Criteria for dendritic cell receptor selection for efficient antibody-targeted vaccination.

Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently, there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study, we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First, using mixed bone marrow chimeras, we established that Ag-targeted, but not nontargeted, DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next, we analyzed parameters of DEC205 (CD205), Clec9A, CD11c, CD11b, and CD40 endocytosis and obtained quantitative measurements of internalization speed, surface turnover, and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays, we showed that receptor expression level, proportion of surface turnover, or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore, the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast, targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation, respectively. Therefore, receptor expression levels, speed of internalization, and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination.

Selective uptake of cylindrical poly(2-oxazoline) brush-antiDEC205 antibody-OVA antigen conjugates into DEC-positive dendritic cells and subsequent T-cell activation.

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well-defined structures with a high biological specificity. A polymeric nanocarrier is presented that consists of a cylindrical brush polymer with poly-2-oxazoline side chains carrying an azide functional group on each of the many side chain ends. After click conjugation of dye and an anti-DEC205 antibody to the periphery of the cylindrical brush polymer, antibody-mediated specific binding and uptake into DEC205(+) -positive mouse bone marrow-derived dendritic cells (BMDC) was observed, whereas binding and uptake by DEC205(-) negative BMDC and non-DC was essentially absent. Additional conjugation of an antigen peptide yielded a multifunctional polymer structure with a much stronger antigen-specific T-cell stimulatory capacity of pretreated BMDC than application of antigen or polymer-antigen conjugate.

Evaluation of nanoparticle aggregation in human blood serum.

In a certain stage of development, the performance of nanoparticle- or polymer-drug conjugates is tested "in vivo", that is, in mice or rats. Besides pharmaceutical and chemical characterization, the structural characterization of such drug carrier systems in terms of size, size distribution, and shape is typically performed in physiological salt solution prior to animal tests. The present work introduces a simple method based on dynamic light scattering to monitor the particle size in blood serum. Utilizing a model system of pegylated poly-l-lysines (PLL-g-PEOx) of various degrees of pegylation, x, it is demonstrated that large aggregates may form in human serum solution that are not observed in isotonic salt solution. Aggregates of a few hundred nanometers in size were found in mixtures of serum solution and PLL-g-PEOx with degrees of pegylation <10%, whereas no aggregates are being observed if the degree of pegylation exceeds 20%. The described method may have the potential to become an easy and routine test for drug carrier systems prior to animal applications.