As shown in Number?2B, 6E10 antibody bound to both peptides: 3A11-PADRE-Thep and N-3A11-PADRE-Thep, whereas rabbit anti-A N-terminus specific antibody recognized only N-3A11-PADRE-Thep (Fig.?2C), demonstrating that transmission sequence cleavage produced a protein with a free aspartic acid at the one position of A. that are currently being carried out in Rhesus monkeys. Keywords: Alzheimer disease, DNA vaccine, T helper epitope, electroporation, humoral immune responses Intro Vaccination methods against AD must be designed to induce strong antibody responses and prevent pro-inflammatory autoreactive T cell reactions that are likely responsible for meningoencephalitis in subset of AD patients enrolled in AN1792 tests.1-8 Therefore, it is crucial to develop a vaccine that is safe enough to be used as an early therapeutic YM-90709 or preventative measure. Previously we reported on immunogenicity, security and restorative effectiveness of an AD DNA epitope vaccine in wild-type and 3xTg-AD mice.9 This vaccine was specifically designed to reduce the risk of T cell-mediated autoimmunity by encoding a non-self T helper cell epitope (PADRE) and a short self B cell epitope from your N-terminus of A. Although this vaccine induced strong humoral B cell reactions SKP1 in mice, the fact that DNA vaccines generally show poor immune reactions in large animals and humans, particularly due to low transfection effectiveness of naked DNA, is another major consideration for the design of novel vaccine strategies. To improve transfection effectiveness of DNA vaccines for humans, numerous DNA delivery systems such as aircraft injectors, gene gun and electroporation (EP) have been developed. EP enhances DNA uptake into cells through the YM-90709 delivery of brief electrical pulses, which transiently destabilize the cell membrane to allow DNA uptake into the cell, probably by electrophoretic movement of the negatively charged DNA within the electrical field.10 EP can increase gene expression in vivo by 100- to 1000-fold compared with needle injection of naked plasmid DNA.11,12 Several electroporation products from VGXi, Inc., Ichor Medical Systems Inc., BTX Harvard Apparatus are now being tested in more than in 30 Phase I-III clinical tests worldwide (http://clinicaltrials.gov/ct2/results?term=electroporation+device). Specifically, a clinical grade EP device (Intramuscular TriGridTM Delivery System, TDS-IM) developed by Ichor Medical Systems is currently being evaluated for DNA vaccine delivery in several clinical tests13 and has been shown to markedly enhance reactions to an HIV vaccine,14 consequently, we aimed to test this delivery system for a novel DNA-based epitope vaccine against AD. With this translational study, we tested TDS-IM and the efficacy of a modified version of the p3A11-PADRE vaccine designed to express 3A11-PADRE protein with free N-terminal aspartic acid fused with eight additional promiscuous Th epitopes (pN-3A11-PADRE-Thep) in rabbits. Results Immunogenicity of second- and third-generation DNA epitope vaccines delivered in rabbits by EP To evaluate whether anti-A reactions to our second-generation DNA epitope vaccine could be scaled up from mice to a larger species, rabbits were immunized intramuscularly with p3A11-PADRE vaccine (Fig.?1A). All 14 animals responded to immunization with concentrations of anti-A antibodies in ranging from 3.1C19.4g/ml (Fig.?1B) and these antibodies were mostly of IgG isotype (Fig.?1C). Next, we used two different approaches to refine the p3A11-PADRE vaccine to enhance its immunogenicity (Fig.?2A and Table 1). First, to enhance the immunogenicity of a vaccine for potential medical use in humans with highly polymorphic classical MHC class II genes, we integrated eight promiscuous foreign Th cell epitopes from standard vaccines into this create (Table 1). Good epitope mapping of sera from individuals enrolled in the AN1792 trial suggested that the free N-terminal aspartic acid of A42 may be essential for induction of antibodies in YM-90709 humans,15 which was also supported by studies in monkeys16 and rabbits.17 Therefore, we next modified p3A11-PADRE-Thep vaccine to generate a construct that would encode an immunogen possessing a free N-terminal aspartic acid following signal sequence cleavage (Fig.?2A). Open in a separate window Number?1. (A) Schematic representation of construct encoding epitope vaccine p3A11-PADRE. (B) p3A11-PADRE induces anti-A antibody reactions in all immunized rabbits. Antibody reactions were analyzed in individual sera after 2nd, 3rd and 4th immunizations by ELISA. Lines show the mean (n = 14). (C) All animals immunized two times with p3A11-PADRE produced anti-A antibodies of IgG isotype. IgG and IgM isotypes of antibodies were analyzed in individual sera of immunized animals at dilution 1:200. Error bars show SD (n = 14). Open in a separate window Number?2. (A) Schematic representation of third generation.