ABSTRACT
Polymer nanoparticles (NP) are of escalating interest for their application as immune stimulatory pharmaceutics. The production of nanosized carrier systems is currently being widely investigated, but commonly used techniques, such as the double emulsion technique, are limited by shortcomings of low encapsulation efficiency and poor control over size distribution. In this study, the electrospray technique was successfully implemented and optimized to produce monodisperse 200-nm poly(lactide-co-glycolide) (PLGA) NP. For cytomegalovirus (CMV) pp65 and IE-1 peptides, a consistent encapsulation efficiency of approximately 85% was achieved. In vitro stimulation of peripheral blood mononuclear cells (PBMCs) from CMV+ donors using electrosprayed pp65489-503 peptide-loaded NP revealed a significantly increased proliferation rate and frequency of antigen-specific CD8+ T cells as compared to the soluble peptide. The results of this study demonstrate the suitability of the electrospray technique for production of monodisperse PLGA NP with high drug encapsulation efficiency as promising peptide-based vaccine carriers.
Subject(s)
CD8-Positive T-Lymphocytes/drug effects , Cell Proliferation/drug effects , Drug Carriers/chemistry , Leukocytes, Mononuclear/drug effects , Nanoparticles/chemistry , Peptides/administration & dosage , Polyglactin 910/chemistry , CD8-Positive T-Lymphocytes/cytology , Cells, Cultured , Cytomegalovirus/chemistry , Humans , Immediate-Early Proteins/administration & dosage , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/pharmacology , Leukocytes, Mononuclear/cytology , Peptides/chemistry , Peptides/pharmacology , Phosphoproteins/administration & dosage , Phosphoproteins/chemistry , Phosphoproteins/pharmacology , Spectrometry, Mass, Electrospray Ionization , Trans-Activators/administration & dosage , Trans-Activators/chemistry , Trans-Activators/pharmacology , Vaccines/administration & dosage , Vaccines/chemistry , Vaccines/pharmacology , Viral Matrix Proteins/administration & dosage , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/pharmacologyABSTRACT
Plasmacytoid dendritic cells (PDCs) express Toll-like receptor (TLR) 9, which mediates recognition of microbial DNA during infection or self-DNA in autoimmune diseases. Triggering TLR-9 in PDC induces either maturation (lysosomal TLR-9 triggering) or type I interferon (IFN-I) production (endosomal TLR-9 triggering). PDCs also express BDCA-2 (CD303), a C-type lectin receptor (CLR) unique to these cells. CLRs appear to function in innate immunity and microbial recognition, and may cooperate with TLRs to fine-tune inflammatory responses. It has been shown that anti-BDCA-2 monoclonal antibody is internalized by PDC for antigen presentation and inhibits TLR-9 induced IFN-I expression. Here we investigated the cross-talk between BDCA-2 and TLR-9-signaling during PDC maturation and antigen presentation. We found that BDCA-2-induced signaling in PDCs inhibits up-regulation of CD86 and CD40 molecules in CpG-activated PDCs, but not in CD40L-activated PDCs. Furthermore, triggering of BDCA-2 diminished the ability of CpG- and CD40L-stimulated PDCs to process and present antigen to antigen-specific autologous memory T cells. This study demonstrates that BDCA-2 represents an attractive target for clinical immunotherapy of IFN-I dependent autoimmune diseases influencing both, IFN-I production and antigen-specific T-cell stimulation by PDC.
