Vaccinia Virus Defective Particles Lacking the F17 Protein Do Not Inhibit Protein Synthesis: F17, a Double-Edged Sword for Protein Synthesis?

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

An Oil-Based Adjuvant Improves Immune Responses Induced by Canine Adenovirus-Vectored Vaccine in Mice.

There is a significant need for highly effective vaccines against emerging and common veterinary infectious diseases. Canine adenovirus type 2 (CAV2) vectors allow rapid development of multiple vaccines and have demonstrated their potential in animal models. In this study, we compared the immunogenicity of a non-replicating CAV2 vector encoding the rabies virus glycoprotein with and without MontanideTM ISA 201 VG, an oil-based adjuvant. All vaccinated mice rapidly achieved rabies seroconversion, which was associated with complete vaccine protection. The adjuvant increased rabies antibody titers without any significant effect on the anti-CAV2 serological responses. An RT2 Profiler™ PCR array was conducted to identify host antiviral genes modulated in the blood samples 24 h after vaccination. Functional analysis of differentially expressed genes revealed the up-regulation of the RIG-I, TLRs, NLRs, and IFNs signaling pathways. These results demonstrate that a water-in-oil-in-water adjuvant can shape the immune responses to an antigen encoded by an adenovirus, thereby enhancing the protection conferred by live recombinant vaccines. The characterization of early vaccine responses provides a better understanding of the mechanisms underlying the efficacy of CAV2-vectored vaccines.

The antibody response induced FMDV vaccines in sheep correlates with early transcriptomic responses in blood.

Foot and mouth disease (FMD) is a highly contagious viral disease with high economic impact, representing a major threat for cloven-hooved mammals worldwide. Vaccines based on adjuvanted inactivated virus (iFMDV) induce effective protective immunity implicating antibody (Ab) responses. To reduce the biosafety constraints of the manufacturing process, a non-replicative human adenovirus type 5 vector encoding FMDV antigens (Ad5-FMDV) has been developed. Here we compared the immunogenicity of iFMDV and Ad5-FMDV with and without the ISA206VG emulsion-type adjuvant in sheep. Contrasted Ab responses were obtained: iFMDV induced the highest Ab levels, Ad5-FMDV the lowest ones, and ISA206VG increased the Ad5-FMDV-induced Ab responses to protective levels. Each vaccine generated heterogeneous Ab responses, with high and low responders, the latter being considered as obstacles to vaccine effectiveness. A transcriptomic study on total blood responses at 24 h post-vaccination revealed several blood gene module activities correlating with long-term Ab responses. Downmodulation of T cell modules' activities correlated with high responses to iFMDV and to Ad5-FMDV+ISA206VG vaccines as also found in other systems vaccinology studies in humans and sheep. The impact of cell cycle activity depended on the vaccine types, as it positively correlated with higher responses to iFMDV but negatively to non-adjuvanted Ad5-FMDV. Finally an elevated B cell activity at 24 h correlated with high Ab responses to the Ad5-FMDV+ISA206VG vaccine. This study provides insights into the early mechanisms driving the Ab response induced by different vaccine regimens including Ad5 vectors and points to T cell modules as early biomarker candidates of different vaccine-type efficacy across species.

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors.

Translation initiation of hepatitis C virus (HCV) occurs through an internal ribosome entry site (IRES) located at its 5'-end. As a positive-stranded RNA virus, HCV uses its genome as a common template for translation and replication, but the coordination between these two processes remains poorly characterized. Moreover, although genetic evidence of RNA-protein interactions for viral replication is accumulating because of subgenomic replicons and a recent culture system for HCV, such interactions are still contentious in the regulation of translation. To gain insight into such mechanisms, we addressed the involvement of cis and trans viral factors in HCV IRES activity by using a cell-based RNA reporter system. We found that the HCV 3' noncoding region (NCR) strongly stimulates IRES efficiency in cis, depending on the genotype and the cell line. Moreover, we confirmed the role of the core protein in viral gene expression as previously reported in vitro. Surprisingly, we observed a similar effect, i.e. a twofold increase under low amounts of NS5B RNA polymerase, followed by a decrease at higher concentrations. However, no contribution of NS5A to HCV IRES-mediated translation was noted and no cooperative effect could be detected between 3' NCR and viral proteins or between proteins. Collectively, these results suggest that HCV RNA translation is regulated, and that the switch from translation to replication might involve a sequential requirement for both cis and trans viral factors, because of their apparent lack of synergy, probably with the aid of host factors.