Protective immunity against Helicobacter pylori induced by a single dose of an mRNA-based UreB vaccine candidate in mice

Protective immunity against Helicobacter pylori induced by a single dose of an mRNA-based UreB vaccine candidate in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective immunity against Helicobacter pylori induced by a single dose of an mRNA-based UreB vaccine candidate in mice Jing Gao, Yinan Hao, Yujiao Yin, Lei Yuan, Han Lei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8837369/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Helicobacter pylori ( H. pylori ) is a global public health concern because of its strong ability to colonise the gastrointestinal tract. Current clinical therapeutic strategies are limited by antibiotic resistance, reinfection and a high recurrence rate after eradication. Therefore, effective preventive vaccines against H. pylori infections are needed to counter the potential threat of a global pandemic. Recently, mRNA vaccines have emerged as promising platforms for the prevention of major infectious diseases. UreB is regarded as a critical virulence factor in H. pylori and serves as a key vaccine candidate target. This study aimed to investigate the immunogenicity of an mRNA-based agent against H. pylori . Methods We generated an mRNA-based H. pylori vaccine candidate targeting UreB. The expression levels of LNP/mRNA-UreB in transfected HepG2 cells in vitro were characterised via Western blot, immunofluorescence, flow cytometric and sandwich ELISA. BALB/c mice were vaccinated with a single dose of LNP/mRNA-UreB to evaluate its immunogenicity and immune protective efficacy against H. pylori . Both humoral and cellular immune responses induced by LNP/mRNA-UreB were measured via ELISA, followed by challenge with H. pylori to validate the immune protective efficacy. Results LNP/mRNA-UreB was highly expressed in HepG2 cells; additionally, the molecular weight of the UreB antigen ranged from approximately 80–90 kDa, and the quantitative expression concentration was determined to be 902 pg/mL. Furthermore, mice vaccinated with LNP/mRNA-UreB via a single dose exhibited robust UreB-specific humoral immune responses with markedly elevated antibody levels and a significant Th1/Th2 mixed cellular immune response. Finally, in mice vaccinated with LNP/mRNA-UreB and subsequently infected with H. pylori SS1, gastric colonisation and urease levels were effectively reduced, demonstrating that LNP/mRNA-UreB was a promising vaccine candidate against H. pylori infection. Conclusions The LNP/mRNA-UreB vaccine candidate developed in this study represents a proof-of-concept for effective protection against H. pylori infection and mediates balanced humoral and cellular immune responses. Moreover, mRNA-based techniques may constitute a universal approach for the development of H. pylori vaccines. LNP/mRNA-UreB Single dose Immunogenicity Protective efficacy H. pylori infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Helicobacter pylori is a gram-negative microaerophilic bacterium associated with a high prevalence of human peptic ulcers and gastritis [ 1 ]. Statistics indicate that approximately 76% of gastric cancer cases are associated with H. pylori infection [ 2 ]. The World Health Organization (WHO) has declared that H. pylori is a Group 1 carcinogen associated with major global public health issues [ 3 ]. Currently, the standard clinical eradication strategy relies on a 10–14-day regimen combining antibiotics with proton pump inhibitors (PPIs) [ 4 , 5 ]. However, this regimen is subject to antibiotic resistance, which may disrupt the gut microbiota and increase recurrence rates after H. pylori eradication [ 6 – 8 ]. Therefore, vaccination is the most effective measure for preventing and controlling H. pylori infection. H. pylori infection is closely related to the movement of the flagellum and other aetiological factors. Various virulence factors, including urease subunit B (UreB), vacuolating toxin A (VacA), the cagA gene associated with cytotoxin (CagA), neutrophil-activating protein (NAP), heat-shock protein A (HspA), H. pylori adhesin A (HpaA), and lipopolysaccharide (LPS), have been employed in the development of H. pylori vaccine candidate antigens based on the completion of H. pylori whole-genome sequencing [ 9 ]. Notably, UreB has been widely identified as the major vaccine target because of its critical role in H. pylori survival and colonisation processes [ 10 ]. Nasab et al. used a viral-based vector system expressing the UreB antigen in rapeseed ( Brassica napus ), and their results demonstrated rapid and low-cost plant-based H. pylori vaccine antigen production [ 11 ]. Hirota et al. identified a sequence involving 19 UreB-derived amino acids (AAs) as a potential antigenic epitope that induced the production of antibodies to inhibit H. pylori activity [ 12 ]. Moreover, Zhang et al. evaluated the immune efficacy of an attenuated Shigella vector expressing the UreB-HspA fusion protein of H. pylori in a mouse model [ 13 ]. Liu et al. engineered three crucial H. pylori antigen proteins (UreB, CagA, and VacA) onto the surfaces of outer membrane vesicles derived from Salmonella Typhimurium ( S . Typhimurium) using the haemoglobin protease (HBP) autotransporter system [ 14 ]. Our previous study demonstrated that Saccharomyces cerevisiae -based H. pylori oral vaccines (EBY100/pYD1-UreB, EBY100/pYD1-VacA, or EBY100/pYD1-UreB + EBY100/pYD1-VacA) induced a significant immune response and reduced bacterial loads after H. pylori infection [ 15 ]. In summary, UreB or UreB combined with other virulence factors of H. pylori has been widely identified as a promising and potential antigen target for clinical application [ 16 , 17 ]. mRNA-based technology has already emerged as a novel therapeutic platform for emerging infectious diseases due to the successful applications of mRNA-1273 and BNT162b2 against SARS-CoV-2 [ 18 , 19 ]. Although mRNA vaccines demonstrate efficacy in cancer immunotherapy and for the targeting of viral pathogens, the development of mRNA vaccines against bacterial infections has been more challenging, due to the fact that bacteria exhibit greater biological complexity compared to viruses [ 20 ]. Recent evidence has demonstrated that mRNA vaccines represent alternative approaches for the prevention and treatment of bacterial infections against M. tuberculosis [ 21 ], B. burgdorferi [ 22 ], B. pertussis [ 23 ], P. aeruginosa [ 24 ], S. pyogenes [ 25 ], Listeria monocytogenes [ 26 ] and S. aureus [ 27 ]. Among these agents, an mRNA candidate against tuberculosis, which is being tested in a clinical Phase 1 trial (NCT05547464) [ 28 ], as well as an mRNA candidate against Lyme disease (NCT05975099) [ 29 ], have been issued by Moderna and BioNTech, respectively. These preclinical and clinical bacterial mRNA vaccines may increase the availability of commercial applications. Unfortunately, no attempts have been made to develop mRNA vaccines against H. pylori infection. To assess the immune efficacy of an mRNA vaccine encoding the UreB of H. pylori , we generated LNP/mRNA-UreB and investigated both its expression levels in vitro and immunogenicity in vivo in a mouse model by utilising a single dose. The results of the present study strongly suggest that LNP/mRNA-UreB is a promising vaccine candidate that provides an effective alternative approach to prevent H. pylori infection. Methods mRNA production The pUC57-Kan plasmid, which contains the codon-optimised UreB gene of H. pylori (GenBank accession No. 899104) and incorporates the 5' and 3' untranslated regions (UTRs) and 115-nt-long poly(A) tail, was synthesised (GenScript Biotech Corp., Nanjing, China) and termed pUC57/UreB. mRNA-UreB was produced by using a T7 RNA transcription kit (Hzymes Biotechnology Co., Ltd., Wuhan, China) on the linearised plasmid pUC57/UreB and subsequently purified by using lithium chloride (LiCl) precipitation. The mRNA-UreB concentration was adjusted to 1 mg/mL, after which it was stored at -20°C until use. The integrity of the mRNA-UreB was analysed by using a Fragment Analyzer 5200 (Agilent, USA). A scheme of the mRNA-UreB design is presented in Fig. 1 A. LNP formulation The mRNA-UreB in 20 mM citrate buffer (pH 4.0) was formulated into LNPs including ionisable lipids, 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), cholesterol and PEG-DMG 2000 (molar ratios of 50:10:38.5:1.5) at a ratio of 1:2 through INano TM X (Micronanobiologics, Shanghai, China). The formulation was buffer-exchanged into Tris buffer with CH3COONa and sucrose via tangential flow filtration (TFF), 0.22 µm-sterile-filtered, and stored at -80°C until use. The particle size, distribution, mRNA concentration and encapsulation rate of the formulation (known as LNP/mRNA-UreB) were measured via dynamic light scattering (DLS) (Malvern Instruments Ltd. England). The capping efficacy of LNP/mRNA-UreB was determined by using Liquid chromatograph mass spectrometer (LC-MS) (Agilent, USA). Empty LNPs served as negative controls. mRNA transfection and expression analyses HepG2 cells (ATCC, USA) were seeded in 24-well plates at 10 5 cells per well and cultured in EMEM supplemented with 10% FBS, 1% NEAA, and 1% penicillin-streptomycin at 37°C in 5% CO 2 for 12 h. Afterwards, 4 µg of mRNA-UreB was transfected into HepG2 cells per well by using a Fast LNP Transfection Kit (Hzymes Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Cell lysates were harvested with RIPA lysis buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitor cocktail (Roche, Switzerland) at 6 h, 24 h, 48 h and 72 h after transfection. The supernatants derived from the cell lysates were mixed with 5 × SDS-loading buffer after centrifugation at 13000 × g and then loaded for SDS-PAGE. The UreB protein in the cell lysates was detected via Western blotting by using a rabbit polyclonal anti-UreB antibody (1:2000 dilution) as the primary antibody, with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) (Thermo Fisher Scientific, USA) being used as the secondary antibody. Moreover, β-actin (1:10000 dilution) (Thermo Fisher Scientific, USA) was also detected as an internal control (Thermo Fisher Scientific, USA). The blots were developed by using chemiluminescent substrates and detected by using a gel imaging system (Bio-Rad, USA). For immunofluorescence and flow cytometry assays, 100 µL of HepG2 cells transfected with LNP/mRNA-UreB for 24 h were harvested at 6000 × g via centrifugation. The pellets were treated with blocking buffer containing 3% bovine serum albumin (BSA) for 30 min and then incubated with a rabbit polyclonal anti-UreB antibody (1:1000) (CUSABIO, Wuhan, China) at room temperature for 1 h. After being washed with sterile PBS three times, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:5000) (Thermo Fisher Scientific, USA) in the dark at room temperature for 40 min. The cells were then washed with sterile PBS three times and resuspended in 350 µL of sterile PBS. Ten microlitres of cell suspension was observed under a fluorescence microscope (Nikon, Japan). The remaining 340 µL of the sample was used for flow cytometry (BD Biosciences, USA) and analysed by using FlowJo software (BD Biosciences, USA). HepG2 cells without transfection served as the negative controls. Quantitative expression analysis The expression level of the UreB protein was quantitatively measured via sandwich ELISA. Briefly, the transfected HepG2 cell lysates were harvested at 24 h posttransfection. The 96-well high-binding plates were coated with 100 µL of serially diluted polyclonal rabbit anti-UreB antibody (0 pg/mL, 62.5 pg/mL, 250 pg/mL, 500 pg/mL, 1000 pg/mL, 2000 pg/mL and 4000 pg/mL) and incubated overnight at 4°C. The plates were subsequently washed three times with sterile PBST (PBS + 0.01% Tween 20) and then blocked with PBST containing 3% BSA for 1 h at 37°C. After the cells were washed three times with PBST, either 100 µL of the transfected HepG2 cell lysates or the standard antigen UreB (CUSABIO, Wuhan, China) was added to each well and incubated for 2 h at 37°C. The plates were subsequently washed three times with PBST and then incubated with 100 µL/well of a mouse polyclonal anti-UreB antibody for 2 h at 37°C. After being washed three times with PBST, the plates were incubated with 100 µL/well of HRP-conjugated goat anti-mouse antibody for 40 min at 37°C. Finally, the plates were developed with 3,3',5,5'-tetramethylbenzidine (TMB) in the dark for 20 min at room temperature. The reaction was stopped by the addition of 50 µL/well of 2 mol/L sulfuric acid. The absorbance was measured at 450 nm by using a microplate reader (Bio-Rad, USA). Each experiment was performed three times. Vaccination, sample collection and H. pylori infection in mice Female BALB/c mice (6–8-weeks-old) were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., China, and were immunised (10 mice/group) on day 1 with 50 µL of LNP/mRNA-UreB containing 4 µg of mRNA-UreB via intramuscular injections in the rear quadriceps. The same dosage (50 µL) of saline or empty LNPs served as negative controls. The immune schedule is shown in Fig. 1 B. Blood samples (10 mice/group) were obtained from the submandibular vein on days 14 and 28; additionally, serum was isolated to evaluate the antibody response. Moreover, 5 mice/group were anaesthetised via inhalation of isoflurane (4–5% in oxygen), and spleens were obtained on day 28 to evaluate cell-mediated immunity. For protective efficacy analysis, the immunised mice were orally administered 500 µL of H. pylori SS1 (1 × 10⁹ CFU/mL) every other day for a total of three doses. Weight loss was monitored for 14 days. The mice were then euthanised, and the stomach was isolated on day 60 to detect the colonisation of H. pylori . Enzyme-linked immunosorbent assay ELISA was used to determine the IgG titres of mouse sera as previously described [ 15 ]. Briefly, 2 µg of UreB protein was coated into 96-well high binding microtitre plates (Corning, USA) at 4°C overnight. After blocking with 3% BSA in PBST for 2 h, the plates were washed three times with PBST. Serum samples from immunised mice were twofold serially diluted in blocking buffer, after which they were added to the plates and incubated at 37°C for 2 h. The plates were again washed three times with PBST and subsequently incubated with biotinylated anti-mouse IgG (Sigma, USA) at a 1:5000 dilution for 1 h at room temperature. After being washed three times with PBST, the plates were incubated with 100 µL/well of streptavidin-conjugated alkaline phosphatase (ALP) (R&D Systems, USA) at room temperature for 2 h and then washed three times with PBST. Afterwards, 100 µL of 1.25 mM pNPP phosphatase substrate (MP Biomedicals, USA) was added to each well and incubated in the dark for 25 min. The reaction was stopped by the addition of 50 µL of 2N NaOH to each well, and the optical density (OD) at 405 nm and 603 nm was measured with a multimode microplate reader (Bio-Tek, USA). The cutoff value was set as the reciprocal of the highest dilution to obtain an OD value more than twofold that of the negative serum. For IgG1 or IgG2a detection using the abovementioned ELISA, the biotinylated anti-mouse IgG was replaced with IgG1 and IgG2a. ELISpot assays The cell-mediated response was measured via ELISpot as previously described [ 15 ]. Briefly, 96-well ELISpot plates (Corning, USA) were coated with anti-mouse IFN-γ/IL-4 capture Ab (R&D Systems, USA) and incubated overnight at 4°C. The plates were subsequently washed with PBST and blocked with PBST containing 1% BSA for 2 h. A total of 1×10 5 splenocytes from the immunised mice were added to each well and incubated overnight at 37°C in 5% CO 2 with 10 µL of UreB protein (1 µg/ml) stimulation. After 36 h, the plates were washed and then incubated at 37°C for 2 h with biotinylated anti-mouse IFN-γ Ab (R&D Systems, USA). Streptavidin-alkaline phosphatase (R&D Systems, USA) was added to each well after washing with PBST, after which the samples were incubated for 1 h at room temperature. The plates were subsequently washed, after which 50 µL of pNPP was added to each well. Finally, the plates were rinsed with sterile H 2 O and dried at room temperature. The spots were quantified by using an automatic ELISpot reader (CTL Limited, USA). Protective efficacy analysis The protective efficacy was measured by using a rapid urease test kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. The samples obtained from the stomach tissue homogenates were mixed with the urease reaction solution and incubated at room temperature for 2 h. Finally, the absorbance at OD 550nm was measured by using a microplate reader (Bio-Tek, USA). The colonisation load of H. pylori in the gastric tissues of immunised mice was determined by using the bacterial culture method, as previously described [ 15 ]. Briefly, tenfold serial dilutions of the gastric tissue homogenates were inoculated on Columbia blood agar plates. The colonisation number was calculated at 3 days after culture. Statistical analysis Statistical analyses were performed with GraphPad Prism version 10.1.2. The data are presented as the means ± SDs or SEMs. Statistical significance for group comparisons was determined by using one-way ANOVA with Tukey’s post hoc test for comparisons with control groups. P values < 0.05 were considered to indicate statistical significance. Results Construction and characterisation of mRNA-UreB We generated mRNA-UreB by inserting the 5', 3' untranslated regions (UTRs) and poly(A) tail into the flank of the codon-optimised UreB gene (Fig. 1 A). This UreB antigen has previously been observed to be protective as a subunit vaccine candidate target [ 15 ]. The length of mRNA-UreB was observed to be approximately 2,200 nt ( Additional file: Fig. S1 ), which is consistent with the expected size. Furthermore, the integrity of mRNA-UreB was observed to be approximately 97% ( Additional file: Fig. S2 ). The in vitro expression profile was confirmed by subjecting HepG2 cells transfected with mRNA-UreB to Western blotting, immunofluorescence assays and flow cytometric assays. As expected, a specific protein band was detected via Western blotting in the cell lysates of HepG2 cells transfected with mRNA-UreB, which revealed a relative molecular mass of approximately 80 ~ 90 kDa (Fig. 1 B, Lanes 5–8 ). Notably, the highest expression level of UreB protein in the transfected HepG2 cells occurred at 24 h after transfection ( Additional file: Fig. S3) . Similarly, strong fluorescence signals were observed in the mRNA-UreB-transfected HepG2 cells than in the untransfected HepG2 cells (Fig. 1 C, Right ). Subsequent flow cytometry assays also revealed that the positive rate of mRNA-UreB-transfected HepG2 cells was approximately 51.2% (Fig. 1 D, Right ). Furthermore, the quality control of the mRNA-UreB encapsulated by LNPs was analysed, as shown in Additional file: Table 1 . Quantitative expression analysis of mRNA-UreB To determine the quantitative expression of mRNA-UreB, cell lysates from HepG2 cells transfected with mRNA-UreB at 24 h after transfection were analysed via sandwich ELISA. A standard curve was generated by using GraphPad Prism software (Fig. 1 E). The equation for the standard curve was determined as y = 0.0001x + 0.0678, and the R² value was 0.991. Moreover, the OD 450nm value of the mRNA-UreB-transfected HepG2 cells was 0.158, which corresponded to a specific concentration of UreB protein in the HepG2 cells of 902 pg/mL. Antibody response induced by a single dose of LNP/mRNA-UreB To assess humoral immunogenicity, the total anti-UreB IgG antibody response from all mice vaccinated with a single dose of 50 µL of LNP/mRNA-UreB containing 4 µg of mRNA-UreB via intramuscular injection was analysed in the serum samples collected on days 14 and 28; moreover, the same dosage of saline or empty LNP functioned as a control group. At 14 and 28 days after initial immunisation, the ELISA results indicated that a single dose of LNP/mRNA-UreB could elicit robust UreB-specific IgG antibodies; in comparison, the IgG titre on day 28 was significantly greater than that on day 14, whereas all of the control groups (including the saline and LNP groups) exhibited undetectable antibody levels for UreB (Fig. 2 A). These results demonstrate that a single dose of LNP/mRNA-UreB effectively induces efficient humoral immunity. To further assess the IgG subtypes, the levels of IgG1 and IgG2a were also analysed via ELISA. A similar trend as that of the IgG antibody was detected for IgG1 and IgG2a. Specifically, the levels of both IgG1 and IgG2a antibodies induced by the LNP/mRNA-UreB group were significantly greater than the control groups (Fig. 2 B-C) on days 14 and 18 after the initial immunisation. Notably, the IgG1/IgG2a ratio was calculated to be approximately 1.0, thus suggesting that a relatively balanced Th1/Th2 mixed immune response was elicited by a single dose of LNP/mRNA-UreB, which contributed to the balanced adaptative immune response profiles. Cell-mediated immune response induced by a single dose of LNP/mRNA-UreB The secretion levels of IFN-γ and IL-4 cytokines induced by LNP/mRNA-UreB in mouse splenocytes were measured via ELISpot assays at 28 days after the initial immunisation. As shown in Fig. 3 A, the LNP/mRNA-UreB group demonstrated characteristic increases in the secretion levels of IFN-γ and IL-4 compared to the control groups (the saline or LNP group). Notably, a significantly greater secretion of IFN-γ spots was induced by LNP/mRNA-UreB, thus indicating a T-helper-1 (Th1)-biased cell-mediated immune response. Furthermore, the concentrations of IFN-γ and IL-4 secretion spots mediated by CD4 + and CD8 + T cells were measured. As shown in Fig. 3 B, both CD4 + and CD8 + T cells produced increased numbers of IFN-γ spots in response to stimulation with the UreB antigen, whereas the level of secreted IL-4 was relatively low. Consequently, the IFN-γ/IL-4 spot ratio was significantly increased, thereby suggesting that a meaningful mixed Th1/Th2 immune response was induced by a single dose of LNP/mRNA-UreB. Protective efficacy induced by LNP/mRNA-UreB The protective efficacy induced by a single dose of LNP/mRNA-UreB was assessed by using H. pylori SS1 challenge assays on day 42. Body weight changes were monitored for 14 days. As shown in Fig. 4 A, body weight loss in the control groups (the saline or LNP groups) was observed to be approximately 5% on day 5 after H. pylori SS1 challenge until recovery was observed on day 12. In contrast, the LNP/mRNA-UreB group exhibited a slight decrease in body weight on day 2, with a weight loss rate of approximately 1%; moreover, body weight gradually recovered on day 3, thus suggesting that LNP/mRNA-UreB effectively protected mice from H. pylori SS1 infection. The colonisation numbers of H. pylori in the gastric tissue were also determined on day 60 after the initial immunisation. Compared with the control groups (the saline or LNP groups), the LNP/mRNA-UreB group demonstrated a significant reduction in H. pylori SS1 colonisation in gastric tissues (Fig. 4 B). To further assess the protective efficacy of LNP/mRNA-UreB against H. pylori SS1 infection, urease activity was measured in the gastric tissues isolated from all of the vaccinated mice. The OD 550nm values in the LNP/mRNA-UreB group were much lower than those in the control groups (the saline or LNP groups) (Fig. 4 C), thereby demonstrating that LNP/mRNA-UreB effectively reduced H. pylori SS1 colonisation. Discussion H. pylori infection remains a serious public health concern [ 20 ]. In particular, the increasing rate of H. pylori infection induced by antibiotic resistance is a serious issue for antimicrobial therapy. Therefore, an effective vaccine against H. pylori is urgently needed to prevent malignant gastric tumours and other serious H. pylori -associated diseases [ 9 ]. Most of the current vaccine research on H. pylori conducted in the preclinical or clinical phases has focused on oral administration routes [ 30 ]. To define a more optimised vaccine approach, we first developed an mRNA-based H. pylori vaccine candidate via a single dose of LNP/mRNA-UreB and subsequently investigated its immunogenicity and protective efficacy against H. pylori infection in a mouse model, which represents a promising direction for the development of preventive vaccines against H. pylori infection. To ensure the efficient expression of LNP/mRNA-UreB, we performed a specific design on the key components of mRNA-UreB. First, we utilised codon-optimised software to optimise the UreB antigen, and humans were used as the final host, which provided the foundation for the future clinical application of H. pylori vaccine candidates. Second, the modified 5'-UTR and 3'-UTR derived from the commercial products of mRNA-1273 [ 18 ] and BNT162b [ 19 ], respectively, were incorporated into mRNA-UreB. Notably, both the 5'-UTR and the 3'-UTR originated from human α-globin HBA1 [ 31 ]. Third, we used a strategy similar to that of the Pfizer/BioNTech mRNA vaccine [ 32 ], in which two segmented poly-A tails (30A+75A) with an interval of 10 nucleotides were added into the 3′ end of mRNA-UreB. All of these optimised elements demonstrated superior potential for the high expression level of mRNA-UreB (Fig. 1 B-D). In addition, the current commercial LNP formulations used in the present study mainly consist of four components, including ionisable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids, and the molecular ratio of the four components was optimised to form an ideal encapsulation. Notably, the LNP formulation is being continuously optimised. Su et al. developed three formulations to achieve precise pulmonary targeting and accumulation of mRNA [ 33 ], thereby representing a safe and effective LNP formulation with distinct targeting properties. Immune responses serve as the most important indicators for evaluating the immune efficacy induced by H. pylori vaccine candidates. Thus, the total IgG antibody response is strongly related to the immunogenicity of LNP/mRNA-UreB after single-dose vaccination in a mouse model (Fig. 2 A) and is superior to that observed in our previous study, in which the oral administration of Saccharomyces cerevisiae resulted in UreB [ 15 ]. Furthermore, we detected the ratio of serum IgG1 and IgG2a for the serum IgG subtype (Fig. 2 B-C), in which IgG1 was closely linked to humoral immunity and effectively neutralised target antigens; moreover, IgG2a played a critical role in the cell-mediated immune response against bacterial infections [ 34 ], as reflected by the high secretion level of IFN-γ (Fig. 3 A). In summary, the LNP/mRNA-UreB vaccine developed in this study has been demonstrated to elicit balanced activation of both humoral and cellular immune responses, thus greatly contributing to the fight against intracellular pathogen infection [ 35 ]. Moreover, CD4 + and CD8 + T cells synergise to generate immunity against intracellular bacteria [ 20 ], and the IFN-γ/IL-4 ratio could serve as a clinical substitute marker for Th1/Th2 balance [ 36 ]. The ratio of IFN-γ/IL-4 secretion detected in this study strongly supports the notion that LNP/mRNA-UreB could induce a mixed Th1/Th2-mediated cellular immune response (Fig. 3 B) and support the feasibility of mRNA-based H. pylori vaccine candidates. An important characteristic of H. pylori is its ability to survive in extremely acidic gastric tissues, which easily leads to constant H. pylori infection after eradication therapy. The presence of H. pylori infection is mainly evaluated via urease assays [ 37 , 38 ]. Therefore, the extent of H. pylori colonisation in gastric tissues directly reflects the severity of H. pylori infection and the immune efficacy of H. pylori vaccine candidates. An outer membrane vesicle (OMV)-based or Saccharomyces cerevisiae -based UreB vaccine was observed to decrease the colonisation levels and urease activity by approximately twofold compared with those of the controls [ 14 , 15 ], whereas mice vaccinated with a single dose of LNP/mRNA-UreB in this study exhibited approximately fourfold greater bacterial colonisation and approximately fivefold lower urease activity than the controls (the saline or LNP groups) (Fig. 4 B-C), thus suggesting that an mRNA-based H. pylori vaccine candidate is superior to OMV-based or Saccharomyces cerevisiae -based UreB vaccines. As with different vaccine platforms, the development of mRNA-based H. pylori vaccine candidates is dependent on the reasonable selection and modification of antigens, as well as an efficient immune schedule. It is well known that an mRNA-based vaccine requires only a single dose that elicits robust immune responses [ 39 , 40 ]. Our study clearly demonstrated that a single dose of LNP/mRNA-UreB could induce sufficient immunity against H. pylori infection in a mouse model, thus supporting the advantage of an mRNA-based H. pylori vaccine candidate. Taken together, these findings suggest that LNP/mRNA-UreB elicits balanced immune responses, as well as a Th1-mediated cellular immune response. Importantly, mice vaccinated via a single dose of LNP/mRNA-UreB exhibited effectively reduced bacterial colonisation in gastric tissues after H. pylori infection. H. pylori has multiple important virulence factors (with the exception of UreB), such as VacA, CagA, and Hsp; moreover, UreB may be feasible for serving as a target antigen for the development of an mRNA-based H. pylori vaccine candidate. Therefore, the further design of combined H. pylori vaccine candidates based on mRNA platforms is highly important for the effective control of H. pylori infection and other infectious diseases. Conclusions Overall, we have developed a single-dose mRNA-based H. pylori vaccine candidate that provides significant protection against H. pylori infection by mediating balanced immune responses. An mRNA-based technology platform may represent a promising approach for the development of H. pylori vaccine candidates and speed up the translation from preclinical studies to clinical applications. Abbreviations AA amino acid ALP Alkaline phosphatase BSA Bovine serum albumin CagA Cytotoxin-associated gene A CFU Colony-forming units CGE Capillary gel electrophoresis DLS Dynamic light scattering DSPC 1,2-distearoylsn-glycero-3-phosphocholine ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum FITC Fluorescein isothiocyanate HBP Hemoglobin protease HpaA H. pylori adhesin A H. pylori. Helicobacter pylori HRP Horseradish peroxidase HspA Heat-shock protein A IVT In vitro transcribed LC-MS Liquid chromatograph mass spectrometer LNP Lipid nanoparticles LPS Lipopolysaccharide NAP Neutrophil-activating protein NEAA Non-Essential Amino Acids OD Optical density PBS Phosphate-buffered saline PBST Phosphate buffered saline-Tween PEG Polyethylene glycol PPI Proton pump inhibitor SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TFF Tangential flow filtration Th1 T-helper-1 TMB 3,3',5,5'-Tetramethylbenzidine UreB Urease B UTR Untranslated regions VacA Vacuolating cytotoxin A WHO World Health Organization Declarations Ethics approval and consent to participate This study was carried out in strict accordance with the protocols approved by Southwest Jiaotong University. All mouse experiments were performed according to the guidelines stated by the Institutional Animal Care and Use Committees of Southwest Jiaotong University (Approval No. SWJTU-2503-041). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was supported by the National Natural Science Foundation of China (No. 31360225; No. 32070920), and the Fundamental Research Funds for the Central Universities (No. 2682023ZTPY06387) to H. Lei. Author Contribution HL planned and designed the study. JG, YH, YY, LY and HL contributed to the data analysis and the interpretation of the results. JG and HL wrote the manuscript and produced all the figures. All authors read and approved the final manuscript. Acknowledgements We thank Kui Wang at Hualan Biological Vaccine Co., Ltd, China, for his assistance with analysis of mRNA-UreB. Data Availability The datasets generated and analyzed in the current study are available from the corresponding author upon reasonable request. References Shah SAR, Mumtaz M, Sharif S, Mustafa I, Nayila I. 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Strong immune responses and protection of PcrV and OprF-I mRNA vaccine candidates against Pseudomonas aeruginosa. NPJ vaccines. 2023;8(1):76. https://doi.org/10.1038/s41541-023-00672-4 . Chiarot E, Faralla C, Chiappini N, Tuscano G, Falugi F, Gambellini G, et al. Targeted amino acid substitutions impair streptolysin O toxicity and group A Streptococcus virulence. mBio. 2013;4(1):e00387. https://doi.org/10.1128/mBio.00387-12 . Mayer RL, Verbeke R, Asselman C, Aernout I, Gul A, Eggermont D, et al. Immunopeptidomics-based design of mRNA vaccine formulations against Listeria monocytogenes. Nat Commun. 2022;13(1):6075. https://doi.org/10.1038/s41467-022-33721-y . Arya S, Lin Q, Zhou N, Gao X, Huang JD. Strong Immune Responses Induced by Direct Local Injections of Modified mRNA-Lipid Nanocomplexes. Molecular therapy. Nucleic acids. 2020;19:1098–109. https://doi.org/10.1016/j.omtn.2019.12.044 . BioNTech SE. Safety and Immune Responses After Vaccination With Two Investigational RNA-based Vaccines Against Tuberculosis in BCG Vaccinated Volunteers. ClinicalTrials.gov;2026 [Available from: https://clinicaltrials.gov/study/NCT05547464 ModernaTX I. A Study to Evaluate the Safety and Immunogenicity of mRNA-1975 and mRNA-1982 Against Lyme Disease in Participants 18 Through 70 Years of Age. ClinicalTrials.gov;2025 [Available from: https://clinicaltrials.gov/study/NCT05975099 Liu Z, Li H, Huang X, Liu Q. Advances and challenges in Helicobacter pylori subunit vaccine development: antigen candidates and immunization strategies. J Appl Microbiol. 2025;136(10):lxaf236. https://doi.org/10.1093/jambio/lxaf236 . Xia X. Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines. Vaccines. 2021;9(7):734. https://doi.org/10.3390/vaccines9070734 . Nance KD, Meier JL. Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines. ACS Cent Sci. 2021;7(5):748–56. https://doi.org/10.1021/acscentsci.1c00197 . Su K, Shi L, Sheng T, Yan X, Lin L, Meng C, et al. Reformulating lipid nanoparticles for organ-targeted mRNA accumulation and translation. Nat Commun. 2024;15(1):5659. https://doi.org/10.1038/s41467-024-50093-7 . Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Front Immunol. 2014;5:520. https://doi.org/10.3389/fimmu.2014.00520 . Cho E, Davis MA, Nowak JA, Izzo M, Ferrante AM, Sha F, et al. Modulating antigen processing through metal-organic frameworks to bias adaptive immunity. Proc Natl Acad Sci USA. 2025;122(45):e2409555122. https://doi.org/10.1073/pnas.2409555122 . Zhou P, Zhou Y, Yang L, Liu C, Zuo S, Pei X, et al. Identification of the IFN-γ/IL-4 ratio as a prognostic biomarker and development of an immune-related prognostic model in diffuse large B-cell lymphoma. BMC Cancer. 2025;25(1):1386. https://doi.org/10.1186/s12885-025-14737-1 . Guo BP, Mekalanos JJ. Rapid genetic analysis of Helicobacter pylori gastric mucosal colonization in suckling mice. Proc Natl Acad Sci USA. 2002;99(12):8354–59. https://doi.org/10.1073/pnas.122244899 . Lee JY, Kim N. Diagnosis of Helicobacter pylori by invasive test: histology. Annals translational Med. 2015;3(1):10. https://doi.org/10.3978/j.issn.2305-5839.2014.11.03 . Mulligan MJ, Lyke KE, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature. 2020;586(7830):589–93. https://doi.org/10.1038/s41586-020-2639-4 . Wilson E, Goswami J, Baqui AH, Doreski PA, Perez-Marc G, Zaman K, et al. Efficacy and Safety of an mRNA-Based RSV PreF Vaccine in Older Adults. N Engl J Med. 2023;389(24):2233–44. https://doi.org/10.1056/NEJMoa2307079 . Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8837369","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594193843,"identity":"fb283a11-d7c2-4355-953a-63b4169773dc","order_by":0,"name":"Jing Gao","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Gao","suffix":""},{"id":594193846,"identity":"e91589b6-78d5-4d57-901c-cf35ef64fbf1","order_by":1,"name":"Yinan Hao","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yinan","middleName":"","lastName":"Hao","suffix":""},{"id":594193847,"identity":"3f4c8bfd-b099-4697-a5d3-e69b3ff7725b","order_by":2,"name":"Yujiao Yin","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yujiao","middleName":"","lastName":"Yin","suffix":""},{"id":594193849,"identity":"03175f1b-e4f8-40ff-9812-032e28f3a866","order_by":3,"name":"Lei Yuan","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Yuan","suffix":""},{"id":594193850,"identity":"46a285f7-68f6-49b6-b771-4d899668edd5","order_by":4,"name":"Han Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACA3YGhgMfGBiYwTweorQA1R6cAdTCQ5IWsHLitZgzMx88bPPnMLu9RALjg7dtDPLmhLRYNrMlHM5tO8zMI5HAbDi3jcFwZwMhhx3mMTic23AbpIVNmreNIcHgAEEt/B8OW/wBa2H/TaQWHobDDGwQW5iJ1MJmcLC37T8zz5mHzZJzzkkYbiCo5Xjz4w8//qQls7cnH/zwpsxGnqAtMJDMwMDYAKQliFQPBHbEKx0Fo2AUjIIRBwBzwDuUGtocKwAAAABJRU5ErkJggg==","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Han","middleName":"","lastName":"Lei","suffix":""}],"badges":[],"createdAt":"2026-02-10 06:53:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8837369/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8837369/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103264919,"identity":"822dc51e-b6eb-478d-9ad8-7ecae9e62717","added_by":"auto","created_at":"2026-02-23 19:14:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":395009,"visible":true,"origin":"","legend":"\u003cp\u003emRNA-based \u003cem\u003eH. pylori \u003c/em\u003evaccine candidate design and characterisation in vitro. \u003cstrong\u003eA\u003c/strong\u003e Schematic diagram of an \u003cem\u003eH. pylori \u003c/em\u003evaccine candidate encoding the antigen protein UreB. \u003cstrong\u003eB\u003c/strong\u003e Western blot analysis of mRNA-UreB expression in HepG2 cells at 6, 24, 48, and 72 h after transfection.\u003cstrong\u003e \u003c/strong\u003eLanes 1–4: the lysates of HepG2 cells without transfection; M: proteinmarker; Lanes 5–6: the lysates of HepG2 cells with transfected mRNA-UreB. \u003cstrong\u003eC\u003c/strong\u003eImmunofluorescence assay of mRNA-UreB expression in HepG2 cells at 24 h after transfection. HepG2 cells without transfection (Left); HepG2 cells with transfected mRNA-UreB (Right). \u003cstrong\u003eD\u003c/strong\u003e Flow cytometric analysis of mRNA-UreB expression in HepG2 cells at 24 h after transfection.\u003cstrong\u003e \u003c/strong\u003eHepG2 cells without transfection (Left); HepG2 cells with transfected mRNA-UreB (Right). \u003cstrong\u003eE\u003c/strong\u003e A standardcurve was constructed via sandwich ELISA. The data are shown as the means ± SDs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8837369/v1/0a4ce95722b54657cd5e3ad1.png"},{"id":103264920,"identity":"ea670130-8cb0-4c73-8a0c-02191fbab102","added_by":"auto","created_at":"2026-02-23 19:14:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":299833,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral immune responses detected via ELISA after a single dose of LNP/mRNA-UreB vaccination. \u003cstrong\u003eA\u003c/strong\u003e UreB-specific serumIgG titres (n=10). \u003cstrong\u003eB \u003c/strong\u003eUreB-specific serumIgG1 titres (n=10). \u003cstrong\u003eC\u003c/strong\u003e UreB-specific serumIgG2a titres (n=10). The data are shown as the means ± SEMs. Asterisks indicatestatistically significant differences compared with the controls (the saline or LNP groups), \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8837369/v1/27c0ea0f21873f3708969ef5.png"},{"id":103264922,"identity":"8041c9de-bd2d-45b0-b48b-4faf08fcd66b","added_by":"auto","created_at":"2026-02-23 19:14:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":248339,"visible":true,"origin":"","legend":"\u003cp\u003eCell-mediated immune responses detected via ELISpot assay after a single dose of LNP/mRNA-UreB vaccination. \u003cstrong\u003eA\u003c/strong\u003e Secretion levels of IFN-γ and IL-4 spots in the spleen (n=5). \u003cstrong\u003eB\u003c/strong\u003e The IFN-γ/IL-4 ratio mediated by CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells. The data are shown as the means ± SEMs. Asterisks indicate statistically significant differences compared with the controls (the saline or LNP groups), ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8837369/v1/897c1aa1d7daa66fd4d78c14.png"},{"id":103505620,"identity":"83e3a8b3-4240-4f1e-8437-5775f6f41511","added_by":"auto","created_at":"2026-02-26 13:32:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256955,"visible":true,"origin":"","legend":"\u003cp\u003eThe protective\u003cstrong\u003e \u003c/strong\u003eefficacy\u003cstrong\u003e \u003c/strong\u003econferred by\u003cstrong\u003e \u003c/strong\u003ea single dose of LNP/mRNA-UreB vaccination.\u003cstrong\u003e A \u003c/strong\u003eBody weight changes in mice after \u003cem\u003eH. pylori\u003c/em\u003e SS1 challenge. \u003cstrong\u003eB\u003c/strong\u003e The colonisation levels of \u003cem\u003eH. pylori\u003c/em\u003e SS1 in gastric tissues. \u003cstrong\u003eC\u003c/strong\u003eUrease activity. The data are shown as the means ± SEMs; n = 10. Asterisks indicatestatistically significant differences compared with the controls (the saline or LNP groups), *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8837369/v1/9e0e1157963c6370ea3b0d2a.png"},{"id":104397620,"identity":"ce93a1e9-5b9b-4b42-8942-fc3b62ad721e","added_by":"auto","created_at":"2026-03-11 11:53:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2149285,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8837369/v1/8288a6cd-1d20-4adb-93a5-20686af8ffab.pdf"},{"id":103505228,"identity":"8524e402-a487-462c-ad66-8717fa19d079","added_by":"auto","created_at":"2026-02-26 13:28:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":352375,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8837369/v1/d6b4c364ff3537478a68891f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective immunity against Helicobacter pylori induced by a single dose of an mRNA-based UreB vaccine candidate in mice","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eHelicobacter pylori\u003c/em\u003e is a gram-negative microaerophilic bacterium associated with a high prevalence of human peptic ulcers and gastritis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Statistics indicate that approximately 76% of gastric cancer cases are associated with \u003cem\u003eH. pylori\u003c/em\u003e infection [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The World Health Organization (WHO) has declared that \u003cem\u003eH. pylori\u003c/em\u003e is a Group 1 carcinogen associated with major global public health issues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, the standard clinical eradication strategy relies on a 10\u0026ndash;14-day regimen combining antibiotics with proton pump inhibitors (PPIs) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, this regimen is subject to antibiotic resistance, which may disrupt the gut microbiota and increase recurrence rates after \u003cem\u003eH. pylori\u003c/em\u003e eradication [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, vaccination is the most effective measure for preventing and controlling \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003e \u003cem\u003eH. pylori\u003c/em\u003e infection is closely related to the movement of the flagellum and other aetiological factors. Various virulence factors, including urease subunit B (UreB), vacuolating toxin A (VacA), the cagA gene associated with cytotoxin (CagA), neutrophil-activating protein (NAP), heat-shock protein A (HspA), \u003cem\u003eH. pylori\u003c/em\u003e adhesin A (HpaA), and lipopolysaccharide (LPS), have been employed in the development of \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate antigens based on the completion of \u003cem\u003eH. pylori\u003c/em\u003e whole-genome sequencing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, UreB has been widely identified as the major vaccine target because of its critical role in \u003cem\u003eH. pylori\u003c/em\u003e survival and colonisation processes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nasab et al. used a viral-based vector system expressing the UreB antigen in rapeseed (\u003cem\u003eBrassica napus\u003c/em\u003e), and their results demonstrated rapid and low-cost plant-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine antigen production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hirota et al. identified a sequence involving 19 UreB-derived amino acids (AAs) as a potential antigenic epitope that induced the production of antibodies to inhibit \u003cem\u003eH. pylori\u003c/em\u003e activity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, Zhang et al. evaluated the immune efficacy of an attenuated \u003cem\u003eShigella\u003c/em\u003e vector expressing the UreB-HspA fusion protein of \u003cem\u003eH. pylori\u003c/em\u003e in a mouse model [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Liu et al. engineered three crucial \u003cem\u003eH. pylori\u003c/em\u003e antigen proteins (UreB, CagA, and VacA) onto the surfaces of outer membrane vesicles derived from \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium (\u003cem\u003eS\u003c/em\u003e. Typhimurium) using the haemoglobin protease (HBP) autotransporter system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our previous study demonstrated that \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e-based \u003cem\u003eH. pylori\u003c/em\u003e oral vaccines (EBY100/pYD1-UreB, EBY100/pYD1-VacA, or EBY100/pYD1-UreB\u0026thinsp;+\u0026thinsp;EBY100/pYD1-VacA) induced a significant immune response and reduced bacterial loads after \u003cem\u003eH. pylori\u003c/em\u003e infection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In summary, UreB or UreB combined with other virulence factors of \u003cem\u003eH. pylori\u003c/em\u003e has been widely identified as a promising and potential antigen target for clinical application [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003emRNA-based technology has already emerged as a novel therapeutic platform for emerging infectious diseases due to the successful applications of mRNA-1273 and BNT162b2 against SARS-CoV-2 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although mRNA vaccines demonstrate efficacy in cancer immunotherapy and for the targeting of viral pathogens, the development of mRNA vaccines against bacterial infections has been more challenging, due to the fact that bacteria exhibit greater biological complexity compared to viruses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recent evidence has demonstrated that mRNA vaccines represent alternative approaches for the prevention and treatment of bacterial infections against \u003cem\u003eM. tuberculosis\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003eB. burgdorferi\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], \u003cem\u003eB. pertussis\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eS. pyogenes\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], \u003cem\u003eListeria monocytogenes\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Among these agents, an mRNA candidate against tuberculosis, which is being tested in a clinical Phase 1 trial (NCT05547464) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], as well as an mRNA candidate against Lyme disease (NCT05975099) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], have been issued by Moderna and BioNTech, respectively. These preclinical and clinical bacterial mRNA vaccines may increase the availability of commercial applications. Unfortunately, no attempts have been made to develop mRNA vaccines against \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eTo assess the immune efficacy of an mRNA vaccine encoding the UreB of \u003cem\u003eH. pylori\u003c/em\u003e, we generated LNP/mRNA-UreB and investigated both its expression levels in vitro and immunogenicity in vivo in a mouse model by utilising a single dose. The results of the present study strongly suggest that LNP/mRNA-UreB is a promising vaccine candidate that provides an effective alternative approach to prevent \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003emRNA production\u003c/h2\u003e \u003cp\u003eThe pUC57-Kan plasmid, which contains the codon-optimised UreB gene of \u003cem\u003eH. pylori\u003c/em\u003e (GenBank accession No. 899104) and incorporates the 5' and 3' untranslated regions (UTRs) and 115-nt-long poly(A) tail, was synthesised (GenScript Biotech Corp., Nanjing, China) and termed pUC57/UreB. mRNA-UreB was produced by using a T7 RNA transcription kit (Hzymes Biotechnology Co., Ltd., Wuhan, China) on the linearised plasmid pUC57/UreB and subsequently purified by using lithium chloride (LiCl) precipitation. The mRNA-UreB concentration was adjusted to 1 mg/mL, after which it was stored at -20\u0026deg;C until use. The integrity of the mRNA-UreB was analysed by using a Fragment Analyzer 5200 (Agilent, USA). A scheme of the mRNA-UreB design is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLNP formulation\u003c/h3\u003e\n\u003cp\u003eThe mRNA-UreB in 20 mM citrate buffer (pH 4.0) was formulated into LNPs including ionisable lipids, 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), cholesterol and PEG-DMG 2000 (molar ratios of 50:10:38.5:1.5) at a ratio of 1:2 through INano\u003csup\u003eTM\u003c/sup\u003eX (Micronanobiologics, Shanghai, China). The formulation was buffer-exchanged into Tris buffer with CH3COONa and sucrose via tangential flow filtration (TFF), 0.22 \u0026micro;m-sterile-filtered, and stored at -80\u0026deg;C until use. The particle size, distribution, mRNA concentration and encapsulation rate of the formulation (known as LNP/mRNA-UreB) were measured via dynamic light scattering (DLS) (Malvern Instruments Ltd. England). The capping efficacy of LNP/mRNA-UreB was determined by using Liquid chromatograph mass spectrometer (LC-MS) (Agilent, USA). Empty LNPs served as negative controls.\u003c/p\u003e\n\u003ch3\u003emRNA transfection and expression analyses\u003c/h3\u003e\n\u003cp\u003eHepG2 cells (ATCC, USA) were seeded in 24-well plates at 10\u003csup\u003e5\u003c/sup\u003e cells per well and cultured in EMEM supplemented with 10% FBS, 1% NEAA, and 1% penicillin-streptomycin at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 12 h. Afterwards, 4 \u0026micro;g of mRNA-UreB was transfected into HepG2 cells per well by using a Fast LNP Transfection Kit (Hzymes Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer\u0026rsquo;s instructions. Cell lysates were harvested with RIPA lysis buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitor cocktail (Roche, Switzerland) at 6 h, 24 h, 48 h and 72 h after transfection. The supernatants derived from the cell lysates were mixed with 5 \u0026times; SDS-loading buffer after centrifugation at 13000 \u0026times; g and then loaded for SDS-PAGE. The UreB protein in the cell lysates was detected via Western blotting by using a rabbit polyclonal anti-UreB antibody (1:2000 dilution) as the primary antibody, with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) (Thermo Fisher Scientific, USA) being used as the secondary antibody. Moreover, β-actin (1:10000 dilution) (Thermo Fisher Scientific, USA) was also detected as an internal control (Thermo Fisher Scientific, USA). The blots were developed by using chemiluminescent substrates and detected by using a gel imaging system (Bio-Rad, USA).\u003c/p\u003e \u003cp\u003eFor immunofluorescence and flow cytometry assays, 100 \u0026micro;L of HepG2 cells transfected with LNP/mRNA-UreB for 24 h were harvested at 6000 \u0026times; g via centrifugation. The pellets were treated with blocking buffer containing 3% bovine serum albumin (BSA) for 30 min and then incubated with a rabbit polyclonal anti-UreB antibody (1:1000) (CUSABIO, Wuhan, China) at room temperature for 1 h. After being washed with sterile PBS three times, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:5000) (Thermo Fisher Scientific, USA) in the dark at room temperature for 40 min. The cells were then washed with sterile PBS three times and resuspended in 350 \u0026micro;L of sterile PBS. Ten microlitres of cell suspension was observed under a fluorescence microscope (Nikon, Japan). The remaining 340 \u0026micro;L of the sample was used for flow cytometry (BD Biosciences, USA) and analysed by using FlowJo software (BD Biosciences, USA). HepG2 cells without transfection served as the negative controls.\u003c/p\u003e\n\u003ch3\u003eQuantitative expression analysis\u003c/h3\u003e\n\u003cp\u003eThe expression level of the UreB protein was quantitatively measured via sandwich ELISA. Briefly, the transfected HepG2 cell lysates were harvested at 24 h posttransfection. The 96-well high-binding plates were coated with 100 \u0026micro;L of serially diluted polyclonal rabbit anti-UreB antibody (0 pg/mL, 62.5 pg/mL, 250 pg/mL, 500 pg/mL, 1000 pg/mL, 2000 pg/mL and 4000 pg/mL) and incubated overnight at 4\u0026deg;C. The plates were subsequently washed three times with sterile PBST (PBS\u0026thinsp;+\u0026thinsp;0.01% Tween 20) and then blocked with PBST containing 3% BSA for 1 h at 37\u0026deg;C. After the cells were washed three times with PBST, either 100 \u0026micro;L of the transfected HepG2 cell lysates or the standard antigen UreB (CUSABIO, Wuhan, China) was added to each well and incubated for 2 h at 37\u0026deg;C. The plates were subsequently washed three times with PBST and then incubated with 100 \u0026micro;L/well of a mouse polyclonal anti-UreB antibody for 2 h at 37\u0026deg;C. After being washed three times with PBST, the plates were incubated with 100 \u0026micro;L/well of HRP-conjugated goat anti-mouse antibody for 40 min at 37\u0026deg;C. Finally, the plates were developed with 3,3',5,5'-tetramethylbenzidine (TMB) in the dark for 20 min at room temperature. The reaction was stopped by the addition of 50 \u0026micro;L/well of 2 mol/L sulfuric acid. The absorbance was measured at 450 nm by using a microplate reader (Bio-Rad, USA). Each experiment was performed three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVaccination, sample collection and\u003c/b\u003e \u003cb\u003eH. pylori\u003c/b\u003e \u003cb\u003einfection in mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale BALB/c mice (6\u0026ndash;8-weeks-old) were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., China, and were immunised (10 mice/group) on day 1 with 50 \u0026micro;L of LNP/mRNA-UreB containing 4 \u0026micro;g of mRNA-UreB via intramuscular injections in the rear quadriceps. The same dosage (50 \u0026micro;L) of saline or empty LNPs served as negative controls. The immune schedule is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eBlood samples (10 mice/group) were obtained from the submandibular vein on days 14 and 28; additionally, serum was isolated to evaluate the antibody response. Moreover, 5 mice/group were anaesthetised via inhalation of isoflurane (4\u0026ndash;5% in oxygen), and spleens were obtained on day 28 to evaluate cell-mediated immunity.\u003c/p\u003e \u003cp\u003eFor protective efficacy analysis, the immunised mice were orally administered 500 \u0026micro;L of \u003cem\u003eH. pylori\u003c/em\u003e SS1 (1 \u0026times; 10⁹ CFU/mL) every other day for a total of three doses. Weight loss was monitored for 14 days. The mice were then euthanised, and the stomach was isolated on day 60 to detect the colonisation of \u003cem\u003eH. pylori\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay\u003c/h3\u003e\n\u003cp\u003eELISA was used to determine the IgG titres of mouse sera as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, 2 \u0026micro;g of UreB protein was coated into 96-well high binding microtitre plates (Corning, USA) at 4\u0026deg;C overnight. After blocking with 3% BSA in PBST for 2 h, the plates were washed three times with PBST. Serum samples from immunised mice were twofold serially diluted in blocking buffer, after which they were added to the plates and incubated at 37\u0026deg;C for 2 h. The plates were again washed three times with PBST and subsequently incubated with biotinylated anti-mouse IgG (Sigma, USA) at a 1:5000 dilution for 1 h at room temperature. After being washed three times with PBST, the plates were incubated with 100 \u0026micro;L/well of streptavidin-conjugated alkaline phosphatase (ALP) (R\u0026amp;D Systems, USA) at room temperature for 2 h and then washed three times with PBST. Afterwards, 100 \u0026micro;L of 1.25 mM pNPP phosphatase substrate (MP Biomedicals, USA) was added to each well and incubated in the dark for 25 min. The reaction was stopped by the addition of 50 \u0026micro;L of 2N NaOH to each well, and the optical density (OD) at 405 nm and 603 nm was measured with a multimode microplate reader (Bio-Tek, USA). The cutoff value was set as the reciprocal of the highest dilution to obtain an OD value more than twofold that of the negative serum.\u003c/p\u003e \u003cp\u003eFor IgG1 or IgG2a detection using the abovementioned ELISA, the biotinylated anti-mouse IgG was replaced with IgG1 and IgG2a.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISpot assays\u003c/h2\u003e \u003cp\u003eThe cell-mediated response was measured via ELISpot as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, 96-well ELISpot plates (Corning, USA) were coated with anti-mouse IFN-γ/IL-4 capture Ab (R\u0026amp;D Systems, USA) and incubated overnight at 4\u0026deg;C. The plates were subsequently washed with PBST and blocked with PBST containing 1% BSA for 2 h. A total of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e splenocytes from the immunised mice were added to each well and incubated overnight at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e with 10 \u0026micro;L of UreB protein (1 \u0026micro;g/ml) stimulation. After 36 h, the plates were washed and then incubated at 37\u0026deg;C for 2 h with biotinylated anti-mouse IFN-γ Ab (R\u0026amp;D Systems, USA). Streptavidin-alkaline phosphatase (R\u0026amp;D Systems, USA) was added to each well after washing with PBST, after which the samples were incubated for 1 h at room temperature. The plates were subsequently washed, after which 50 \u0026micro;L of pNPP was added to each well. Finally, the plates were rinsed with sterile H\u003csub\u003e2\u003c/sub\u003eO and dried at room temperature. The spots were quantified by using an automatic ELISpot reader (CTL Limited, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtective efficacy analysis\u003c/h3\u003e\n\u003cp\u003eThe protective efficacy was measured by using a rapid urease test kit (Beyotime Biotechnology, China) according to the manufacturer\u0026rsquo;s instructions. The samples obtained from the stomach tissue homogenates were mixed with the urease reaction solution and incubated at room temperature for 2 h. Finally, the absorbance at OD\u003csub\u003e550nm\u003c/sub\u003e was measured by using a microplate reader (Bio-Tek, USA).\u003c/p\u003e \u003cp\u003eThe colonisation load of \u003cem\u003eH. pylori\u003c/em\u003e in the gastric tissues of immunised mice was determined by using the bacterial culture method, as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, tenfold serial dilutions of the gastric tissue homogenates were inoculated on Columbia blood agar plates. The colonisation number was calculated at 3 days after culture.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with GraphPad Prism version 10.1.2. The data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs or SEMs. Statistical significance for group comparisons was determined by using one-way ANOVA with Tukey\u0026rsquo;s post hoc test for comparisons with control groups. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConstruction and characterisation of mRNA-UreB\u003c/h2\u003e \u003cp\u003eWe generated mRNA-UreB by inserting the 5', 3' untranslated regions (UTRs) and poly(A) tail into the flank of the codon-optimised UreB gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This UreB antigen has previously been observed to be protective as a subunit vaccine candidate target [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The length of mRNA-UreB was observed to be approximately 2,200 nt (\u003cb\u003eAdditional file: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), which is consistent with the expected size. Furthermore, the integrity of mRNA-UreB was observed to be approximately 97% (\u003cb\u003eAdditional file: Fig. S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe in vitro expression profile was confirmed by subjecting HepG2 cells transfected with mRNA-UreB to Western blotting, immunofluorescence assays and flow cytometric assays. As expected, a specific protein band was detected via Western blotting in the cell lysates of HepG2 cells transfected with mRNA-UreB, which revealed a relative molecular mass of approximately 80\u0026thinsp;~\u0026thinsp;90 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eLanes 5\u0026ndash;8\u003c/b\u003e). Notably, the highest expression level of UreB protein in the transfected HepG2 cells occurred at 24 h after transfection (\u003cb\u003eAdditional file: Fig. S3)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSimilarly, strong fluorescence signals were observed in the mRNA-UreB-transfected HepG2 cells than in the untransfected HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cb\u003eRight\u003c/b\u003e). Subsequent flow cytometry assays also revealed that the positive rate of mRNA-UreB-transfected HepG2 cells was approximately 51.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cb\u003eRight\u003c/b\u003e). Furthermore, the quality control of the mRNA-UreB encapsulated by LNPs was analysed, as shown in \u003cb\u003eAdditional file: Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative expression analysis of mRNA-UreB\u003c/h2\u003e \u003cp\u003eTo determine the quantitative expression of mRNA-UreB, cell lysates from HepG2 cells transfected with mRNA-UreB at 24 h after transfection were analysed via sandwich ELISA. A standard curve was generated by using GraphPad Prism software (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The equation for the standard curve was determined as y\u0026thinsp;=\u0026thinsp;0.0001x\u0026thinsp;+\u0026thinsp;0.0678, and the R\u0026sup2; value was 0.991. Moreover, the OD\u003csub\u003e450nm\u003c/sub\u003e value of the mRNA-UreB-transfected HepG2 cells was 0.158, which corresponded to a specific concentration of UreB protein in the HepG2 cells of 902 pg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntibody response induced by a single dose of LNP/mRNA-UreB\u003c/h2\u003e \u003cp\u003eTo assess humoral immunogenicity, the total anti-UreB IgG antibody response from all mice vaccinated with a single dose of 50 \u0026micro;L of LNP/mRNA-UreB containing 4 \u0026micro;g of mRNA-UreB via intramuscular injection was analysed in the serum samples collected on days 14 and 28; moreover, the same dosage of saline or empty LNP functioned as a control group. At 14 and 28 days after initial immunisation, the ELISA results indicated that a single dose of LNP/mRNA-UreB could elicit robust UreB-specific IgG antibodies; in comparison, the IgG titre on day 28 was significantly greater than that on day 14, whereas all of the control groups (including the saline and LNP groups) exhibited undetectable antibody levels for UreB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results demonstrate that a single dose of LNP/mRNA-UreB effectively induces efficient humoral immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the IgG subtypes, the levels of IgG1 and IgG2a were also analysed via ELISA. A similar trend as that of the IgG antibody was detected for IgG1 and IgG2a. Specifically, the levels of both IgG1 and IgG2a antibodies induced by the LNP/mRNA-UreB group were significantly greater than the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C) on days 14 and 18 after the initial immunisation. Notably, the IgG1/IgG2a ratio was calculated to be approximately 1.0, thus suggesting that a relatively balanced Th1/Th2 mixed immune response was elicited by a single dose of LNP/mRNA-UreB, which contributed to the balanced adaptative immune response profiles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell-mediated immune response induced by a single dose of LNP/mRNA-UreB\u003c/h2\u003e \u003cp\u003eThe secretion levels of IFN-γ and IL-4 cytokines induced by LNP/mRNA-UreB in mouse splenocytes were measured via ELISpot assays at 28 days after the initial immunisation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the LNP/mRNA-UreB group demonstrated characteristic increases in the secretion levels of IFN-γ and IL-4 compared to the control groups (the saline or LNP group). Notably, a significantly greater secretion of IFN-γ spots was induced by LNP/mRNA-UreB, thus indicating a T-helper-1 (Th1)-biased cell-mediated immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the concentrations of IFN-γ and IL-4 secretion spots mediated by CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells were measured. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells produced increased numbers of IFN-γ spots in response to stimulation with the UreB antigen, whereas the level of secreted IL-4 was relatively low. Consequently, the IFN-γ/IL-4 spot ratio was significantly increased, thereby suggesting that a meaningful mixed Th1/Th2 immune response was induced by a single dose of LNP/mRNA-UreB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtective efficacy induced by LNP/mRNA-UreB\u003c/h2\u003e \u003cp\u003eThe protective efficacy induced by a single dose of LNP/mRNA-UreB was assessed by using \u003cem\u003eH. pylori\u003c/em\u003e SS1 challenge assays on day 42. Body weight changes were monitored for 14 days. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, body weight loss in the control groups (the saline or LNP groups) was observed to be approximately 5% on day 5 after \u003cem\u003eH. pylori\u003c/em\u003e SS1 challenge until recovery was observed on day 12. In contrast, the LNP/mRNA-UreB group exhibited a slight decrease in body weight on day 2, with a weight loss rate of approximately 1%; moreover, body weight gradually recovered on day 3, thus suggesting that LNP/mRNA-UreB effectively protected mice from \u003cem\u003eH. pylori\u003c/em\u003e SS1 infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe colonisation numbers of \u003cem\u003eH. pylori\u003c/em\u003e in the gastric tissue were also determined on day 60 after the initial immunisation. Compared with the control groups (the saline or LNP groups), the LNP/mRNA-UreB group demonstrated a significant reduction in \u003cem\u003eH. pylori\u003c/em\u003e SS1 colonisation in gastric tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo further assess the protective efficacy of LNP/mRNA-UreB against \u003cem\u003eH. pylori\u003c/em\u003e SS1 infection, urease activity was measured in the gastric tissues isolated from all of the vaccinated mice. The OD\u003csub\u003e550nm\u003c/sub\u003e values in the LNP/mRNA-UreB group were much lower than those in the control groups (the saline or LNP groups) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), thereby demonstrating that LNP/mRNA-UreB effectively reduced \u003cem\u003eH. pylori\u003c/em\u003e SS1 colonisation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eH. pylori\u003c/em\u003e infection remains a serious public health concern [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In particular, the increasing rate of \u003cem\u003eH. pylori\u003c/em\u003e infection induced by antibiotic resistance is a serious issue for antimicrobial therapy. Therefore, an effective vaccine against \u003cem\u003eH. pylori\u003c/em\u003e is urgently needed to prevent malignant gastric tumours and other serious \u003cem\u003eH. pylori\u003c/em\u003e-associated diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Most of the current vaccine research on \u003cem\u003eH. pylori\u003c/em\u003e conducted in the preclinical or clinical phases has focused on oral administration routes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To define a more optimised vaccine approach, we first developed an mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate via a single dose of LNP/mRNA-UreB and subsequently investigated its immunogenicity and protective efficacy against \u003cem\u003eH. pylori\u003c/em\u003e infection in a mouse model, which represents a promising direction for the development of preventive vaccines against \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eTo ensure the efficient expression of LNP/mRNA-UreB, we performed a specific design on the key components of mRNA-UreB. First, we utilised codon-optimised software to optimise the UreB antigen, and humans were used as the final host, which provided the foundation for the future clinical application of \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates. Second, the modified 5'-UTR and 3'-UTR derived from the commercial products of mRNA-1273 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and BNT162b [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], respectively, were incorporated into mRNA-UreB. Notably, both the 5'-UTR and the 3'-UTR originated from human α-globin HBA1 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Third, we used a strategy similar to that of the Pfizer/BioNTech mRNA vaccine [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], in which two segmented poly-A tails (30A+75A) with an interval of 10 nucleotides were added into the 3\u0026prime; end of mRNA-UreB. All of these optimised elements demonstrated superior potential for the high expression level of mRNA-UreB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eIn addition, the current commercial LNP formulations used in the present study mainly consist of four components, including ionisable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids, and the molecular ratio of the four components was optimised to form an ideal encapsulation. Notably, the LNP formulation is being continuously optimised. Su et al. developed three formulations to achieve precise pulmonary targeting and accumulation of mRNA [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], thereby representing a safe and effective LNP formulation with distinct targeting properties.\u003c/p\u003e \u003cp\u003eImmune responses serve as the most important indicators for evaluating the immune efficacy induced by \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates. Thus, the total IgG antibody response is strongly related to the immunogenicity of LNP/mRNA-UreB after single-dose vaccination in a mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and is superior to that observed in our previous study, in which the oral administration of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e resulted in UreB [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, we detected the ratio of serum IgG1 and IgG2a for the serum IgG subtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C), in which IgG1 was closely linked to humoral immunity and effectively neutralised target antigens; moreover, IgG2a played a critical role in the cell-mediated immune response against bacterial infections [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], as reflected by the high secretion level of IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In summary, the LNP/mRNA-UreB vaccine developed in this study has been demonstrated to elicit balanced activation of both humoral and cellular immune responses, thus greatly contributing to the fight against intracellular pathogen infection [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells synergise to generate immunity against intracellular bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and the IFN-γ/IL-4 ratio could serve as a clinical substitute marker for Th1/Th2 balance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The ratio of IFN-γ/IL-4 secretion detected in this study strongly supports the notion that LNP/mRNA-UreB could induce a mixed Th1/Th2-mediated cellular immune response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and support the feasibility of mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates.\u003c/p\u003e \u003cp\u003eAn important characteristic of \u003cem\u003eH. pylori\u003c/em\u003e is its ability to survive in extremely acidic gastric tissues, which easily leads to constant \u003cem\u003eH. pylori\u003c/em\u003e infection after eradication therapy. The presence of \u003cem\u003eH. pylori\u003c/em\u003e infection is mainly evaluated via urease assays [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, the extent of \u003cem\u003eH. pylori\u003c/em\u003e colonisation in gastric tissues directly reflects the severity of \u003cem\u003eH. pylori\u003c/em\u003e infection and the immune efficacy of \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates. An outer membrane vesicle (OMV)-based or \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e-based UreB vaccine was observed to decrease the colonisation levels and urease activity by approximately twofold compared with those of the controls [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], whereas mice vaccinated with a single dose of LNP/mRNA-UreB in this study exhibited approximately fourfold greater bacterial colonisation and approximately fivefold lower urease activity than the controls (the saline or LNP groups) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C), thus suggesting that an mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate is superior to OMV-based or \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e-based UreB vaccines.\u003c/p\u003e \u003cp\u003eAs with different vaccine platforms, the development of mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates is dependent on the reasonable selection and modification of antigens, as well as an efficient immune schedule. It is well known that an mRNA-based vaccine requires only a single dose that elicits robust immune responses [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our study clearly demonstrated that a single dose of LNP/mRNA-UreB could induce sufficient immunity against \u003cem\u003eH. pylori\u003c/em\u003e infection in a mouse model, thus supporting the advantage of an mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate. Taken together, these findings suggest that LNP/mRNA-UreB elicits balanced immune responses, as well as a Th1-mediated cellular immune response. Importantly, mice vaccinated via a single dose of LNP/mRNA-UreB exhibited effectively reduced bacterial colonisation in gastric tissues after \u003cem\u003eH. pylori\u003c/em\u003e infection. \u003cem\u003eH. pylori\u003c/em\u003e has multiple important virulence factors (with the exception of UreB), such as VacA, CagA, and Hsp; moreover, UreB may be feasible for serving as a target antigen for the development of an mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate. Therefore, the further design of combined \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates based on mRNA platforms is highly important for the effective control of \u003cem\u003eH. pylori\u003c/em\u003e infection and other infectious diseases.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOverall, we have developed a single-dose mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate that provides significant protection against \u003cem\u003eH. pylori\u003c/em\u003e infection by mediating balanced immune responses. An mRNA-based technology platform may represent a promising approach for the development of \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidates and speed up the translation from preclinical studies to clinical applications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eamino acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eALP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlkaline phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCagA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytotoxin-associated gene A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCFU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColony-forming units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCGE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCapillary gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDLS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDynamic light scattering\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDSPC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,2-distearoylsn-glycero-3-phosphocholine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eELISA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFITC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescein isothiocyanate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHBP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHemoglobin protease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHpaA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eH. pylori\u003c/em\u003e adhesin A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eH. pylori.\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHelicobacter pylori\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHRP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHspA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat-shock protein A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIVT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIn vitro transcribed\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLC-MS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiquid chromatograph mass spectrometer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLNP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipid nanoparticles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLPS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNAP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutrophil-activating protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNEAA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-Essential Amino Acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBST\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffered saline-Tween\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePEG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyethylene glycol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePPI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProton pump inhibitor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSDS-PAGE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTFF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTangential flow filtration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTh1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT-helper-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTMB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,3',5,5'-Tetramethylbenzidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eUreB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrease B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eUTR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUntranslated regions\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVacA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVacuolating cytotoxin A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eWHO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThis study was carried out in strict accordance with the protocols approved by Southwest Jiaotong University. All mouse experiments were performed according to the guidelines stated by the Institutional Animal Care and Use Committees of Southwest Jiaotong University (Approval No. SWJTU-2503-041).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 31360225; No. 32070920), and the Fundamental Research Funds for the Central Universities (No. 2682023ZTPY06387) to H. Lei.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHL planned and designed the study. JG, YH, YY, LY and HL contributed to the data analysis and the interpretation of the results. JG and HL wrote the manuscript and produced all the figures. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Kui Wang at Hualan Biological Vaccine Co., Ltd, China, for his assistance with analysis of mRNA-UreB.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShah SAR, Mumtaz M, Sharif S, Mustafa I, Nayila I. Helicobacter pylori and gastric cancer: current insights and nanoparticle-based interventions. 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N Engl J Med. 2023;389(24):2233\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMoa2307079\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa2307079\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"LNP/mRNA-UreB, Single dose, Immunogenicity, Protective efficacy, H. pylori infection","lastPublishedDoi":"10.21203/rs.3.rs-8837369/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8837369/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground Helicobacter pylori\u003c/h2\u003e \u003cp\u003e(\u003cem\u003eH. pylori\u003c/em\u003e) is a global public health concern because of its strong ability to colonise the gastrointestinal tract. Current clinical therapeutic strategies are limited by antibiotic resistance, reinfection and a high recurrence rate after eradication. Therefore, effective preventive vaccines against \u003cem\u003eH. pylori\u003c/em\u003e infections are needed to counter the potential threat of a global pandemic. Recently, mRNA vaccines have emerged as promising platforms for the prevention of major infectious diseases. UreB is regarded as a critical virulence factor in \u003cem\u003eH. pylori\u003c/em\u003e and serves as a key vaccine candidate target. This study aimed to investigate the immunogenicity of an mRNA-based agent against \u003cem\u003eH. pylori\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe generated an mRNA-based \u003cem\u003eH. pylori\u003c/em\u003e vaccine candidate targeting UreB. The expression levels of LNP/mRNA-UreB in transfected HepG2 cells in vitro were characterised via Western blot, immunofluorescence, flow cytometric and sandwich ELISA. BALB/c mice were vaccinated with a single dose of LNP/mRNA-UreB to evaluate its immunogenicity and immune protective efficacy against \u003cem\u003eH. pylori\u003c/em\u003e. Both humoral and cellular immune responses induced by LNP/mRNA-UreB were measured via ELISA, followed by challenge with \u003cem\u003eH. pylori\u003c/em\u003e to validate the immune protective efficacy.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLNP/mRNA-UreB was highly expressed in HepG2 cells; additionally, the molecular weight of the UreB antigen ranged from approximately 80\u0026ndash;90 kDa, and the quantitative expression concentration was determined to be 902 pg/mL. Furthermore, mice vaccinated with LNP/mRNA-UreB via a single dose exhibited robust UreB-specific humoral immune responses with markedly elevated antibody levels and a significant Th1/Th2 mixed cellular immune response. Finally, in mice vaccinated with LNP/mRNA-UreB and subsequently infected with \u003cem\u003eH. pylori\u003c/em\u003e SS1, gastric colonisation and urease levels were effectively reduced, demonstrating that LNP/mRNA-UreB was a promising vaccine candidate against \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe LNP/mRNA-UreB vaccine candidate developed in this study represents a proof-of-concept for effective protection against \u003cem\u003eH. pylori\u003c/em\u003e infection and mediates balanced humoral and cellular immune responses. Moreover, mRNA-based techniques may constitute a universal approach for the development of \u003cem\u003eH. pylori\u003c/em\u003e vaccines.\u003c/p\u003e","manuscriptTitle":"Protective immunity against Helicobacter pylori induced by a single dose of an mRNA-based UreB vaccine candidate in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 19:14:45","doi":"10.21203/rs.3.rs-8837369/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"78930d73-ec0c-401d-b2b7-d27387947dc6","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T11:42:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 19:14:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8837369","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8837369","identity":"rs-8837369","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}