Background Urease subunit B (UreB), a conserved and key virulence factor of Helicobacter pylori ( H. pylori ), can induce the host CD4 + T cell immune responses to provide protection, but less is known regarding CD8 + T cell responses. The characteristics of H. pylori -specific CD8 + T cell responses and the mechanism underlying antigen processing and presentation pathways remain unclear. This study was focus on protective antigen recombinant UreB (rUreb) to detect specific CD8 + T cell responses in vitro and elucidate the mechanism of UreB antigen processing and presentation. Methods The peripheral blood mononuclear cells (PBMCs) collected from H. pylori -infected individuals were stimulated with rUreB in vitro to detect specific CD8 + T cell responses after co-culture with rUreB-pulsed autologous hMDCs. Through blocking assay, we investigated the potential pathway of UreB antigen processing and presentation via the cytosolic pathway or vacuolar pathway. The cytokines production of UreB specific CD8 + T cell were evaluated as well. Results We demonstrated UreB can induce specific CD8 + T cell immune responses in H. pylori infected individuals. Importantly, we characterized that UreB were mainly processed by proteasome instead of lysosomal proteases and presented through cytosolic pathway of cross-presentation, which requires endoplasmic reticulum–Golgi transport and newly synthesized MHC-I molecules, to induce functional-specific CD8 + T cell (IFN-γ + TNF-α + Grz A+ Grz B+) responses. Conclusions These results suggest that H. pylori UreB induces specific CD8 + T cell responses through cytosolic pathway of cross-presentation in infected individuals.
Mucosal vaccines are considered an ideal choice for combating mucosal pathogens due to their ability to neutralize pathogens at the first line of defense. However, the development of mucosal subunit vaccines is constricted by rigorous challenges, such as low immunogenicity, poor antigen delivery efficiency, and mucosal tolerance. Here, a buccal microneedle patch incorporated with engineered nanoparticles loaded with urease B subunit (rUreB) was developed to overcome the above challenges. Specifically, an engineered nanocarrier was developed to protect the antigen and modulate its release profile. Then, the nanoparticles were enriched to form microneedle tips with superior mechanical and waterproof properties, allowing effective penetration of the buccal mucosa and resistance to salivary washout. Besides, the microneedles demonstrated an S-curve antigen release pattern which was crucial for the recruitment of antigen presenting cells (APCs) and the downregulation of mucosal tolerogenic DCs and Treg cells. The buccal microneedle vaccine without any immune stimulators induced potent systemic and mucosal immune responses, resulting in superior protection of mice from the oral challenge of Helicobacter pylori . These results suggested that the rationally designed buccal microneedle vaccine can effectively overcome mucosal delivery barriers and mucosal tolerance, providing a promising alternative strategy for mucosal vaccination of subunit antigens.
Chen Ma,Dingnan Lu,Fangyuan Zhou,Jiayue Luo,Huihui Gan,Weidong He,Pradeep U. Kurup,David Z. Zhu
Due to the almost-complete dependence on clinical diagnosis, current Helicobacter pylori (H. pylori) epidemiological statistics suffer from fragmented geographical coverage and lagged data updating worldwide. Acquiring information based on the quantitative detection of biomarkers from domestic wastewater provides a promising way for the future H. pylori epidemiological study. Here, we report wastewater-adapted electrochemical competitive immunosensor (WECI). This method focuses explicitly on detecting H. pylori cytoplasmic ureB in wastewater and contains a series of designs to overcome the challenges brought by the complicated analytical matrix. The developed WECI achieved a practical detection range from 10 pg mL $^{-{1}}$ to 5.0 ng mL $^{-{1}}$ ( ${R}^{{2}}$ = 0.996) and showed the corresponding limit of detection (LoD) and limit of quantification (LoQ) of 0.37 and 1.4 pg mL $^{-{1}}$ , respectively. It can be prepared within 6 h and kept highly sensitive for at least six weeks. When testing real wastewater samples, WECI resulted in an average biomarkers recovery rate of 70.9%, which was remarkably higher than the average of 47.0% achieved using qPCR, indicating the excellent applicability toward actual wastewater detection. Our reported WECI is the first study to quantitatively detect H. pylori ureB from wastewater, opening up new vistas for H. pylori epidemiological study.
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