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Peng X, Zhang R, Wang C, Yu F, Yu M, Chen S, Fan Q, Xi Y, Duan G. E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti- H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions. Cells 2019; 8:982. [PMID: 31461854 PMCID: PMC6770474 DOI: 10.3390/cells8090982] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2019] [Revised: 08/23/2019] [Accepted: 08/23/2019] [Indexed: 12/11/2022] Open
Abstract
Current studies indicate that the anti-H. pylori protective efficacy of oral vaccines to a large extent depends on using mucosal adjuvants like E. coli heat-lable enterotoxin B unit (LtB). However, the mechanism by which Th17/Th1-driven cellular immunity kills H. pylori and the role of LtB remains unclear. Here, two L.lactis strains, expressing H. pylori NapA and LtB, respectively, were orally administrated to mice. As observed, the administration of LtB significantly enhanced the fecal SIgA level and decreased gastric H. pylori colonization, but also markedly aggravated gastric inflammatory injury. Both NapA group and NapA+LtB group had elevated splenocyte production of IL-8, IL-10, IL-12, IL-17, IL-23 and INF-γ. Notably, gastric leukocytes' migration or leakage into the mucus was observed more frequently in NapA+LtB group than in NapA group. This report is the first that discusses how LtB enhances vaccine-induced anti-H. pylori efficacy by aggravating gastric injury and leukocytes' movement into the mucus layer. Significantly, it brings up a novel explanation for the mechanism underlying mucosal cellular immunity destroying the non-invasive pathogens. More importantly, the findings suggest the necessity to further evaluate LtB's potential hazards to humans before extending its applications. Thus, this report can provide considerable impact on the fields of mucosal immunology and vaccinology.
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Affiliation(s)
- Xiaoyan Peng
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
- Department of Basic Medicine, Chuxiong Medical College, Chuxiong 675005, China
| | - Rongguang Zhang
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China.
| | - Chen Wang
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
| | - Feiyan Yu
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
| | - Mingyang Yu
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
| | - Shuaiyin Chen
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
| | - Qingtang Fan
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
| | - Yuanlin Xi
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
| | - Guangcai Duan
- Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
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Espinosa-Ramos D, Caballero-Hernández D, Gomez-Flores R, Trejo-Chávez A, Pérez-Limón LJ, de la Garza-Ramos MA, Tamez-Guerra R, Tamez-Guerra P, Rodriguez-Padilla C. Immunization with a Synthetic Helicobacter pylori Peptide Induces Secretory IgA Antibodies and Protects Mice against Infection. THE CANADIAN JOURNAL OF INFECTIOUS DISEASES & MEDICAL MICROBIOLOGY = JOURNAL CANADIEN DES MALADIES INFECTIEUSES ET DE LA MICROBIOLOGIE MEDICALE 2019; 2019:8595487. [PMID: 31065302 PMCID: PMC6466936 DOI: 10.1155/2019/8595487] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/05/2018] [Revised: 02/05/2019] [Accepted: 02/11/2019] [Indexed: 01/20/2023]
Abstract
Helicobacter pylori is a spiral Gram-negative bacterium associated with inflammation of the gastric mucosa, peptic ulcer, and gastric adenocarcinoma, whose treatment has failed due to antibiotic resistance and side effects. Furthermore, because there are no vaccines effective against H. pylori, an appropriate vaccine design targeting conserved/essential genes must be identified. In the present study, a H. pylori 50-52 kDa immunogen-derived peptide antigen with the sequence Met-Val-Thr-Leu-Ile-Asn-Asn-Glu (MVTLINNE) was used to immunize against H. pylori infection. For this, mice received an intraperitoneal injection of 100 μg of H. pylori peptide on the first week, followed by two weekly subcutaneous reinforcements and further 109 bacteria administration in the drinking water for 3 weeks. Thymic cells proliferative responses to concanavalin A, serum levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α cytokines, and IgG1, IgG2a, IgG2b, IgG3 IgM, and IgA immunoglobulins were evaluated. Significant (p < 0.05) increases on lymphoproliferation and spleen weights after immunization were observed. In contrast, infection significantly (p < 0.05) decreased lymphoproliferation, which was recovered in immunized mice. In addition, levels of serum TH1 and TH2 cytokines were not altered after immunization, except for the significant increase in IL-6 production in immunized and/or infected animals. Moreover, immunization correlated with plasma secretory IgA and IgG, whereas infection alone only elicited IgM antibodies. Peptide immunization protected 100% of mice against virulent H. pylori. MVTLINNE peptide deserves further research as an approach to the prophylaxis of H. pylori infection.
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Affiliation(s)
- David Espinosa-Ramos
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
| | - Diana Caballero-Hernández
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
| | - Ricardo Gomez-Flores
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
| | - Armando Trejo-Chávez
- Universidad Autónoma de Nuevo León, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Patobiología, Campus de Ciencias Agropecuarias, Escobedo, NL. C.P. 66050, Mexico
| | - Luis Jerónimo Pérez-Limón
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
| | - Myriam Angélica de la Garza-Ramos
- Universidad Autónoma de Nuevo León, Facultad de Odontología y Centro de Investigación y Desarrollo en Ciencias de la Salud, Unidad de Odontología Integral y Especialidades, Av. Dr. Aguirre Pequeño y Silao S/N, Monterrey, NL. C.P. 64460, Mexico
| | - Reyes Tamez-Guerra
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
| | - Patricia Tamez-Guerra
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
| | - Cristina Rodriguez-Padilla
- Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología, San Nicolás de los Garza, NL. C.P. 66450, Mexico
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The Human Stomach in Health and Disease: Infection Strategies by Helicobacter pylori. Curr Top Microbiol Immunol 2017; 400:1-26. [PMID: 28124147 DOI: 10.1007/978-3-319-50520-6_1] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Helicobacter pylori is a bacterial pathogen which commonly colonizes the human gastric mucosa from early childhood and persists throughout life. In the vast majority of cases, the infection is asymptomatic. H. pylori is the leading cause of peptic ulcer disease and gastric cancer, however, and these outcomes occur in 10-15% of those infected. Gastric adenocarcinoma is the third most common cause of cancer-associated death, and peptic ulcer disease is a significant cause of morbidity. Disease risk is related to the interplay of numerous bacterial host and environmental factors, many of which influence chronic inflammation and damage to the gastric mucosa. This chapter summarizes what is known about health and disease in H. pylori infection, and highlights the need for additional research in this area.
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Zhou Z, Dong H, Huang Y, Yao S, Liang B, Xie Y, Long Y, Mai J, Gong S. Recombinant Bacillus subtilis spores expressing cholera toxin B subunit and Helicobacter pylori urease B confer protection against H. pylori in mice. J Med Microbiol 2017; 66:83-89. [DOI: 10.1099/jmm.0.000404] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Affiliation(s)
- Zhenwen Zhou
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Hui Dong
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Yanmei Huang
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Shuwen Yao
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Bingshao Liang
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Yongqiang Xie
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Yan Long
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Jialiang Mai
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
| | - Sitang Gong
- Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou,Guangdong 510120, PR China
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Suganya K, Prem Kumar A, Sekar B, Sundaran B. Protection of mice against gastric colonization of Helicobacter pylori by therapeutic immunization with systemic whole cell inactivated vaccines. Biologicals 2017; 45:39-46. [DOI: 10.1016/j.biologicals.2016.10.002] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2016] [Revised: 09/06/2016] [Accepted: 10/04/2016] [Indexed: 10/20/2022] Open
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Fuenmayor-Boscán AD, Hernández IM, Valero KJ, Paz AM, Sandrea LB, Rivero Z. Association between Helicobacter pylori and intestinal parasites in an Añu indigenous community of Venezuela. Indian J Gastroenterol 2016; 35:106-12. [PMID: 27138927 DOI: 10.1007/s12664-016-0641-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Accepted: 03/13/2016] [Indexed: 02/04/2023]
Abstract
BACKGROUND Helicobacter pylori (Hp) and enteroparasite infections are highly prevalent in populations with poor living conditions, like the Amerindian communities. Identifying associations between both types of infectious agents could help to detect shared risk factors or transmission routes in these minority ethnic groups. Therefore, the prevalence and association between Hp and enteroparasites were investigated in an indigenous community whose living conditions favor such infectious diseases. METHODS Seropositivity (anti-Hp-specific IgG) and active infection (stool antigen test), intestinal parasitosis (direct and concentrated coproparasitological test, methylene blue, and Kinyoun stains), and risk factors for fecal-oral transmission were determined in 167 children and 151 adults of the Añu indigenous community living at the Sinamaica Lagoon, in Venezuela. RESULTS A high rate of Hp infection (seropositivity and active infection) and enteroparasitosis was evidenced, as expected. Some significant associations were detected: direct associations between Hp and polyparasitic infection, helminths, and protozoan (particularly in children); inverse association between Hp and Giardia lamblia. No shared epidemiological factors were identified for Hp and the detected intestinal parasites, probably due to overlapping factors. CONCLUSION Direct associations detected support the participation of the fecal-oral route in the transmission of the involved infectious agents. Inverse relationship (Hp) and G. lamblia may suggest the existence of antagonistic interactions between them. Further research is required to elucidate the mechanisms underlying these associations.
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Affiliation(s)
- Alisbeth D Fuenmayor-Boscán
- Facultad de Medicina, Departamento de Microbiología. Escuela de Bioanálisis, Universidad del Zulia, Maracaibo, Venezuela.
| | - Ileana M Hernández
- Facultad de Medicina Instituto de Investigaciones Biológicas, Universidad del Zulia, Maracaibo, Venezuela
| | - Kutchynskaya J Valero
- Facultad de Medicina, Departamento de Microbiología. Escuela de Bioanálisis, Universidad del Zulia, Maracaibo, Venezuela
| | - América M Paz
- Facultad de Medicina, Departamento de Microbiología. Escuela de Bioanálisis, Universidad del Zulia, Maracaibo, Venezuela
| | - Lisette B Sandrea
- Facultad de Medicina, Departamento de Microbiología. Escuela de Bioanálisis, Universidad del Zulia, Maracaibo, Venezuela
| | - Zulbey Rivero
- Facultad de Medicina, Departamento de Microbiología. Escuela de Bioanálisis, Universidad del Zulia, Maracaibo, Venezuela
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Moyat M, Velin D. Immune responses to Helicobacter pylori infection. World J Gastroenterol 2014; 20:5583-5593. [PMID: 24914318 PMCID: PMC4024767 DOI: 10.3748/wjg.v20.i19.5583] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Revised: 12/13/2013] [Accepted: 02/20/2014] [Indexed: 02/06/2023] Open
Abstract
Helicobacter pylori (H. pylori) infection is one of the most common infections in human beings worldwide. H. pylori express lipopolysaccharides and flagellin that do not activate efficiently Toll-like receptors and express dedicated effectors, such as γ-glutamyl transpeptidase, vacuolating cytotoxin (vacA), arginase, that actively induce tolerogenic signals. In this perspective, H. pylori can be considered as a commensal bacteria belonging to the stomach microbiota. However, when present in the stomach, H. pylori reduce the overall diversity of the gastric microbiota and promote gastric inflammation by inducing Nod1-dependent pro-inflammatory program and by activating neutrophils through the production of a neutrophil activating protein. The maintenance of a chronic inflammation in the gastric mucosa and the direct action of virulence factors (vacA and cytotoxin-associated gene A) confer pro-carcinogenic activities to H. pylori. Hence, H. pylori cannot be considered as symbiotic bacteria but rather as part of the pathobiont. The development of a H. pylori vaccine will bring health benefits for individuals infected with antibiotic resistant H. pylori strains and population of underdeveloped countries.
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Li Y, Jiang Y, Xi Y, Zhang L, Luo J, He D, Zeng S, Ning Y. Identification and characterization of H-2d restricted CD4+ T cell epitopes on Lpp20 of Helicobacter pylori. BMC Immunol 2012; 13:68. [PMID: 23234363 PMCID: PMC3534527 DOI: 10.1186/1471-2172-13-68] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2012] [Accepted: 11/30/2012] [Indexed: 12/12/2022] Open
Abstract
Background Previous investigation has demonstrated that CD4+ T cells play a crucial role in effective immunity against Helicobacter pylori (H.pylori) infection. It has been well proved that Lpp20 is one of major protective antigens that induce immune responses after H.pylori invades host. Therefore it is valuable to identify CD4+ T cell epitopes on Lpp20, which is uncharacterized. Methods Putative epitopes of H-2d restricted CD4+ T cell on Lpp20 of H.pylori were predicted by the SYFPEITHI algorithm and then eight hypothetical epitope peptides were synthesized. After BALB/c mice were primed with recombinant Lpp20, splenic CD4+ T cells were isolated and stimulated with synthesized peptides to measure T cell proliferation and MHC restriction. Cytokine profile was determined by ELISA and real-time PCR. Two identified epitopes were used to immunize mice to investigate CD4+ T cell response by flow cytometry. Results Two of eight peptides were able to stimulate CD4+ T cell proliferation and were mapped to residues 83-97aa and 58-72aa on Lpp20 respectively. These two peptides additively stimulated Th1 cells to secrete IFN-γ. The percentage of CD4+ T cell from mice immunized with two identified epitopes respectively was higher than the control group. Conclusion The identification and characterization of two CD4+ T cell epitopes of Lpp20 helps understand the protective immunity of Lpp20 in H.pylori infection and design effective epitope vaccines against H.pylori.
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Affiliation(s)
- Yan Li
- Institute of Biotherapy, School of Biotechnology, Southern Medical University, North1838 Guangzhou Road, Guangzhou 510515, PR China.
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Huang CF, Wu TC, Wu CC, Lee CC, Lo WT, Hwang KS, Hsu ML, Peng HJ. Sublingual vaccination with sonicated Salmonella proteins and mucosal adjuvant induces mucosal and systemic immunity and protects mice from lethal enteritis. APMIS 2011; 119:468-478. [PMID: 21635554 DOI: 10.1111/j.1600-0463.2011.02761.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Salmonella enteritidis is one of the most common pathogens of enteritis. Most experimental vaccines against Salmonella infection have been applied through injections. This is a new trial to explore the effect of sublingual administration of Salmonella vaccines on systemic and mucosal immunity. Adult BALB/c mice were sublingually vaccinated with sonicated Salmonella proteins (SSP) alone, or plus adjuvant CpG DNA (CpG) or cholera toxin (CT). They were boosted 2 weeks later. Saliva specific secretory IgA (SIgA) antibody responses were significantly stimulated in the mice vaccinated with SSP only or together with CpG or CT. Whereas the mice sublingually vaccinated with SSP and CpG had higher spleen cell IFN-γ production and serum specific IgG2a antibody responses, those receiving SSP and CT showed enhanced spleen cell IL-4, IL-5 and IL-6 production, and serum specific IgG1 antibody responses. After oral challenge with live S. enteritidis, the same strain of the source of SSP, immune protection in those sublingually vaccinated with SSP and CpG or CT was found to prevent intestinal necrosis and to render a higher survival rate. In conclusion, sublingual vaccination together with mucosal adjuvant CpG or CT is a simple but effective way against enteric bacterial pathogens.
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Affiliation(s)
- Ching-Feng Huang
- Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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Abstract
A vaccination against Helicobacter pylori may represent both prophylactic and therapeutic approaches to the control of H. pylori infection. Different protective H. pylori-derived antigens, such as urease, vacuolating cytotoxin A, cytotoxin-associated antigen, neutrophil-activating protein and others can be produced at low cost in prokaryote expression systems and most of these antigens have already been administered to humans and shown to be safe. The recent development by Graham et al. of the model of H. pylori challenge in humans, the recent published clinical trials and the last insight generated in animal models of H. pylori infection regarding the immune mechanisms leading to vaccine-induced Helicobacter clearance will facilitate the evaluation of immunogenicity and efficacy of H. pylori vaccine candidates in Phase II and III clinical trials.
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Affiliation(s)
- Dominique Velin
- Service de Gastro-entérologie et d'Hépatologie, Centre Hospitalier Universitaire Vaudois and University of Lausanne, BH18-521, Rue du Bugnon 46, CH-1011 Lausanne, Switzerland.
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Inoue K, Shiota S, Yamada K, Gotoh K, Suganuma M, Fujioka T, Ahmed K, Iha H, Nishizono A. Evaluation of a new tumor necrosis factor-alpha-inducing membrane protein of Helicobacter pylori as a prophylactic vaccine antigen. Helicobacter 2009; 14:135-43. [PMID: 19751439 DOI: 10.1111/j.1523-5378.2009.00713.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND Tumor necrosis factor (TNF)-alpha-inducing protein (Tip alpha) is a newly identified carcinogenic factor present in Helicobacter pylori. Tip alpha has the unique function of inducing TNF-alpha production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tip alpha against H. pylori infection and analyzed the immune responses. METHODS C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tip alpha + CpG, and recombinant del-Tip alpha (a mutant of Tip alpha) + CpG. Eight weeks after the mice were infected with H. pylori (5 x 10(7) CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tip alpha-specific IgG and IgA antibodies in mouse serum were measured by an enzyme-linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)-10, TNF-alpha and Interferon (IFN)-gamma in gastric mucosa was also measured by real time-PCR. RESULTS Levels of Tip alpha-specific antibodies were significantly higher in Tip alpha-immunized and del-Tip alpha-immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tip alpha-immunized mice (4.29 x 10(5) CFU/g) and del-Tip alpha immunized mice (2.5 x 10(5 )CFU/g) compared with infection control mice (5.7 x 10(6) CFU/g). The levels of IFN-gamma and IL-10 were significantly higher in del-Tip alpha-immunized mice than the infection control group. CONCLUSION Vaccinations with Tip alpha and del-Tip alpha were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell-mediated immunity.
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Affiliation(s)
- Kunimitsu Inoue
- Department of Microbiology, Faculty of Medicine, Oita University, Oita, Japan
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Lü L, Cao HD, Zeng HQ, Wang PL, Wang LJ, Liu SN, Xiang TX. Recombinant Mycobacterium smegmatis mc(2)155 vaccine expressing outer membrane protein 26 kDa antigen affords therapeutic protection against Helicobacter pylori infection. Vaccine 2008; 27:972-8. [PMID: 19111590 DOI: 10.1016/j.vaccine.2008.12.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2008] [Revised: 12/04/2008] [Accepted: 12/05/2008] [Indexed: 11/30/2022]
Abstract
Orally administered recombinant Mycobacterium smegmatis (rM. smegmatis) vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, in the present study, we evaluated the capacity of the outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori (H. pylori) to induce therapeutic protection against H. pylori infection in mice. Omp26 was cloned and expressed in M. smegmatis mc(2)155 as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of the up-regulated M. fortuitum beta-lactamase promoter, pBlaF. The rM. smegmatis strain was shown to be relatively stable in vitro in terms of plasmid stability and bacterial persistence. We found that oral immunization of H. pylori-infected mice with rM. smegmatis-Omp26 induced protection, i.e., significant reduction in bacterial colonization in the stomach. The protection was strongly related to serum specific antibodies with a Th(1) and Th(2) profile as well as to local cytokines in the stomach and spleen. These findings suggest that Omp26 is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori. The rM. smegmatis expressing Omp26 antigen could constitute an effective, low-cost combined vaccine against H. pylori.
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Affiliation(s)
- Lin Lü
- Department of Gastroenterology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China
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Li HX, Mao XH, Shi Y, Ma Y, Wu YN, Zhang WJ, Luo P, Yu S, Zhou WY, Guo Y, Wu C, Guo G, Zou QM. Screening and identification of a novel B-cell neutralizing epitope from Helicobacter pylori UreB. Vaccine 2008; 27:5013-9. [PMID: 18948159 DOI: 10.1016/j.vaccine.2009.05.009] [Citation(s) in RCA: 97] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Revised: 04/24/2009] [Accepted: 05/04/2009] [Indexed: 02/08/2023]
Abstract
Urease plays a crucial role in the survival and pathogenesis of Helicobacter pylori (H. pylori), and antibody neutralizing the urease activity may be implicated for the protection against H. pylori infection. Previously, a neutralizing monoclonal antibody (MAb) 6E6 against UreB of H. pylori was developed. In this work, we try to identify the B-cell epitope recognized by neutralizing MAb 6E6. Following screening a series of truncated proteins of UreB, an epitope was primarily localized in the aa 200-230 of UreB. Subsequently, we screened the overlapping synthetic peptides covering the aa 200-230 and identified a novel B-cell epitope (U(211-225), IEAGAIGFKIHEDWG) that was recognized by specific MAb 6E6. The newly identified epitope may help understanding of the protective immunity against H. pylori and be implicated for vaccine development.
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Affiliation(s)
- Hai-Xia Li
- Department of Clinical Microbiology and Clinical Immunology, The Third Military Medical University, Chongqing 400038, People's Republic of China
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Morihara F, Hifumi E, Yamada M, Nishizono A, Uda T. Therapeutic effects of molecularly designed antigen UREB138 for mice infected withHelicobacter pylori. Biotechnol Bioeng 2008; 100:634-43. [DOI: 10.1002/bit.21804] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Wu C, Shi Y, Guo H, Zou WY, Guo G, Xie QH, Mao XH, Tong WD, Zou QM. Protection against Helicobacter pylori infection in mongolian gerbil by intragastric or intramuscular administration of H. pylori multicomponent vaccine. Helicobacter 2008; 13:191-9. [PMID: 18466394 DOI: 10.1111/j.1523-5378.2008.00609.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Development of Helicobacter pylori vaccine would be a new effective strategy for prevention and treatment of H. pylori infection. Recombinant H. pylori vaccine comprising a single subunit antigen can only induce immune response with limited protection efficiency. In this study, the protective effect of H. pylori multicomponent vaccines consisting of three recombinant subunit antigens was investigated using the Mongolian gerbil model. MATERIALS AND METHODS Mongolian gerbils were immunized with different formulations of three recombinant H. pylori antigens (UreB, HspA, and HpaA) with two different adjuvants (Al(OH)3, LT(R72DITH)) by intragastric (i.g.) or intramuscular (i.m.) routes. The protective effects of multicomponent vaccines were assessed after H. pylori challenge in different studies. The specific IgG antibodies in serum were monitored by ELISA, and the mRNA expressions of IL-4 and IFN-gamma in spleen tissue were detected by reverse transcribed polymerase chain reaction (RT-PCR). RESULTS The protective effect against H. pylori challenge in gerbils immunized with three recombinant antigens and LT(R72DITH) or Al(OH)3 was significantly higher than that in single- or double-antigen vaccine-immunized and control gerbils. Furthermore, the protective effect of the triple-antigen vaccine combined with the LT(R72DITH) adjuvant (average 86.3%) was significantly greater than that of vaccine combined with the Al(OH)3 adjuvant (average 53.4%). After the first immunization, the anti-UreB/HspA/HpaA serum IgG level in gerbils immunized with triple-antigen vaccine combined with Al(OH)3 was higher than that in gerbils immunized with the vaccine combined with LT(R72DITH). Splenic interferon (IFN)-gamma and interleukin (IL)-4 transcript levels were significantly increased in LT(R72DITH) vaccine-immunized gerbils as compared to the Al(OH)3 vaccine group. Moreover, splenic IL-4 mRNA levels were higher than IFN-gamma in gerbils immunized with triple-antigen vaccine with either LT(R72DITH) or Al(OH)3. CONCLUSIONS This study indicated that the recombinant multicomponent vaccine provided effective protection against H. pylori infection as compared to the single-antigen vaccine. This protective immunity would be closely associated with a predominant Th2-type response.
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Affiliation(s)
- Chao Wu
- Department of Clinical Microbiology and Immunology, College of Medical Laboratory Science, Third Military Medical University, Chongqing, China
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16
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Hifumi E, Morihara F, Hatiuchi K, Okuda T, Nishizono A, Uda T. Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori. J Biol Chem 2007; 283:899-907. [PMID: 17991752 DOI: 10.1074/jbc.m705674200] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.
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Affiliation(s)
- Emi Hifumi
- Research Center for Applied Medical Engineering, Oita University, Dan-noharu 700, Oita-shi, Oita 870-1192, Japan
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17
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Taylor JM, Ziman ME, Canfield DR, Vajdy M, Solnick JV. Effects of a Th1- versus a Th2-biased immune response in protection against Helicobacter pylori challenge in mice. Microb Pathog 2007; 44:20-7. [PMID: 17683897 PMCID: PMC2234601 DOI: 10.1016/j.micpath.2007.06.006] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2007] [Accepted: 06/13/2007] [Indexed: 12/16/2022]
Abstract
The roles that T helper type 1 (Th1) and T helper type 2 (Th2) Helicobacter pylori-specific immune responses play in protection from H. pylori challenge are poorly understood. It is expected that Th2 immune responses are required for protection against extracellular bacteria, such as H. pylori. However, recent studies have suggested that Th1 immunity is required for protection. The mechanisms by which this might occur are unknown. Our goal in this study was to more clearly define the effects of a Th1- versus a Th2-promoting H. pylori vaccine on immunity and protection. Therefore, we tested a Th1 vaccine consisting of an H. pylori sonicate and CpG oligonucleotides (CpG) and a Th2 vaccine consisting of a lipopolysaccharide (LPS)-depleted H. pylori sonicate combined with cholera toxin (CT). We demonstrate that although the Th2-promoting vaccine induced stronger systemic and local immune responses, only the Th1-promoting vaccine was protective.
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Affiliation(s)
- Jennifer M. Taylor
- Center for Comparative Medicine, University of California, Davis CA 95616
| | - Melanie E. Ziman
- Center for Comparative Medicine, University of California, Davis CA 95616
| | - Don R. Canfield
- Center for Comparative Medicine, University of California, Davis CA 95616
- California National Primate Research Center, University of California, Davis CA 95616
| | - Michael Vajdy
- Departments of Internal Medicine and Medical Microbiology and Immunology, University of California, Davis CA 95616
| | - Jay V. Solnick
- Center for Comparative Medicine, University of California, Davis CA 95616
- Departments of Internal Medicine and Medical Microbiology and Immunology, University of California, Davis CA 95616
- *Corresponding author Jay V. Solnick, Center for Comparative Medicine, University of California, Davis, Davis, CA 95616, (530) 752-1333 (phone), (530) 752-7914 (fax)
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18
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Shi Y, Wu C, Zhou WY, Mao XH, Guo G, Zou QM. Identification of H-2d restricted Th epitopes in Urease B subunit of Helicobacter pylori. Vaccine 2007; 25:2583-90. [PMID: 17240487 DOI: 10.1016/j.vaccine.2006.12.024] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2006] [Revised: 12/03/2006] [Accepted: 12/12/2006] [Indexed: 12/28/2022]
Abstract
CD4+ T cells play important roles in protection against Helicobacter pylori (H. pylori) infection. In order to better understand the immune responses of H. pylori infection and improve immune interventions against this pathogen, we identified the Th epitopes in UreB of H. pylori, an excellent vaccine candidate antigen. By using the RANKPEP prediction algorithm, we have identified and characterized three Th epitopes within the UreB antigen, which can be recognized by CD4+ T cells from BALB/c (H-2d) mice. They were U(546-561), U(229-244), and U(237-251). These epitopes have important value for studying the immune response of H. pylori infection and for designing effective vaccine against H. pylori.
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Affiliation(s)
- Yun Shi
- Department of Clinical Microbiology and Immunology, College of Medical Laboratory Science, The Third Military Medical University, Chongqing 400038, People's Republic of China
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Robinson K, Argent RH, Atherton JC. The inflammatory and immune response to Helicobacter pylori infection. Best Pract Res Clin Gastroenterol 2007; 21:237-59. [PMID: 17382275 DOI: 10.1016/j.bpg.2007.01.001] [Citation(s) in RCA: 130] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Lifelong Helicobacter pylori infection and its associated gastric inflammation underlie peptic ulceration and gastric carcinogenesis. The immune and inflammatory responses to H. pylori are doubly responsible: gastric inflammation is the main mediator of pathology, and the immune and inflammatory response is ineffective, allowing lifelong bacterial persistence. However, despite inducing gastric inflammation, most infections do not cause disease, and bacterial, host and environmental factors determine individual disease risk. Although H. pylori avoids many innate immune receptors, specific virulence factors (including those encoded on the cag pathogenicity island) stimulate innate immunity to increase gastric inflammation and increase disease risk. An acquired T helper 1 response upregulates local immune effectors. The extent to which environmental factors (including parasite infection), host factors and H. pylori itself influence T-helper differentiation and regulatory T-cell responses remains controversial. Finally, effective vaccines have still not been developed: a better understanding of the immune response to H. pylori may help.
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Affiliation(s)
- Karen Robinson
- Wolfson Digestive Diseases Centre, University of Nottingham, C Floor, South Block, Queen's Medical Centre Campus, Nottingham University Hospital NHS Trust, Nottingham NG7 2UH, UK.
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20
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Otsu S, Gotoh K, Yamashiro T, Yamagata J, Shin K, Fujioka T, Nishizono A. Transfer of antigen-pulsed dendritic cells induces specific T-Cell proliferation and a therapeutic effect against long-term Helicobacter pylori infection in mice. Infect Immun 2006; 74:984-93. [PMID: 16428744 PMCID: PMC1360321 DOI: 10.1128/iai.74.2.984-993.2006] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Helicobacter pylori causes persistent infection of the stomach and results in chronic gastritis and peptic ulcers. Jaws II cells, derived from mouse bone marrow, were pulsed with live or formalin-killed or whole-cell sonicates (WCS) of H. pylori. Representative cell surface molecules were expressed at substantial levels on Jaws II cells, indicating that appropriate maturation of the cells was achieved with the three H. pylori antigens without any significant differences. H. pylori WCS-pulsed Jaws II cells secreted a significant amount of tumor necrosis factor alpha into the culture supernatant. The naïve T cells exposed to the WCS-pulsed Jaws II cells showed significant proliferation and gamma interferon (IFN-gamma) and interleukin-10 (IL-10) production in vitro. A 2-log reduction in the number of colonizing bacteria was observed in the mice treated with the WCS-pulsed Jaws II cells; however, no significant reductions were achieved in mice treated with Jaws II cells pulsed with other H. pylori antigens. Up-regulated production of IFN-gamma and IL-10 was observed in the stomachs of the mice treated with the WCS-pulsed Jaws II cells, which is consistent with the result obtained in vitro. There were no differences in gastritis scores or H. pylori-specific antibody titers among the mice treated with Jaws II cells pulsed with the three different H. pylori antigens. The results suggest that Th1 cell-mediated immunity in combination with Th2 cell-mediated immunity plays a role in reducing colonizing bacterial numbers in mice with chronic H. pylori infections.
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Affiliation(s)
- Satoshi Otsu
- Department of Infectious Diseases (Microbiology), Faculty of Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Oita 879-5593, Japan
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21
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Hikichi T, Kobayashi H, Oyama H, Yamamoto G, Watanabe H, Irisawa A, Obara K, Sato Y. Effectiveness of intragastric immunization with protein and oligodeoxynucleotides containing a CpG motif for inducing a gastrointestinal mucosal immune response in mice. Fukushima J Med Sci 2005; 51:19-31. [PMID: 16167670 DOI: 10.5387/fms.51.19] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
PURPOSE To investigate a new modality of mucosal vaccines, we evaluated the effectiveness of intragastric immunization for inducing a mucosal immune response in the gastrointestinal tract. METHODS Mice were immunized with beta-galactosidase (beta-gal) and synthesized oligodeoxynucleotides containing a CpG motif (CpG-DNA) by intragastric injection, and the immune response was compared with those induced by 3 other immunization forms: intranasal, oral, and intradermal. RESULTS Intragastric immunization with beta-gal and CpG-DNA induced significant anti-beta-gal fecal IgA production at 2 weeks; however, at 4 weeks the response was lacking. In contrast, intranasal immunization with beta-gal and CpG-DNA induced the highest anti-beta-gal fecal IgA production at 4 weeks. CONCLUSION Although intragastric immunization with protein and CpG-DNA induces a mucosal immune response in the gastrointestinal tract, intranasal immunization is the most effective to induce both mucosal and systemic immune responses. This finding may increase the possibility for developing vaccines against mucosal pathogens, especially Helicobacter pylori.
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Affiliation(s)
- Takuto Hikichi
- Department of Internal Medicine II, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan.
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22
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Affiliation(s)
- Sean P Harbison
- Temple University School of Medicine, Philadelphia, Pennsylvania, USA
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23
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Czinn SJ, Nedrud JG. Peptic Ulcers and Gastritis. Mucosal Immunol 2005. [DOI: 10.1016/b978-012491543-5/50073-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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24
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Watanabe K, Murakami K, Sato R, Okimoto T, Maeda K, Nasu M, Nishizono A, Fujioka T. CTLA-4 blockade inhibits induction of Helicobacter pylori-associated gastritis in mice. Clin Exp Immunol 2004; 135:29-34. [PMID: 14678261 PMCID: PMC1808916 DOI: 10.1111/j.1365-2249.2004.02338.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The balance between Th1 and Th2 response determines the outcome of Helicobacter pylori infection. Interferon (IFN)-gamma plays an inductive role in gastric inflammation, whereas interleukin (IL)-4 counterbalances Th1 response and suppresses the development of gastritis. Th cell response is regulated by co-stimulatory factors. A co-stimulatory molecule, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), plays an inhibitory role in IL-2-dependent cell growth and mediates an optimal inhibitory signal to Th1 and Th2 cells. We administered anti-CTLA-4 monoclonal antibody (MoAb), which blocks CTLA-4 signalling, to examine the relative role for this signalling during maturation of Th1 and Th2 cells in H. pylori infection in mice. Mice treated by anti-CTLA-4 MoAb within the first week of infection showed an inhibition of gastric inflammation, accompanied by an increasing ratio of H. pylori-specific IgG1/IgG2a in serum following infection. Furthermore, the treatment resulted in the higher ratio of IL-4/IFN-gamma by splenocytes in response to H. pylori antigen at 6 weeks after infection, compared with untreated mice. These results suggest that the predominance of Th2 response by CTLA-4 blockade leads to an inhibition of the development of gastric inflammation. CTLA-4 signalling could contribute to the regulation of Th subsets and the development of gastric inflammation in H. pylori infection.
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Affiliation(s)
- K Watanabe
- Department of Infectious Diseases and General Medicine, Oita University, Oita, Japan.
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25
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Jiang Z, Pu D, Huang AL, Tao XH, Wang PL. Cloning, expression and antigenic analysis of heat shock protein A gene of human Helicobacter pylori. Shijie Huaren Xiaohua Zazhi 2003; 11:1480-1484. [DOI: 10.11569/wcjd.v11.i10.1480] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To construct a recombinant vector containing gene encoding heat shock protein A with a Mr of 13 000 from human Helicobacter pylori (H pylori) and express it in E. coli BL21, and to explore the antigenicity.
METHODS The target gene was amplified from H pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) digested by restrictive endonuclease enzymes of kpn I, BamH I simultaneously. The recombinant vector was transformed and expressed in E.coli BL21.The antigenicity of recombinant fusion protein was analysed by Western blot.
RESULTS Enzyme digestion and sequencing analysis showed that the target gene has been inserted into the recombinant vector, but as compared with the gene reported by GenBank, 1.6% of gene mutation and 1.6% of amino acid residues change in H pylori occurred, respectively. SDS-PAGE analysis showed that the recombinant vector could be expressed in E.coli BL21, the relative molecular mass (Mr) of expressed product was 33×103, while Mr of protein expressed by pET32a (+) was about 20×103, and soluble fusion expression product accounted for 18.96% of total bacterial protein. After purification with Ni+-NTA agarose resin, the purity of recombinant fusion protein was about 95%. Western blot result showed that recombinant fusion protein could be recognized by anti-H pylori positive serum, suggesting that the protein had good antigenicity.
CONCLUSION The gene encoding H pylori heat shock protein A has been cloned and expressed successfully. The results lay the foundation for development of H pylori protein vaccine and a quick diagnostic kit for detection of H pylori infection.
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Affiliation(s)
- Zheng Jiang
- Department of Gastroenterology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Dan Pu
- Department of Gastroenterology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Ai-Long Huang
- Institute of Viral Hepatitis, Chongqing Medical University, Chongqing 400010, China
| | - Xiao-Hong Tao
- Department of Gastroenterology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Pi-Long Wang
- Department of Gastroenterology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
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26
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Yan J, Liang SH, Mao YF, Li LW, Li SP. Construction of expression systems for flaA and flaB genes of Helicobacter pylori and determination of immunoreactivity and antigenicity of recombinant proteins. World J Gastroenterol 2003; 9:2240-50. [PMID: 14562386 PMCID: PMC4656471 DOI: 10.3748/wjg.v9.i10.2240] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To clone flagellin genes A (flaA) and B (flaB) from a clinical strain of Helicobacter pylori (H pylori) and to construct prokaryotic expression systems of the genes and identify immunity of the fusion proteins.
METHODS: The flaA and flaB genes from a clinical H pylori isolate Y06 were amplified by high fidelity PCR. The nucleotide sequences of target DNA amplification fragments from the two genes were sequenced after T-A cloning. The recombinant expression vector pET32a inserted with flaA and flaB genes was constructed, respectively. The expressions of FlaA and FlaB fusion proteins in E. coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations were examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and immunodiffusion assay using self-prepared rabbit antiserum against FlaA (rFlaA) or FlaB (rFlaB) recombinant proteins were applied to the determination of the fusion proteins immunity. ELISA was used to detect the antibodies against rFlaA and rFlaB in sera of 125 H pylori infected patients and to examine rFlaA and rFlaB expression in 98 clinical isolates of H pylori, respectively.
RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homologies of the cloned flaA and flaB genes were from 96.28%-97.13% and 96.31%-97.73%, and their putative amino acid sequence homologies were 99.61%-99.80% and 99.41%-100% for the two genes, respectively. The output of rFlaA and rFlaB expressed by pET32a-flaA-BL21DE3 and pET32a-flaB-BL21DE3 systems was as high as 40%-50% of the total bacterial proteins. Both rFlaA and rFlaB were able to combine with the commercial antibodies against whole cell of H pylori and to induce rabbits to produce specific antibodies with the same 1:2 immunodiffusion titers after the animals were immunized with the two recombinant proteins. Ninety-eight and zero point 4 and 92.80% of the serum samples from 125 patients infected with H pylori were positive for rFlaA and rFlaB antibodies, respectively. One hundred percent and 98.98% of the 98 tested isolates of H pylori were detectable for rFlaA and rFlaB epitopes, respectively.
CONCLUSION: Two prokaryotic expression systems with high efficiency of H pylori flaA and flaB genes were successfully established. The expressed rFlaA and rFlaB showed satisfactory immunoreactivity and antigenicity. High frequencies of FlaA and FlaB expression in different H pylori clinical strains and the general existence of specific antibodies against FlaA and FlaB in H pylori infected patients strongly indicate that FlaA and FlaB are excellent antigen candidates for developing H pylori vaccine.
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Affiliation(s)
- Jie Yan
- Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, 353 Yan an Road, Hangzhou 310031, Zhejiang Province, China.
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27
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Mao YF, Yan J, Li LW, Li SP. Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein. World J Gastroenterol 2003; 9:1529-36. [PMID: 12854157 PMCID: PMC4615498 DOI: 10.3748/wjg.v9.i7.1529] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein.
METHODS: The hpaA gene from a clinical isolate Y06 of H. pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.coli BL21(DE3) induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H. pylori as well as immunodiffusion assay with self-prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H. pylori and to examine HpaA expression of 109 clinical isolates of H. pylori.
RESULTS: In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25%-97.32% and 95.38%-98.46%, respectively. The output of HpaA fusion protein in its expression system of pET32a-hpaA-BL21(DE3) was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H. pylori and to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein. 81.6% of the serum samples from 125 patients infected with H. pylori (102/125) were positive for HpaA antibody and all of the tested isolates of H. pylori (109/109) were detectable for HpaA.
CONCLUSION: A prokaryotic expression system with high efficiency of H. pylorihpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H. pylori clinical strains and specific antibody production in H. pylori infected patients indicate that HpaA is an excellent and ideal antigen for developing H. pylori vaccine.
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Affiliation(s)
- Ya-Fei Mao
- Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
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Bai Y, Huang W, Lin HJ, Wang JD, Chen Y, Zhang ZS, Zhou DY, Zhang YL. Construction of clone expressing adhesin Hsp60 of Helicobacter pylori. Shijie Huaren Xiaohua Zazhi 2003; 11:547-550. [DOI: 10.11569/wcjd.v11.i5.547] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To construct a recombinant vector containing gene encoding Hsp60 gene of Helicobacter pylori and to express the vector in E.coli BL21.
METHODS The Hsp60 gene was amplified from H.pylori chromosome by PCR and inserted into the prokaryotie expression vector pET-22b (+). The recombinant vector was transformed and expressed in E.coli BL21 (DE3). Recombinant Hsp60 protein immunogenicity was studied by Western blot.
RESULTS The 1.6 kb Hsp60 gene was successfully isolated. Recombinant E.coli strains expressed Hsp60 were obtained, the expression protein amounted to 27.2% of the total bacterial protein after induced with IPTG for 3 h at 37 ℃, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 76.6% of the insoluble protein. Western blot analysis of rHsp60 confirmed that it could be specially recognized by serum from Hp infected patients.
CONCLUSION The gene coding for Hp Hsp60 is cloned and expressed successfully. The results obtained lay the foundation for constructing the H.pylori vaccine.
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Affiliation(s)
- Yang Bai
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
| | - Wen Huang
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
| | - Huan-Jian Lin
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
| | - Ji-De Wang
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
| | - Ye Chen
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
| | - Zhao-San Zhang
- Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China
| | - Dian-Yuan Zhou
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
| | - Ya-Li Zhang
- PLA Institute for Digestive Medicine, the First Military Medical Univercity, Guangzhou 510515, Guangdong Province, China
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Xia HHX, Talley NJ, Blum AL, O'Morain CA, Stolte M, Bolling-Sternevald E, Mitchell HM. Clinical and pathological implications of IgG antibody responses to Helicobacter pylori and its virulence factors in non-ulcer dyspepsia. Aliment Pharmacol Ther 2003; 17:935-943. [PMID: 12656696 DOI: 10.1046/j.1365-2036.2003.01525.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
AIM To determine whether pre-treatment antibody response to Helicobacter pylori virulence factors predicts eradication success and symptom relief 12 months after triple therapy in non-ulcer dyspepsia. METHODS H. pylori-positive patients with non-ulcer dyspepsia received 1-week omeprazole-based triple therapy, or omeprazole plus placebos. Symptoms were assessed using a validated Likert scale. Gastric biopsies taken before and 12 months after treatment were used for histological examination. Pre-treatment blood samples were used for the detection of anti-H. pylori immunoglobulin G (IgG) antibodies, and specific IgG antibodies to 19.5-, 26.5-, 30-, 35-, 89- (VacA) and 116-kDa (CagA) antigens of H. pylori. RESULTS IgG antibodies to the six antigens were detected in 62%, 96%, 88%, 47%, 54% and 78% of patients, respectively. The presence of antibody to 19.5-, 26.5- or 30-kDa antigen was associated with an increased anti-H. pylori IgG absorbance index. IgG absorbance indices were greater in those with H. pylori eradication (vs. persistent infection). The prevalence of antibodies to the six antigens was not significantly different between those with symptom relief vs. those without. The 19.5-kDa antigen (P = 0.018) and VacA (P = 0.001) were independent risk factors for body gastritis. CONCLUSIONS An increased pre-treatment anti-H. pylori IgG absorbance index may be a useful predictor of the success of eradication therapy. Although the 19.5-kDa antigen and VacA were associated with body gastritis, none of the six antigens tested predicted symptom relief after triple therapy.
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Affiliation(s)
- H H-X Xia
- Department of Medicine, University of Sydney, Nepean Hospital, Penrith, NSW, Australia
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Watanabe K, Murakami K, Maeda K, Fujioka T, Nasu M, Nishizono A. Intraperitoneal immunization led to T cell hyporesponsiveness to Helicobacter pylori infection in mice. Microbiol Immunol 2003; 46:441-7. [PMID: 12222930 DOI: 10.1111/j.1348-0421.2002.tb02718.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori-challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori-challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized-challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL-2, IFN-gamma, and IL-4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL-2 and IFN-gamma. CTLA-4 has been documented to be a negative regulator of IL-2 production and lymphoproliferation that induces peripheral tolerance and functions 24-72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized-challenged mice showed higher levels of CTLA-4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori-associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA-4 following challenge.
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Garhart CA, Heinzel FP, Czinn SJ, Nedrud JG. Vaccine-induced reduction of Helicobacter pylori colonization in mice is interleukin-12 dependent but gamma interferon and inducible nitric oxide synthase independent. Infect Immun 2003; 71:910-21. [PMID: 12540573 PMCID: PMC145373 DOI: 10.1128/iai.71.2.910-921.2003] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Previous studies with mice have shown that major histocompatibility complex class II (MHC-II) is required for protection from Helicobacter pylori, while MHC-I and antibodies are not. Thus, CD4(+) T cells are presumed to play an essential role in protective immunity via secretion of cytokines. To determine which cytokines are associated with a reduction of bacterial load in immunized mice, gastric cytokine expression was examined by semiquantitative reverse transcription-PCR in protected (defined as > or =2-log-unit decrease in bacterial load) and unprotected mice 4 weeks after challenge. Elevated levels of mRNA for interleukin-12p40 (IL-12p40), gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) were associated with protection in immunized-challenged (I/C) mice, but Th2 cytokine (IL-4, IL-5, IL-10, and IL-13) and chemokine (KC, MIP-2, and MCP-1) expression was not associated with protection. Despite the association of IFN-gamma and iNOS message with protection, I/C mice genetically lacking either of these products were able to reduce the bacterial load as well as the wild-type I/C controls. The I/C mice lacking IL-12p40 were not protected compared to unimmunized-challenged mice. All I/C groups developed gastritis. We conclude that neither IFN-gamma nor iNOS is essential for vaccine-induced protection from H. pylori infection. The p40 subunit of IL-12, which is a component of both IL-12 and IL-23, is necessary for protection in immunized mice. These findings suggest a novel IFN-gamma-independent function of IL-12p40 in effective mucosal immunization against H. pylori.
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Affiliation(s)
- Christine A Garhart
- Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
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Maeda K, Yamashiro T, Minoura T, Fujioka T, Nasu M, Nishizono A. Evaluation of therapeutic efficacy of adjuvant Helicobacter pylori whole cell sonicate in mice with chronic H. pylori infection. Microbiol Immunol 2003; 46:613-20. [PMID: 12437028 DOI: 10.1111/j.1348-0421.2002.tb02742.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Successful prophylactic administration of Helicobacter pylori whole cell sonicate (WCS) plus complete Freund's adjuvant (CFA) or aluminum hydroxide (ALM) against subsequent H. pylori infection was reported recently. Here we tested the effect of WCS plus TiterMax Gold (TMX) or ALM in mice with chronic H. pylori infection. Mice with chronic (18 weeks) H. pylori infection were injected intraperitoneally with H. pylori (Sydney strain) WCS plus ALM or TMX once weekly for three times. The number of colonizing H. pylori in the stomach, IgG1 and IgG2a levels, and local inflammatory status were determined after therapeutic immunization. H. pylori specific IgG1, but not IgG2a, was significantly induced in mice immunized with H. pylori WCS plus TMX or ALM. Immunization did not result in reduction of bacterial count or recruiting inflammatory cells to the stomach. Adjuvant H. pylori WCS resulted in induction of CD4+ Th2 cell-mediated immunity although it did not reduce bacterial density in mice with chronic H. pylori infection. Our results implied that CD4+ Th1 cell-mediated immunity, rather than Th2 cell dominant immunity, might play a role in reducing the number of bacteria in chronic H. pylori infection.
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Affiliation(s)
- Kosaku Maeda
- Department of Infectious Diseases, Oita Medical University, Japan
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Abstract
OBJECTIVES Effective eradication of Helicobacter pylori (H. pylori) infection has often proved more difficult than expected. Antimicrobial resistance incompletely explains eradication failure. This study tests the hypothesis that an impaired immune response may contribute to failed eradication after standard antibiotic therapy. METHODS Parameters of host immunity were assessed as blood T lymphocyte production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) being surrogate markers of mucosal Th1 and Th2 responses, respectively. The validity of using circulating T cell cytokines as surrogate markers of mucosal immunity was established (unstimulated lymphocyte IL-4 level correlation r2 = 0.549, p < 0.001; antigen-stimulated lymphocyte correlation r2 = 0.62, p < 0.001). RESULTS A total of 52 dyspeptic patients and 11 patients with previous H. pylori eradication failure were recruited into the study. There was no significant difference in secretion of IFN-gamma from peripheral blood T cells, in either unstimulated or antigen-stimulated cultures, between clinical groups. There was, however, a significant reduction in secretion of IL-4 from blood T cells in subjects failing to eradicate H. pylori compared with those who successfully eradicated the infection in both unstimulated and stimulated cultures. A significant difference in IL-4 secretion was also detected in antigen-stimulated cultures compared with that in H. pylori-positive subjects (p < 0.05). Low levels of IL-4 secretion were detected irrespective of the number of courses of antibiotic therapy. Lower levels of IgG anti-H. pylori antibody were detected in both serum and saliva of subjects with persistent H. pylori infection after use of antibiotics compared with untreated H. pylori-positive subjects (difference not statistically significant). CONCLUSIONS These results support the hypothesis that impaired mucosal immunity, particularly involving the secretion of IL-4, may contribute to H. pylori eradication failure. Measurement of whole blood secretion of IL-4 may predict which patients are more likely to fail standard antibiotic therapy.
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Affiliation(s)
- Tom Borody
- Centre for Digestive Diseases, Sydney, New South Wales, Australia
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Raghavan S, Hjulström M, Holmgren J, Svennerholm AM. Protection against experimental Helicobacter pylori infection after immunization with inactivated H. pylori whole-cell vaccines. Infect Immun 2002; 70:6383-8. [PMID: 12379718 PMCID: PMC130438 DOI: 10.1128/iai.70.11.6383-6388.2002] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The protective effect of therapeutic oral immunization with homologous and heterologous formalin-inactivated Helicobacter pylori cells given together with cholera toxin as an adjuvant was evaluated with C57BL/6 mice infected with H. pylori Sydney strain 1 (SS1). The bacteria used for immunization were strains that were either homologous or heterologous with regard to the O antigen (i.e., the Lewis antigen [Le antigen]) expressed by the lipopolysaccharide of the infecting H. pylori SS1 strain. We found that repeated oral immunization with inactivated H. pylori SS1 cells can significantly inhibit an existing infection (P < 0.001) and that the protection induced by such therapeutic immunization extends to protection against reinfection (P < 0.001). A similar level of protection was also achieved by immunization with another inactivated H. pylori strain having the same O antigen (Le antigen) as the infecting H. pylori SS1 strain. In contrast, immunization with inactivated strains expressing a heterologous O antigen, Le(x), provided less protection or no protection. Immunization with H. pylori lysate preparations, on the other hand, resulted in significant comparable protection whether the lysates were prepared from an Le(x) strain or an Le(y) strain. Postimmunization gastritis was seen in mice that were protected after vaccination but not in unimmunized or unprotected mice. In conclusion, therapeutic immunization with inactivated H. pylori whole-cell vaccines may provide strong protection both against experimental H. pylori infection and against later reinfection.
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Affiliation(s)
- S Raghavan
- Department of Medical Microbiology and Immunology and Göteborg University Vaccine Research Institute (GUVAX), Göteborg University, S 41346 Göteborg, Sweden
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Raghavan S, Svennerholm AM, Holmgren J. Effects of oral vaccination and immunomodulation by cholera toxin on experimental Helicobacter pylori infection, reinfection, and gastritis. Infect Immun 2002; 70:4621-7. [PMID: 12117975 PMCID: PMC128197 DOI: 10.1128/iai.70.8.4621-4627.2002] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Therapeutic vaccination is an attractive strategy to control infection and disease caused by Helicobacter pylori. In mice infected with H. pylori we have studied the protective effect of oral immunization with an H. pylori lysate preparation given together with the mucosal adjuvant cholera toxin (CT), both against the initial infection and against a later reinfection challenge. We have also examined the effects of treatment with the CT adjuvant alone on H. pylori infection and reinfection. Specific immunization with lysate was found to result in a sixfold reduction of the extent (bacterial load) of the primary infection and also to provide similar levels of protection against reinfection. However, these effects were associated with severe postimmunization gastritis. In contrast, oral treatment with CT alone at the time of initial infection, while unable to suppress the initial infection, gave rise to a 20-fold reduction in bacterial load upon reinfection without causing any associated gastric inflammation. Both the infected animals that were specifically immunized and those that were treated with CT only displayed increased in vitro proliferative responses of mononuclear cells to H. pylori antigens. Antibody levels in response to H. pylori were on the other hand only marginally increased after treatment with CT, whereas they were markedly elevated after immunization with lysate plus CT, with a rise in both (Th2-driven) immunoglobulin G1 (IgG1) and, especially, (Th1-driven) IgG2a antibodies. The results illustrate the complex balance between protection and harmful inflammation after postinfection vaccination against H. pylori as studied in a mouse model.
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Affiliation(s)
- S Raghavan
- Department of Medical Microbiology and Immunology and Göteborg University Vaccine Research Institute, Göteborg University, Sweden
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Abstract
AIM: To construct a recombinant vector which can express Mr26000 outer membrane protein (OMP) from Helicobacter pylori (H. pylori), and to obtain the vaccine protecting against H. pylori infection and a diagnostic reagent kit quickly detecting H. pylori infection.
METHODS: The gene encoding the structural Mr26000 outer membrane protein of H. pylori was amplified from H. pylori chromosomal DNA by PCR, and inserted in the prokaryotic expression vector pET32a(+), which was transformed into the Top10 E. coli strain. Recombinant vector was selected, identified and transformed into BL-21(DE3) E. coli strain. The recombinant fusion proteins were expressed. The antigenicity of recombinant protein was studied by ELISA or immunoblotting and immunized Balb/c mice.
RESULTS: The gene of Mr26000 OMP was amplified to be 594 base pairs, 1.1% of the cloned genes was mutated and 1.51% of amino acid residues was changed, but there was homogeneity between them. The recombinant fusion protein encoded objective polypeptides of 198 amino acid residues, corresponding to calculated molecular masses of Mr26000. The level of soluble expression products was about 38.96% of the total cell protein. After purification by Ni-NTA agarose resin columniation, the purity of objective protein became about 90%. The ELISA results showed that recombinant fusion protein could be recognized by patient serum infected with H. pylori and rabbit serum immunized with the recombinant protein. Furthermore, Balb/c mice immunized with the recombinant protein were protected against H. pylori infection.
CONCLUSION: Mr26000 OMP may be a candidate vaccine preventing H. pylori infection.
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Affiliation(s)
- Zheng Jiang
- Department of Gastroenterology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China.
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Koesling J, Lucas B, Develioglou L, Aebischer T, Meyer TF. Vaccination of mice with live recombinant Salmonella typhimurium aroA against H. pylori: parameters associated with prophylactic and therapeutic vaccine efficacy. Vaccine 2001; 20:413-20. [PMID: 11672904 DOI: 10.1016/s0264-410x(01)00355-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Previously we described a recombinant attenuated Salmonella typhimurium aroA strain (SL3261[pYZ97]) with constitutive expression of plasmid encoded Helicobacter pylori urease subunits A and B (UreAB). Single dose oral vaccination effectively induced prophylactic immunity against bacterial challenge in BALB/c mice. Here we successfully extended this approach to several mouse strains with allelic differences in NRAMP-1 and H-2 genes. The respective host determinants are known to influence the immune response against S. typhimurium. A comparative analysis of the vaccine efficacy in C57BL/6 and BALB/c mice showed that the live vaccine confers long lasting immunity in both strains (>18 weeks). In C57BL/6 mice, protection was still observed 54 weeks while not all vaccinated BALB/c were immune when challenged after this time. BALB/c mice also needed higher doses of SL3261[pYZ97] for full protection. We also demonstrate a therapeutic potential of SL3261[pYZ97] in H. pylori infected BALB/c and C57BL/6 mice. Urease- and carrier-specific serum antibody responses as well as the level of colonization by the Salmonella were analyzed in both mouse strains after immunization with low (4 x 10(7)CFU) or high (1 x 10(9)CFU) vaccine doses. The results are discussed in the context of inoculum size and the mode of antigen supply required for effective vaccination with recombinant Salmonella.
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Affiliation(s)
- J Koesling
- Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstr. 21/22, D-10117 Berlin, Germany
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Kim JS, Chang JH, Chung SI, Yum JS. Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy. FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 2001; 31:41-6. [PMID: 11476980 DOI: 10.1111/j.1574-695x.2001.tb01584.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.
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MESH Headings
- Adjuvants, Immunologic
- Administration, Oral
- Animals
- Antibodies, Bacterial/analysis
- Antigens, Bacterial/therapeutic use
- Bacterial Vaccines/therapeutic use
- Colony Count, Microbial
- Disease Models, Animal
- Female
- Gastric Mucosa/microbiology
- Gastrointestinal Diseases/drug therapy
- Gastrointestinal Diseases/genetics
- Gastrointestinal Diseases/microbiology
- Helicobacter Infections/drug therapy
- Helicobacter Infections/genetics
- Helicobacter Infections/microbiology
- Helicobacter pylori/immunology
- Helicobacter pylori/isolation & purification
- Immunoglobulin A, Secretory/analysis
- Immunoglobulin G/analysis
- Immunotherapy, Active
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C57BL
- Species Specificity
- Specific Pathogen-Free Organisms
- Time Factors
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Affiliation(s)
- J S Kim
- H. pylori team, Mogam Biotechnology Research Institute, 341 Pojung-ri, Koosung-myon, Yongin-city, 449-910, Kyonggi-do, South Korea.
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Kotloff KL, Sztein MB, Wasserman SS, Losonsky GA, DiLorenzo SC, Walker RI. Safety and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine with adjuvant among volunteers with or without subclinical infection. Infect Immun 2001; 69:3581-90. [PMID: 11349017 PMCID: PMC98341 DOI: 10.1128/iai.69.6.3581-3590.2001] [Citation(s) in RCA: 145] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Helicobacter pylori infection of the gastric mucosa can be found in approximately 50% of the world's population and is associated with a range of pathology, including peptic ulcer, atrophic gastritis, and gastric cancer. To explore immunization as a strategy for preventing and treating H. pylori-associated disease, we assessed the safety and immunogenicity in healthy adults of a formalin-inactivated, oral H. pylori whole-cell (HWC) vaccine, administered with or without mutant Escherichia coli heat-labile toxin (LT(R192G)) as a mucosal adjuvant. In a dose-response study, 23 subjects with or without H. pylori infection were vaccinated with either 2.5 x 10(6) HWC, 2.5 x 10(8) HWC, or 2.5 x 10(10) HWC, plus 25 microg of LT(R192G). Thereafter, a randomized study was conducted in which 18 H. pylori-infected subjects were assigned, in a double-blind fashion, to receive either 2.5 x 10(10) HWC plus placebo-adjuvant, placebo-vaccine plus 25 microg of LT(R192G), placebo-vaccine plus placebo-adjuvant, or 2.5 x 10(10) HWC plus 25 microg of LT(R192G). Diarrhea (six subjects), low-grade fever (five subjects), and vomiting (two subjects) were observed, usually after the first dose. Significant rises in geometric mean mucosal (fecal and salivary) anti-HWC immunoglobulin A antibodies occurred among H. pylori-infected and uninfected subjects following inoculation with 2.5 x 10(10) HWC plus 25 microg of LT(R192G). Moreover, among H. pylori-negative volunteers, this regimen induced significant lymphoproliferative responses in 5 of 10 subjects and gamma interferon production responses to H. pylori sonicate in 7 of 10 subjects. There was no evidence that vaccination eradicated H. pylori in infected volunteers. These results suggest that it is possible to stimulate mucosal and systemic immune responses in humans to H. pylori antigens by using an HWC vaccine.
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Affiliation(s)
- K L Kotloff
- Division of Infectious Disease and Tropical Pediatrics, Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201, USA.
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40
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Affiliation(s)
- J G Nedrud
- Institute of Pathology, Case Western Reserve University, Cleveland, OH, USA.
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41
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Lucas B, Bumann D, Walduck A, Koesling J, Develioglu L, Meyer TF, Aebischer T. Adoptive transfer of CD4+ T cells specific for subunit A of Helicobacter pylori urease reduces H. pylori stomach colonization in mice in the absence of interleukin-4 (IL-4)/IL-13 receptor signaling. Infect Immun 2001; 69:1714-21. [PMID: 11179348 PMCID: PMC98077 DOI: 10.1128/iai.69.3.1714-1721.2001] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Protection in the murine model of Helicobacter pylori infection may be mediated by CD4+ T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterized H. pylori urease-specific CD4+ T cells from BALB/c mice immunized with Salmonella enterica serovar Typhimurium expressing H. pylori urease (subunits A and B). The CD4+ T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-gamma), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-gamma and IL-10. Adoptive transfer of the UreA-specific CD4+ T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+ T cells into IL-4 receptor alpha chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis of H. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.
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Affiliation(s)
- B Lucas
- Max-Planck-Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, 10117 Berlin, Germany
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Abstract
Helicobacter pylori causes several gastroduodenal diseases. Various antibiotic regimens are available that eradicate H. pylori in 80 to 90% of patients, but no regimen cures all patients. Dual therapy is now obsolete. Triple therapy with two antibiotics and either a proton pump inhibitor or bismuth is the regimens of choice. Metronidazole and clarithromycin are the two key antibiotics. Antibiotic resistance against these two drugs is becoming more problematic and should be taken into consideration when choosing a regimen. Antibiotic resistance is usually induced after failure. Quadruple therapy has been used as a salvage regimen in failed cases but it is also the most complicated regimen. Several new agents are being studied including a single capsule that contains bismuth, metronidazole, and tetracycline.
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Affiliation(s)
- J S Hoffman
- Division of Gastroenterology, St. Elizabeth's Medical Center, Boston, Massachusetts 02135, USA
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Aebischer T, Laforsch S, Hurwitz R, Brombacher F, Meyer TF. Immunity against Helicobacter pylori: significance of interleukin-4 receptor alpha chain status and gender of infected mice. Infect Immun 2001; 69:556-8. [PMID: 11119552 PMCID: PMC97918 DOI: 10.1128/iai.69.1.556-558.2001] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Vaccination of interleukin-4 (IL-4) receptor alpha (IL-4Ralpha) chain-deficient BALB/c mice with Helicobacter pylori urease and cholera toxin or with urease-expressing, live attenuated Salmonella enterica serovar Typhimurium cells revealed that protection against H. pylori infection is independent of IL-4- or IL-13-mediated signals. A comparison of male and female mice suggests a sexual dimorphism in the extent of bacterial colonization that is particularly evident in the absence of the IL-4Ralpha chain.
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Affiliation(s)
- T Aebischer
- Department of Molecular Biology, Max Planck Institute for Infection Biology, 10117 Berlin, Germany.
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