The early cellular signatures of protective immunity induced by live viral vaccination.

Here, we have used primary vaccination of healthy donors with attenuated live yellow fever virus 17D (YFV-17D) as a model to study the generation of protective immunity. In short intervals after vaccination, we analyzed the induction of YFV-17D specific T- and B-cell immunity, bystander activation, dendritic cell subsets, changes in serum cytokine levels, and YFV-17D-specific antibodies. We show activation of innate immunity and a concomitant decline of numbers of peripheral blood T and B cells. An early peak of antigen-specific T cells at day 2, followed by mobilization of innate immune cells, preceded the development of maximal adaptive immunity against YFV-17D at day 14 after vaccination. Interestingly, potent adaptive immunity as measured by high titers of neutralizing YFV-17D-specific antibodies, correlated with early activation and recruitment of YFV-17D-specific CD4(+) T cells and higher levels of sIL-6R. Thus our data might provide new insights into the interplay of innate and adaptive immunity for the induction of protective immunity.

A simple fluorescence-spectroscopic membrane translocation assay.

We have established a combination of fluorescence-spectroscopic uptake, release, and dilution experiments as a powerful tool for studying the translocation of fluorescent compounds across lipid membranes, demonstrating this through intrinsic tryptophan fluorescence for the interaction of the cell-penetrating peptide penetratin with phospholipid membranes, for which conflicting results have been reported. We found that penetratin is not membrane-permeant under the conditions used here. To confirm this finding and to validate the approach, we also employed an established titration-calorimetric method, the results of which were in excellent agreement with a thermodynamic analysis of the fluorescence-spectroscopic experiments. Further support was provided by a comparison with published data obtained under similar conditions by using a variety of techniques. Unlike these methods, however, the new approach allows consistent and simultaneous assessment of membrane binding and transbilayer movement without depending on extrinsic labels attached to the molecule of interest or on reporter moieties inserted into the lipid membrane.