Helicobacter pylori virulence factors and the host immune response: implications for therapeutic vaccination

Helicobacter pylori colonizes the human gastric mucosa and is associated with specific gastric disease. Virulence factors, such as urease, the vacuolating toxin (VacA), the cytotoxin-associated antigen CagA or blood-group-antigen-binding adhesin (BabA), an adherence factor, might account for the development of different diseases. Vaccination trials exploiting the antigenic properties of some of these proteins have not been successful in preventing infection in humans. A more in-depth understanding of the immune response to H. pylori infection as well as additional information on suitable epitopes and adjuvants will be required before a successful vaccine can be developed.     

The quest for a vaccine against Helicobacter pylori: how to move from mouse to man?

Several lines of evidence from experimental animal models of infection have clearly demonstrated the feasibility of a prophylactic and therapeutic vaccine against Helicobacter pylori. However, comparatively few clinical studies have been carried out to evaluate whether the positive results obtained in animals can be reproduced in humans. The preliminary results obtained with single component, mucosally delivered vaccines have shown very limited results thus far. Very good immunogenicity and safety profiles are now being obtained with parenterally delivered, aluminium hydroxide-adjuvanted multicomponent candidate vaccines. For sure, better vaccine formulations, better antigen preparation(s), better adjuvants, and better delivery systems have to be designed and tested for safety and immunogenicity. These studies are also needed for deciphering those aspects of the effector immune responses that correlate with protection against H. pylori infection and disease.     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .     

Therapeutic effects of molecularly designed antigen UREB138 for mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori) is a bacteria that is well known as the principal cause of chronic gastritis and peptic ulcer disease in humans. Because no effective vaccine has yet been established, we designed a new biomolecule as a vaccination antigen capable of preventing the infection of H. pylori. The designed biomolecule involves a 138 stretch (aa 201-aa 338 of beta-subunit of H. pylori urease), which is the functionally important region for urease activity. The region was expressed as a recombinant protein, called UREB138. The therapeutic vaccination was performed using UREB138 in mice persistently infected with H. pylori. The subcutaneous administration of UREB138 remarkably reduced the number of bacteria in the mice stomach compared with the control. Immunization with UREB138 enhanced the urease-specific IgA and IgG1 in the serum. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in UREB138-immunized mice than in control mice. Furthermore, the expression of interferon-gamma mRNA in the gastric tissues with eradicated bacteria was higher than in the non-eradicated group. The combination of Th1- and Th2-mediated immunity plays a role in reducing the colonization of bacterial numbers of H. pylori.     

Phagocytosis and persistence of Helicobacter pylori

Helicobacter pylori is a spiral-shaped, flagellated, microaerophilic Gram-negative bacterium that colonizes the gastric epithelium of humans. All persons infected with H. pylori have gastritis, and some will develop severe disease such as peptic ulcers or gastric cancer. A characteristic feature of this infection is the pronounced accumulation of phagocytes, particularly neutrophils, in the gastric mucosa. H. pylori thrives in a phagocyte-rich environment, and we describe here how this organism uses an array of novel virulence factors to manipulate chemotaxis, phagocytosis, membrane trafficking and the respiratory burst as a means to evade elimination by the innate immune response.     

Monocyte and Macrophage Killing of Helicobacter pylori: Relationship to Bacterial Virulence Factors

Background:  Helicobacter pylori infection is an important health problem, as it involves approximately 50% of the world's population, causes chronic inflammatory disease and increases the risk of gastric cancer development. H. pylori infection elicits a vigorous immune response, but this does not usually result in bacterial clearance. We have investigated whether the persistence of H. pylori in the host could be partly due to an inability of macrophages to kill this bacterium.
Materials and Methods:  Monocytes and macrophages isolated from the peripheral blood of normal human controls were infected in vitro with five H. pylori isolates. The isolates were characterized for known H. pylori virulence factors; vacuolating cytotoxin (VacA), the cag pathogenicity island ( cag PAI), urease, and catalase by Western blot and polymerase chain reaction analysis. The ability of primary human monocytes and macrophages to kill each of these H. pylori strains was then defined at various time points after cellular infection.
Results:  The five H. pylori strains showed contrasting patterns of the virulence factors. There were different rates of killing for the bacterial strains. Macrophages had less capacity than monocytes to kill three H. pylori strains. There appeared to be no correlation between the virulence factors studied and differential killing in monocytes.
Conclusions:  Primary human monocytes had a higher capacity to kill certain strains of H. pylori when compared to macrophages. The VacA, cag PAI, urease, and catalase virulence factors were not predictive of the capacity to avoid monocyte and macrophage killing, suggesting that other factors may be important in H. pylori intracellular pathogenicity.     

Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model

Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA(183-203)) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.     

幽门螺杆菌表面抗原免疫保护作用的体外与活体研究

目的：调查幽门螺杆菌（Hp)几种表面蛋白体外对T细胞增殖的影响和在小鼠体内的免疫保护作用。方法：评价Hp全菌抗原、尿原酶（Urease)、黏附素（hpaA)、外膜蛋白25（Hop25)和38（Hop38)对人外周血T细胞及小鼠CD4^ T细胞增殖的影响;与佐剂合用,评价上述重组蛋白对小鼠Hp感染的免疫预防作用。结果：Urease和Hop25可刺激人及鼠T细胞增殖,hapA只能刺激Hp^ PBL增殖,而Hop38则有毒性作用;Hop25和Hop38均可产生60％的完全保护,hpaA可产生100％的部分保护即降低细菌定植密度,而Urease只能产生40％的部分保护。结论：外膜蛋白可能是一组高效的Hp疫苗免疫原;其长期免疫效果及对T细胞功能的活体调节作用尚需进一步评价。国际上尚未见相关报道。     

Helicobacter pylori: immunoproteomics related to different pathologies

Helicobacter pylori is a Gram-negative bacterium that causes ulcer, atrophic gastritis, adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Moreover, an ongoing controversial role of this bacterium infection has been suggested in the etiopathogenesis of some extradigestive diseases. The humoral response to H. pylori during a natural infection can be used for diagnostic purposes and as a basis for vaccine development. Host-pathogen interactions may be investigated by means of immunoproteomics, which provides global information about relevant specific and nonspecific antigens, and thus might be suitable to identify novel vaccine candidates or serological markers of H. pylori infection as well as of different related diseases. In this review, we describe how several research groups used H. pylori proteomics combined with western blotting analysis, using sera from patients affected with different H. pylori-related pathologies, to investigate potential associations between host immune response and clinical outcomes of H. pylori infection, resulting in the rapid identification of novel, highly immunoreactive antigens.     

Development and evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent experimental infection in the mouse model

BACKGROUND: The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). METHODS: The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 micro g each) and intranasal (16 micro g) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. RESULTS: Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. CONCLUSION: A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations.     

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.     

Cell envelope characteristics of Helicobacter pylori: their role in adherence to mucosal surfaces and virulence

Abstract Helicobacter pylori colonises the gastric mucosa of humans and causes both antral gastritis and duodenal ulcer disease. Exactly how H. pylori causes disease is not known but several pathogenic determinants have been proposed for the organism. These include adhesins, cytotoxins and a range of different enzymes including urease, catalase and superoxide dismutase. Surface molecules of H. pylori such as flagella, lipopolysaccharide, the urease enzyme and outer membrane proteins are putative adhesin molecules. While phosphatidylethanolamine and the Lewis^b blood group antigen have been proposed as receptor molecules for the organism the exact mechanism by which H. pylori adheres to the gastric mucosa has still to be identified. Characterisation of the adhesins of H. pylori could lead to the development of adhesin analogues for use in the inhibition of colonisation and improved therapy for ulcer disease. In vivo studies with isogenic mutants which are incapable of adhering to the gastric mucosa would greatly clarify the significance of adherence. Such mutants could possibly be useful as a vaccine against infection with wild-type organisms.     

Progress in vaccine development against Helicobacter pylori

Based on the very high prevalence of diseases caused by Helicobacter pylori, particularly in the developing world, and the rapid emergence of antibiotic resistance among clinical isolates, there is a strong rationale for an effective vaccine against H. pylori. In this review we describe recent promising candidate vaccines and prophylactic or therapeutic immunization strategies for use against H. pylori, as well as studies to identify immune responses that are related to protection in experimental animals. We also describe identification of different types of immune responses that may be related to protection against symptoms based on comparisons of H. pylori-infected patients with duodenal ulcers or gastric cancer and asymptomatic carriers. We conclude that there is still a strong need to clarify the main protective immune mechanisms against H. pylori as well as to identify a cocktail of strong protective antigens, or recombinant bacterial strains that express such antigens, that could be administered by a regimen that gives rise to effective immune responses in humans.     

Inflammation, immunity, and vaccine development for Helicobacter pylori

The immune response to Helicobacter pylori entails both innate effectors and a complex mix of Th1, Th17, and Treg adaptive immune responses. The clinical outcome of infection may well depend to a large degree on the relative balance of these responses. Vaccination with a wide range of antigens, adjuvants, and delivery routes can produce statistically significant reductions in H. pylori colonization levels in mice, though rarely sterilizing immunity. Whether similar reductions in bacterial load can be achieved in humans, and whether they would be clinically significant, is still unclear. However, progress in understanding the role of Th1, Th17, and most recently Treg cells in protection against H. pylori infection provides reason for optimism.     

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.     

Cytokines as adjuvants for avian vaccines

The worldwide trend towards a reduced reliance on in-feed antibiotics has increased the pressure to develop alternative strategies to manage infectious diseases in poultry. With this in mind, there is a great emphasis on vaccine use and the enhancement of existing vaccines to provide long-term protection. Currently existing adjuvants for poultry can have deleterious side-effects, such as inflammation, resulting in the down-grading of meat quality and a subsequent reduction in profits. Therefore, to enhance the use of vaccination, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants in poultry is attracting considerable attention, and their potential role as such has been addressed by several studies. The recent identification of a number of chicken cytokine genes has provided the possibility to study their effectiveness in enhancing the immune response during infection and vaccination. This review focuses on the recent studies involving the assessment of cytokines as vaccine adjuvants.     

The combined recombinant cathepsin L1H and cathepsin B3 vaccine against Fasciola gigantica infection

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n = 10 per group) by subcutaneous injection with 50 μg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15 F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals.     

Enhancement of Helicobacter pylori outer inflammatory protein DNA vaccine efficacy by co-delivery of interleukin-2 and B subunit heat-labile toxin gene encoded plasmids

Development of an effective vaccine for controlling H. pylori-associated infection, which is present in about half the people in the world, is a priority. The H. pylori outer inflammatory protein (oipA) has been demonstrated to be a potential antigen for a vaccine. In the present study, use of oipA gene encoded construct (poipA) for C57BL/6 mice vaccination was investigated. Whether co-delivery of IL-2 gene encoded construct (pIL-2) and B subunit heat-labile toxin of Escherichia coli gene encoded construct (pLTB) can modulate the immune response and enhance DNA vaccine efficacy was also explored. Our results demonstrated that poipA administered intradermally ('gene gun' immunization) promoted a strong Th2 immune response, whereas co-delivery of either pIL-2 or pLTB adjuvant elicited a Th1-biased immune response. PoipA administered with both pIL-2 and pLTB adjuvants promoted a strong Th1 immune response. Regardless of the different immune responses promoted by the various vaccination regimes, all immunized mice had smaller bacterial loads after H. pylori challenge than did PBS negative and pVAX1 mock controls. Co-delivery of adjuvant(s) enhances poipA DNA vaccine efficacy by shifting the immune response from being Th2 to being Th1-biased, which results in a greater reduction in bacterial load after H. pylori challenge. Both prophylactic and therapeutic vaccination can achieve sterile immunity in some subjects.