We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of anH. pyloriurease D panthenol T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. D panthenol Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine againstH. pyloriinfection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform. == Introduction == Helicobacter pyloriis a gram negative microaerophilic bacterium which infects the gastric mucosa of approximately half of the world’s population and is a risk factor for both peptic ulcer disease and gastric cancers[1],[2]. The bacteria live in the mucus layer overlying gastric epithelial cells, an environment from which it is able to provoke host inflammatory and immune responses. These host responses are unable to eradicate the infection, however, so that without treatment, the infection can persist for decades or even the life of the host. Although pharmacologic agents can cure the infection, multi-drug regimens which can have significant side effects are required. Using combinations of antibiotics and agents such as proton pump inhibitors it is possible to achieve eradication rates as high as D panthenol 8090%, but failures can lead to antibiotic resistance and re-infection is not uncommon[3],[4]. An alternative and more attractive approach is vaccination which not only leads to more vigorous immune responses than infection but it is also likely to provide herd immunity, dramatically reducing spread of infection. Several candidate vaccines and mucosal vaccines, in particular, have been shown in animal models to reduce or eliminate bacterial load and disease in the stomach[5],[6]. Although an abundance of purified/recombinantH. pyloriantigens and vaccine adjuvants have been successfully used in animal models ofH. pylorivaccination, bacterial lysates and whole cell vaccines combined with the holotoxins cholera toxin (CT) or the closely relatedE. coliheat labile toxin (LT) as mucosal adjuvants have been the gold standard in animal models ofH. pylorivaccination[5]. Most vaccine regimens require an adjuvant and work best intranasally (i.n)[7]or sublingually[8]. Numerous studies in animal models have also demonstrated that antibodies are not required for (but may participate in or even impair) protective immunity, but, in contrast, specific CD4 T cell responses are required forH. pylorivaccine efficacy[9],[10],[11],[12],[13]. Among subunit and vector vaccines,H. pyloriurease has been a leading candidate[14],[15],[16]and both CD4 T cell and B cell peptide epitopes have been defined[17],[18]. Cholera toxin D panthenol or LT have been the most effective and widely used adjuvants forH. pylorimucosal vaccines in animal models. These bacterial toxins are well tolerated when used at adjuvant effective doses in mice and other small animal models ofH. pyloriinfection. CT and LT are too toxic for humans, however, and in a human clinicalH. pylorivaccine trial, the use of holotoxin LT resulted in significant diarrhea in 2/3 of the vaccine recipients[19]. Mutations targeting the active sites of these molecules can reduce the toxicity while retaining adjuvant function and these mutant toxins have been used with some success as mucosal adjuvants forH. pylorivaccines[20],[21]. Our approach has been to create chimeric CT-derived molecules which retain the full enzymatic activity of the holotoxin, but which specifically target immune cells instead of all nucleated GM1-receptor carrying cells, including nerve cells[22]. In this approach we have linked the enzymatically active CTA1 fragment of CT to two copies of the D-fragment ofStaphylococcus aureusprotein A, a strong immunoglobulin binding domain, to create an adjuvant that we have named CTA1-DD[23]. We have shown that this molecule is nontoxic when delivered i.n to mice and non-human primates[24],[25]and significantly reduces the bacterial burden when used as a mucosal adjuvant for anH. pylorilysate Igfbp1 vaccine in mice[26],[27]. We recently demonstrated a related approach in which a peptide from influenza virus, the M2e peptide, was inserted into CTA1-DD (CTA1-M2e-DD) and found to effectively protect against infection in mice[28],[29]. We now report that an MHC class II restrictedH. pyloripeptide inserted into CTA1-DD also can successfully immunize and protect Balb/c mice againstH. pyloriinfection[18]. == Materials and Methods == == H. pylorigrowth, mice, immunization, and challenge == H. pylorifor infection and preparation of bacterial lysate was grown as previously published[30]. Balb/c female mice aged 68 weeks were obtained from Charles River Laboratories (Portage, MI) or from Harlan Laboratories (the Netherlands) and were housed in microisolater cages under specific pathogen free (spf) conditions with free.