DESCRIPTION

      Helicobacter pylori (H. pylori) represent one of the most widespread bacterial infections globally. Current evidence suggests that nearly half of the global population is chronically infected with H. pylori[1], with prevalence exceeding 70% in developing regions. It is the main pathogen of chronic gastritis, duodenal ulcer and other diseases. It is closely related to the occurrence of gastric cancer and malignant lymphoma of gastric mucosa-associated lymphoid tissue. Its transmission is linked to socioeconomic factors and geographical variations.

      Since the discovery of H. pylori, the primary treatment strategy has relied on antibiotic-based therapies. However, conventional therapeutic regimens, including triple therapy and bismuth-based quadruple therapy—a complex combination comprising a proton pump inhibitor (PPI), bismuth, and two antibiotics—remain inadequate for the complete eradication of Helicobacter pylori infection.[3, 4]. At the same time, today's antibiotic treatment methods still have disadvantages such as high treatment costs, antibiotic resistance and side effects related to adverse treatments[5]. The existence of these limitations has made H. pylori infection a global public health issue that cannot be ignored. And it is emergent to solve the problem.

      H. pylori is a urease-producing gram-negative bacterium[6]. It engages in highly complex interactions with gastric mucosal epithelial cells. Virulence factors released by H. pylori (e.g., VacA cytotoxin and CagA oncoprotein) disrupt mucosal integrity, triggering chronic inflammation, acid dysregulation, and peptic ulcers[7, 8]. While most carriers with normal immune systems remain asymptomatic throughout their lives, H. pylori have been classified by the World Health Organization (WHO) as a Group 1 carcinogen due to its unequivocal association with gastric cancer[9, 10].

      Urease is essential for H. pylori colonization, as it catalyzes the hydrolysis of urea into ammonia and carbon dioxide, enabling the bacteria can survive in the extremely acidic environment of the stomach[11]. At the same time, urease cooperates with sports factors and adhesin to directly destroy host cells and create a favorable environment for the survival of Helicobacter pylori. Urease plays an important role in the escape of Helicobacter pylori from the host's immune system, enabling Helicobacter pylori to settle in the host for a long time. Urease is formed of two distinct subunits, UreA and UreB, with the UreB subunit containing the catalytic active site of H. pylori[12]. UreB, as a key subunit of urease, facilitates the efficient incorporation of Ni²⁺ into the enzyme's active site and stabilizes the site against metal chelation[13]. Additionally, UreB interacts with other subunits to confer structural stability, protecting urease from proteolytic degradation. These properties make UreB a valuable target for validating urease function and developing anti-H. pylori strategies[14]. We also plan to take it as the source of inspiration for our subsequent product development.

      In order to solve the problem of Helicobacter pylori infection, innovative therapeutic strategies are imperative. Nano-antibody therapy is the focus of targeted therapy at present, so in this study, we want to treat Helicobacter pylori infection through nano-antibody. Then through what method to synthesize nano-antibodies? By consulting the literature, we found that probiotics may be a good way for us to synthesize nano-antibodies.[15]. Escherichia coli Nissle 1917 (EcN) is a probiotic isolated from intestinal tract. What' more, Escherichia coli Nissle 1917 (EcN) possess distinctive characteristics, including non-virulence, antagonistic activity against other pathogens, immunomodulatory properties, and probiotic potential[16]. These features enable their application in treating a broad spectrum of gastrointestinal disorders. Currently, probiotics have been successfully employed as safe delivery vehicles for therapeutic molecules in both in vivo and in vitro applications[17]. In some literatures, it has been found that some nano-antibodies targeting Ureb play an important role in the treatment of Helicobacter pylori infection, so it may be an important way to treat Helicobacter pylori infection by synthesizing and delivering nano-antibodies targeting Ureb through probiotics. [18].

      In this study, we developed a novel nanobody-based therapeutic strategy targeting urease subunit B (UreB) of H. pylori. Using an Escherichia coli expression system, we engineered recombinant nanobodies with potent urease inhibitory activity, which were then functionally expressed in food-grade probiotic delivery vehicles. This live bacterial delivery system enables direct in vivo neutralization of urease activity. The approach capitalizes on the host immune system while offering multiple therapeutic advantages: (1) high specificity for UreB, (2) superior binding affinity, (3) broad epitope accessibility, and (4) synergistic effects between the nanobodies and probiotic carrier.

      This integrated strategy represents a promising alternative to conventional antibiotic therapies for H. pylori infection, potentially overcoming limitations such as antibiotic resistance while enhancing treatment efficacy through combined mechanisms of action.

The critical aspects of this study lie in the selection of target genes and vector construction.

Due to the consideration of biological safety, we can't use Ureb of Helicobacter pylori directly for the experiment, so our experiment uses Ureb from Bacillus subtilis instead. Based on literature reports, we selected three recombinant nanobody genes specifically targeting the urease subunit (UreB): Nb-human (GenBank: LC375193.1), Nb-scFv (GenBank: LC373564.1), and UreB-Nb6. During vector construction, we incorporated a signal peptide to facilitate nanobody secretion into the extracellular space, thereby obtaining the desired nanobody products to solve the problem of H. pylori infection. Using synthetic biology approaches, we constructed a recombinant nanobody expression system targeting UreB. Initially, we employed an Escherichia coli-based genetic engineering platform to produce functional recombinant nanobodies demonstrating potent urease inhibitory activity. Furthermore, we investigated the expression of these nanobodies in food-grade probiotic strains. This innovative strategy aims to utilize probiotic bacteria as live delivery vehicles for in vivo antibody transport. In the final drug product, our sustained-release capsules deliver probiotics engineered with the Nb-human antibody gene into the human body which can prevent antibodies from being decomposed by gastric acid. The preparation contains a certain amount of IPTG as an inducer to trigger the probiotic expression of nano-antibody Nb-human.

Once expressed, Nb-human binds to urease produced by H. pylori in the gastrointestinal tract, effectively reducing urease secretion. This mechanism disrupts H. pylori's ability to neutralize gastric acid and limits its access to nitrogen sources, thereby impairing bacterial survival. Ultimately, this approach will serve both prophylactic and therapeutic functions against H. pylori infection.

Based on synthetic biology approaches, we selected three candidate nanobodies and comparatively evaluated their performance following signal peptide incorporation. According to our experimental results, the nanobody demonstrating superior binding affinity was subsequently expressed in the Escherichia coli Nissle 1917 (EcN) strain. In the final drug product, our sustained-release capsules deliver probiotics engineered with the Nb-human antibody gene into the human body which can prevent antibodies from being decomposed by gastric acid. The formulation includes trace amounts of IPTG as an inducer to trigger probiotic expression of the nanobody Nb-human. Once expressed, Nb-human binds to urease produced by H. pylori in the gastrointestinal tract, effectively reducing urease secretion. This mechanism disrupts H. pylori's ability to neutralize gastric acid and limits its access to nitrogen sources, thereby impairing bacterial survival. Ultimately, this approach serves both prophylactic and therapeutic functions against H. pylori infection. Compared with conventional approaches, antibodies specifically targeting H. pylori antigens not only effectively combat infection but also overcome the development of bacterial resistance. Furthermore, this study establishes an important foundation for developing immunotherapeutic strategies against H. pylori.

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