Epitope-loaded nanoemulsion delivery system with ability of extending antigen release elicits potent Th1 response for intranasal vaccine against Helicobacter pylori.

BACKGROUND: Helicobacter pylori (H. pylori) infection remains a global public health issue, especially in Asia. Due to the emergence of antibiotic-resistant strains and the complexity of H. pylori infection, conventional vaccination is the best way to control the disease. Our previous study found that the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) is an effective protective antigen for vaccination against H. pylori infection, and intranasal immunization with the immunodominant HpaA epitope peptide (HpaA 154-171, P22, MEGVLIPAGFIKVTILEP) in conjunction with a CpG adjuvant decreased bacterial colonization in H. pylori-infected mice. However, to confer more robust and effective protection against H. pylori infection, an optimized delivery system is needed to enhance the P22-specific memory T cell response. RESULTS: In this study, an intranasal nanoemulsion (NE) delivery system offering high vaccine efficacy without obvious cytotoxicity was designed and produced. We found that this highly stable system significantly prolonged the nasal residence time and enhanced the cellular uptake of the epitope peptide, which powerfully boosted the specific Th1 responses of the NE-P22 vaccine, thus reducing bacterial colonization without CpG. Furthermore, the protection efficacy was further enhanced by combining the NE-P22 vaccine with CpG. CONCLUSION: This epitope-loaded nanoemulsion delivery system was shown to extend antigen release and elicit potent Th1 response, it is an applicable delivery system for intranasal vaccine against H. pylori.

Intranasal immunization with immunodominant epitope peptides derived from HpaA conjugated with CpG adjuvant protected mice against Helicobacter pylori infection.

HpaA is considered to be an effective protective antigen for vaccination against Helicobacter pylori (H. pylori) infection. Oral immunization with HpaA significantly decreases bacterial colonization in H. pylori infected mice. In this study, we investigated whether subcutaneous or intranasal immunization with HpaA could protect against H. pylori infection. Mice immunized subcutaneously with HpaA in Complete Freund's adjuvant, but not mice intranasally immunized with HpaA in CpG adjuvant acquired protection against H. pylori infection. However, intranasal immunization with immunodominant epitope peptides in CpG adjuvant protected mice against H. pylori infection, and immunodominant epitope peptides stimulated stronger Th1 responses and mediated more robust protection against H. pylori infection than subdominant ones. Our results suggest that the length of a candidate antigen is critical for particular vaccination routes, and that immunodominant epitope peptides are promising candidates for protection against H. pylori infection through nasal vaccination.

Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice.

Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1- and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

Small Bowel Endoscopy Diagnostic Yield and Reasons of Obscure GI Bleeding in Chinese Patients.

Aim. To investigate the diagnostic yield and etiologies of patients with obscure gastrointestinal bleeding (OGIB) using capsule endoscopy (CE) or double-balloon enteroscopy (DBE). Method. We studied the data of 532 consecutive patients with OGIB that were referred to Xinqiao Hospital in Chongqing from December 2005 to January 2012. A lesion that was believed to be the source of the bleeding (ulceration, mass lesion, vascular lesion, visible blood, inflammation, or others) was considered to be a positive finding. We analyzed the diagnostic yield of CE and SBE and the etiologies of OGIB. Result. CE and SBE have similar diagnostic yields, at 71.9% (196/231) and 71.8% (251/304), respectively. The most common etiology was erosions/ulceration (27.1%) followed by mass lesion (19.4%) and angiodysplastic/vascular lesions (13.9%). By stratified analysis, we found that erosions/ulceration (27.1%) was the most common etiology for the 21-40-year age group. Mass lesion was the most common etiology in the 41-60-year age group. However, in the >60 years age group, angiodysplastic/vascular lesions were significantly increased compared with the other groups, even though erosions/ulceration was most common. Conclusion. In this study, we found that CE and SBE have similar diagnostic yields and erosions/ulceration was the most common reason for OGIB, followed by mass lesion and angiodysplasias.

Bone morphogenetic protein-2 gene controls tooth root development in coordination with formation of the periodontium.

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.

Identification of two novel immunodominant UreB CD4(+) T cell epitopes in Helicobacter pylori infected subjects.

An epitope-based vaccine is a promising option for treating Helicobacter pylori (H. pylori) infection. Epitope mapping is the first step in designing an epitope-based vaccine. A pivotal role of CD4(+) T cells in protection against H. pylori has been accepted, but few Th epitopes have been identified. In this study, two novel UreB CD4(+) T cell epitopes were identified using PBMCs obtained from two H. pylori infected subjects. We determined the restriction molecules by antibody blocking and used various Epstein-Barr virus-transformed B lymphocyte cell lines (BLCLs) with different HLA alleles as APCs to present peptides to CD4(+) T cells. These epitopes were DRB1*1404-restricted UreB(373-385) and DRB1*0803-restricted UreB(438-452). The T cells specific to these epitopes not only recognized autologous DCs loaded with recombinant UreB but also those pulsed with H. pylori whole cell lysates, suggesting that these epitope peptides are naturally processed. These epitopes have important value for designing an effective H. pylori vaccine.

A dominant CD4(+) T-cell response to Helicobacter pylori reduces risk for gastric disease in humans.

BACKGROUND & AIMS: Immunodominance is an important feature of antiviral, antitumor, and antibacterial cellular immune responses, but it is not well demonstrated in the immune responses against Helicobacter pylori. Antigen-specific CD4(+) T cells protect mice against infection with H pylori. We investigated the immunodominant CD4(+) T-cell response to neuraminyllactose-binding hemagglutinin (HpaA), which is a conserved, H pylori-specific colonization factor that is being investigated as an antigen for vaccination strategies. METHODS: HpaA-specific CD4(+) T cells were expanded with autologous peripheral blood mononuclear cells that had been incubated with recombinant HpaA and characterized using overlapping synthetic peptides. We compared the percentage of CD4(+) T cells with specificity for HpaA(88-100), restricted to HLA-DRB1*1501, among 59 H pylori-infected subjects with different gastric diseases. RESULTS: We identified and characterized several immunodominant CD4(+) T-cell epitopes derived from HpaA. The immunodominant CD4(+) T-cell responses specific to HpaA(88-100) were observed in most H pylori-infected individuals who expressed HLA-DRB1*1501 and were significantly more abundant in patients with less severe diseases (P < .05). CONCLUSIONS: The HLA-DRB1*1501-restricted immunodominant CD4(+) T-cell response to HpaA(88-100) is associated with reduced risk of severe gastric diseases. Further study of these and other immunodominant CD4(+) T-cell responses to H pylori will provide insight into mechanisms of protective immunity and aid in vaccine design.

Systemic immunization with an epitope-based vaccine elicits a Th1-biased response and provides protection against Helicobacter pylori in mice.

Vaccine-mediated Th1-biased CD4+ T cell responses have been shown to be crucial for protection against Helicobacter pylori (H. pylori). In this study, we investigated whether a vaccine composed of CD4+ T cell epitopes together with Th1 adjuvants could confer protection against H. pylori in a mouse model. We constructed an epitope-based vaccine, designated Epivac, which was composed of predicted immunodominant CD4+ T cell epitopes from H. pylori adhesin A (HpaA), urease B (UreB) and cytotoxin-associated gene A product (CagA). Together with four different Th1 adjuvants, Epivac was administered subcutaneously and the prophylactic potential was examined. Compared to non-immunized mice, immunization with Epivac alone or with a Th1 adjuvant significantly reduced H. pylori colonization, and better protection was observed when an adjuvant was used. Immunized mice exhibited a strong local and systemic Th1-biased immune response, which may contribute to the inhibition of H. pylori colonization. Though a significant specific antibody response was induced by the vaccine, no correlation was found between the intensity of the humoral response and the protective effect. Our results suggest that a vaccine containing CD4+ T cell epitopes is a promising candidate for protection against H. pylori infection.