Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5-triphosphate inhibits the activity of the dengue virus 2-O-methyltransferase NS5 domain (NS5MTase DV ). Along with several other guanosine 5-triphosphate analogues such as acyclovir, 5-ethynyl-1-␤-D-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5-triphosphate competes with GTP to bind to NS5MTase DV . A structural view of the binding of ribavirin 5-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase DV , ribavirin 5-triphosphate, and S-adenosyl-L-homocysteine at a resolution of 2.6 Å. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.

The protective Ag of Shigella, the Gram-negative enteroinvasive bacterium causing bacillary dysentery, or shigellosis, is its O-specific polysaccharide (O-SP) domain of the LPS, the major bacterial surface component. As an alternative to the development of detoxified LPS-based conjugate vaccines, recent effort was put into the investigation of neoglycoproteins encompassing synthetic oligosaccharides mimicking the protective Ags of the O-SP. We previously reported that when coupled to tetanus toxoid via single point attachment, a synthetic pentadecasaccharide representing three biological repeating units of the O-SP of Shigella flexneri 2a (SF2a), one of the most common Shigella serotypes, elicits a better serum anti-LPS 2a Ab response in mice than shorter synthetic O-SP sequences. In this study, we show that the pentadecasaccharide-induced anti-LPS 2a Abs protect passively administered naive mice from Shigella infection. Therefore, this three repeating units sequence, which is recognized by anti-SF2a sera from infected patients, acts as a functional mimic of the native polysaccharide Ag. Analyses of parameters influencing immunogenicity revealed that an investigational SF2a vaccine displaying a pentadecasaccharide:tetanus toxoid molar loading of 14:1 triggers a high and sustained anti-LPS Ab response, without inducing anti-linker Ab, when administered four times at a dose corresponding to 1 g of carbohydrate. In addition, the profile of the anti-LPS Ab response, dominated by IgG1 production (Th2-type response), mimics that observed in human upon natural SF2a infection. This synthetic carbohydrate-based conjugate may be a candidate for a SF2a vaccine.

[reaction: see text] The nature of a linker used for preparing glycoconjugate vaccines is of utmost importance as it may lead to immunogenic biomolecules. We report the conjugation of carbohydrate haptens to protein carriers leading to potential vaccines using the traceless Staudinger ligation. The ligation relies on the selective transfer of a phosphane substituent to an azide to form a native amide bond in the final product upon release of an oxidized phosphane byproduct. We designed new phosphino-functionalized cross-linkers suitable for protein carrier derivatization. We evaluated their utility in preparing conjugates using both synthetic and purified bacterial carbohydrates. The use of a borane-protected phosphane which is deprotected at the time of the ligation reaction led to the best results observed thus far in terms of stability toward oxidation and reactivity.