. For example, a single intramuscular injection of 0.1 μg SFV-LacZ replicon RNA generated antigen-specific antibody and CD8+ T cell responses in immunized mice [37]. Immunization with SFV-LacZ RNA prior to challenges by colon tumors provided protection in mice. Moreover, the therapeutic vaccination of animals with pre-existing tumors resulted in prolonged survival. Interestingly, the levels of antigen production for RNA replicons in vitro were not significantly higher than those observed for conventional DNA vaccines, but in vivo the enhanced efficacy correlated with a caspase-dependent apoptotic cell death. In another approach, a SIN RNA replicon expressing the rabies virus glycoprotein gene was applied for immunization studies with 10 μg of SIN-Rab-G RNA in comparison to a conventional rabies DNA vaccine and the commercial cell culture vaccine Rabipur [38]. The SIN-Rab-G RNA immunization elicited similar cellular and humoral IgG responses in comparison to the rabies DNA vaccine. Moreover, the alphavirus RNA vaccine provided similar protection to the rabies DNA vaccine against challenges with the lethal rabies virus CVS strain.

In addition to naked RNA delivery, alphavirus vectors have also been subjected to nanoparticle encapsulation procedures [39]. An in vivo expression comparison of 1 × 106 IU recombinant VEE particles, 1 μg of naked replicon RNA, 1 μg of replicon RNA encapsulated in lipid nanoparticles (RNA/LNPs), 10 μg of conventional plasmid DNA, and 10 μg of replicon DNA expressing firefly luciferase was carried out in mice 7 days after bilateral intramuscular administration. The luciferase levels were similar for RNA/LNPs and VEE particles, but significantly higher than for naked replicon RNA, replicon DNA, and plasmid DNA. The immunogenicity of delivery modes was evaluated by heterologous expression of the respiratory syncytial virus fusion protein (RSV-F) after intramuscular administration. The F-specific IgG response to 1 <sup>μ</sup>g RNA/LNPs was equivalent to that of <sup>1</sup> × 106 IU of VEE particles. In contrast, plasmid DNA/LNPs at a dose of 0.1 μg and 20 μg of electroporated plasmid DNA elicited much lower IgG titers. RNA/LNPs, replicon RNA, VEE particles and an RSV-F subunit vaccine were evaluated for protection against viral challenges after intranasal RSV challenges in cotton rats. All replicon RNA vaccines protected animals for RSV challenges reducing the viral load more than 1000-fold in the lungs. The RNA/LNPs (1 μg) elicited similar responses as VEE particles. However, the recombinant F subunit vaccine formulated with alum showed the highest potency. In another study, naked RNA from SFV replicon (rSFV-NP) and poliovirus (rDELTA1-E-NP) vectors expressing the influenza type A virus nucleoprotein (NP) were intramuscularly administered in C57BL/6 mice [40]. Both rSFV-NP and rDELTA1-E-NP elicited antibodies against the influenza virus NP, but CTL responses against the immunodominant H-2D(b) epitope NP366 was only obtained with the SFV replicon RNA. Furthermore, reduced virus load was demonstrated for rSFV-NP after challenges with a mouse-adapted influenza A/PR/8/34 virus in immunized mice. The protective potential for RNA replicon immunization was similar to what has previously been achieved for plasmid DNA immunizations.
