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Keywords:MERS HPV CrPV IRES RNA Adjuvant Vaccine Th1/Th2 Alum a b s t r a c t An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as liveattenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3 untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3 terminal sequence. Our investigators previously demonstrated that the 3 terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.The study of GB virus B (GBV-B) has generated considerable interest because it is the virus most closely related to hepatitis C virus (HCV) (16,18). In humans, infection with HCV is one of the leading causes of chronic liver disease, which in many cases will progress to liver cirrhosis and liver cancer (9). The natural host of GBV-B remains unknown, but it was demonstrated that this virus does not infect chimpanzees, a surrogate of humans, suggesting that it is not a human virus (3). However, experimental infection of tamarins with GBV-B causes acute hepatitis. Thus, experimental GBV-B infection of tamarins could potentially serve as a surrogate model for the study of HCV, which can only be studied in chimpanzees.The GBV-B virus contains a positive-sense, single-stranded RNA genome of about 9.5 kb (4, 24). Like the other members of the Flaviviridae virus family, it has a single long open reading frame (ORF), bracketed by 5Ј and 3Ј untranslated regions (UTR). Based on hydropathy plots and known motifs, it was predicted to encode the same kinds of proteins as HCV (16). However, it should be emphasized that the overall homology of the predicted polyproteins between GBV-B and HCV is only about 25 to 30% (16). Significant homology was observed between GBV-B and HCV in the NS3 serine protease, the NS3 RNA helicase, and the NS5 RNA-dependent RNA polymerase regions (16,34,35). Furthermore, functional simi...