GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

Background: The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a worldwide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques. Methodology/Principal Findings: Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particlemediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1-3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine. Conclusions/Significance: These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skindelivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract. © 2010 Loudon et al

Die Funktionelle Reife der Neurohypophyse bei neonaten Nestfl\ufcchtern und Nesthockern

Volume: 69Start Page: 297End Page: 30

Acid-base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M2+ = Zn2+ , Cd2+), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H+ in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH2 group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximate to 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S- unit (formation degree above 99.99 chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH2 group (pK(a) = 12.65) is dramatically acidified (pK (a) approximate to 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleoside

Influence of a Decreasing Solvent Polarity on the Stability and Structure of Mixed-Ligand Complexes Formed by Copper(II), 2,2'-Bipyridine or 1,10-Phenanthroline and Guanosine 5'-Diphosphate

The stability consts. of the 1:1 complexes formed between Cu(Arm) 2+, where Arm = 2,2'-bipyridine or 1,10-phenanthroline, and (GDP)3- or its monoprotonated form H(GDP)2- were detd. by potentiometric pH titrns. in water and in water contg. 30 or 50 vol.% of 1,4-dioxane (25°; I = 0.1 M, NaNO3). The stability of the binary Cu(GDP)- complex is enhanced due to macro-chelate formation of the diphosphate-coordinated Cu2+ with N7 of the guanine residue as previously shown. In Cu(Arm)(GDP)- the N7 is released from Cu2+ and the stability enhancement of more than one log unit in aq. soln. is clearly attributable to intramol. stack formation between the arom. rings of Arm and the guanine moiety. Indeed, stacked isomers occur to more than 90% in equil. with open unstacked forms. Surprisingly, the same formation degrees of the stacks have previously been obsd. for Cu(Arm)(dGMP) complexes, where dGMP2- = 2'-deoxyguanosine 5'-monophosphate, despite the fact that the overall stability of the latter species is by about 2.7 log units lower. In 1,4-dioxane-water mixts. stack formation is drastically reduced, probably due to hydrophobic solvation of the arom. rings by the ethylene bridges of 1,4-dioxane. The relevance of these results regarding biol. systems is indicated