Probing the expression and adhesion of glycans involved in Helicobacter pylori infection.

Helicobacter pylori infects approximately half the human population and has an unusual infective niche of the human stomach. Helicobacter pylori is a major cause of gastritis and has been classified as a group 1 carcinogen by the WHO. Treatment involves triple or quadruple antibiotic therapy, but antibiotic resistance is becoming increasingly prevalent. Helicobacter pylori expresses certain blood group related antigens (Lewis system) as a part of its lipopolysaccharide (LPS), which is thought to assist in immune evasion. Additionally, H. pylori LPS participates in adhesion to host cells alongside several adhesion proteins. This study profiled the carbohydrates of H. pylori reference strains (SS1 and 26695) using monoclonal antibodies (mAbs) and lectins, identifying interactions between two carbohydrate-targeting mAbs and multiple lectins. Atomic force microscopy (AFM) scans were used to probe lectin and antibody interactions with the bacterial surfaces. The selected mAb and lectins displayed an increased adhesive force over the surface of the curved H. pylori rods. Furthermore, this study demonstrates the ability of anti-carbohydrate antibodies to reduce the adhesion of H. pylori 26695 to human gastric adenocarcinoma cells via AFM. Targeting bacterial carbohydrates to disrupt crucial adhesion and immune evasion mechanisms represents a promising strategy for combating H. pylori infection.

Nanocapsules Comprised of Purified Protein: Construction and Applications in Vaccine Research.

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

Schistosome Transgenesis: The Long Road to Success.

As research on parasitic helminths has entered the post-genomic era, research efforts have turned to deciphering the function of genes in the public databases of genome sequences. It is hoped that, by understanding the role of parasite genes in maintaining their parasitic lifestyle, critical insights can be gained to develop new intervention and control strategies. Methods to manipulate and transform parasitic worms are now developed to a point where it has become possible to gain a comprehensive understanding of the molecular mechanisms underlying host-parasite interplay, and here, we summarise and discuss the advances that have been made in schistosome transgenesis over the past 25 years. The ability to genetically manipulate schistosomes holds promise in finding new ways to control schistosomiasis, which ultimately may lead to the eradication of this debilitating disease.

Solid Lipid Nanoparticles Delivering a DNA Vaccine Encoding Helicobacter pylori Urease A Subunit: Immune Analyses before and after a Mouse Model of Infection.

In this study, novel solid lipid particles containing the adjuvant lipid monophosphoryl lipid A (termed 'SLN-A') were synthesised. The SLN-A particles were able to efficiently bind and form complexes with a DNA vaccine encoding the urease alpha subunit of Helicobacter pylori. The resultant nanoparticles were termed lipoplex-A. In a mouse model of H. pylori infection, the lipoplex-A nanoparticles were used to immunise mice, and the resultant immune responses were analysed. It was found that the lipoplex-A vaccine was able to induce high levels of antigen-specific antibodies and an influx of gastric CD4+ T cells in vaccinated mice. In particular, a prime with lipoplex-A and a boost with soluble UreA protein induced significantly high levels of the IgG1 antibody, whereas two doses of lipoplex-A induced high levels of the IgG2c antibody. In this study, lipoplex-A vaccination did not lead to a significant reduction in H. pylori colonisation in a challenge model; however, these results point to the utility of the system for delivering DNA vaccine-encoded antigens to induce immune responses and suggest the ability to tailor those responses.

An Evaluation of Urease A Subunit Nanocapsules as a Vaccine in a Mouse Model of Helicobacter pylori Infection.

Using removable silica templates, protein nanocapsules comprising the A subunit of Helicobacter pylori urease (UreA) were synthesised. The templates were of two sizes, with solid core mesoporous shell (SC/MS) silica templates giving rise to nanocapsules of average diameter 510 nm and mesoporous (MS) silica templates giving rise to nanocapsules of average diameter 47 nm. Both were shown to be highly monodispersed and relatively homogenous in structure. Various combinations of the nanocapsules in formulation were assessed as vaccines in a mouse model of H. pylori infection. Immune responses were evaluated and protective efficacy assessed. It was demonstrated that vaccination of mice with the larger nanocapsules combined with an adjuvant was able to significantly reduce colonisation.

Enhancing the photoluminescence and cellular uptake of fluorescent carbon nanodots via cubosome lipid nanocarriers.

Carbon nanodots (C-dots) have attracted much attention for their use in the fields of bioimaging, drug delivery, and sensing due to their excellent fluorescent and photoluminescent properties, photostability, biocompatibility, and amenability to surface modification. Herein, we report a nanocomposite formulation of C-dots (<5 nm) encapsulated in lipid-based lyotropic liquid crystalline nanoparticles (∼250 nm) via either passive diffusion or electrostatic mechanisms. The physicochemical properties of the nanocomposite formulation including particle size, surface charge, internal cubic nanostructures, and pH-dependent fluorescent properties were characterised. Upon loading of C-dots into lipid nanoparticles, the highly ordered inverse bicontinuous cubic mesophase existed in the internal phase of the nanoparticles, demonstrated by synchrotron small angle X-ray scattering, molecular dynamic simulation and cryogenic transmission electron microscopy. The pH-dependent fluorescent property of the C-dots was modified via electrostatic interaction between the C-dots and cationic lipid nanoparticles, which further enhanced the brightness of C-dots through self-quenching prevention. The cytotoxicity and cellular uptake efficiency of the developed nanocomposites were also examined in an epithelial gastric adenocarcinoma cell line (AGS) and a macrophage cell line (stimulated THP-1). Compared to free C-dots, the uptake and cell imaging potential of the C-dot nanocomposites was significantly improved, by several orders of magnitude as demonstrated by cytoplasmic fluorescent intensities using confocal microscopy. Loading C-dots into mesoporous lipid nanocarriers presents a new way of modifying C-dot physicochemical and fluorescent properties, alternative to direct chemical surface modification, and advances the bioimaging potential of C-dots by enhancing cellular uptake efficiency and converging C-dot light emission.

Enhancement of live vaccines by co-delivery of immune modulating proteins.

Vaccines are very effective in providing protection against many infectious diseases. However, it has proven difficult to develop highly efficacious vaccines against some pathogens and so there is a continuing need to improve vaccine technologies. The first successful and widely used vaccines were based on attenuated pathogens (e.g., laboratory passaged Pasteurella multocida to vaccinate against fowl cholera) or closely related non-pathogenic organisms (e.g., cowpox to vaccinate against smallpox). Subsequently, live vaccines, either attenuated pathogens or non-pathogenic microorganisms modified to deliver heterologous antigens, have been successfully used to induce protective immune responses against many pathogens. Unlike conventional killed and subunit vaccines, live vaccines can deliver antigens to mucosal surfaces in a similar manner and context as the natural infection and hence can often produce a more appropriate and protective immune response. Despite these advantages, there is still a need to improve the immunogenicity of some live vaccines. The efficacy of injectable killed and subunit vaccines is usually enhanced using adjuvants such mineral salts, oils, and saponin, but such adjuvants cannot be used with live vaccines. Instead, live vaccines can be engineered to produce immunomodulatory molecules that can stimulate the immune system to induce more robust and long-lasting adaptive immune responses. This review focuses on research that has been undertaken to engineer live vaccines to produce immunomodulatory molecules that act as adjuvants to increase immunogenicity. Adjuvant strategies with varying mechanisms of action (inflammatory, antibody-mediated, cell-mediated) and delivery modes (oral, intramuscular, intranasal) have been investigated, with varying degrees of success. The goal of such research is to define adjuvant strategies that can be adapted to enhance live vaccine efficacy by triggering strong innate and adaptive immune responses and produce vaccines against a wider range of pathogens.

Helicobacter pylori and the Role of Lipopolysaccharide Variation in Innate Immune Evasion.

Helicobacter pylori is an important human pathogen that infects half the human population and can lead to significant clinical outcomes such as acute and chronic gastritis, duodenal ulcer, and gastric adenocarcinoma. To establish infection, H. pylori employs several mechanisms to overcome the innate and adaptive immune systems. H. pylori can modulate interleukin (IL) secretion and innate immune cell function by the action of several virulence factors such as VacA, CagA and the type IV secretion system. Additionally, H. pylori can modulate local dendritic cells (DC) negatively impacting the function of these cells, reducing the secretion of immune signaling molecules, and influencing the differentiation of CD4+ T helper cells causing a bias to Th1 type cells. Furthermore, the lipopolysaccharide (LPS) of H. pylori displays a high degree of phase variation and contains human blood group carbohydrate determinants such as the Lewis system antigens, which are proposed to be involved in molecular mimicry of the host. Lastly, the H. pylori group of outer membrane proteins such as BabA play an important role in attachment and interaction with host Lewis and other carbohydrate antigens. This review examines the various mechanisms that H. pylori utilises to evade the innate immune system as well as discussing how the structure of the H. pylori LPS plays a role in immune evasion.

Gastric organoids: Advancing the study of H. pylori pathogenesis and inflammation.

For decades, traditional in vitro and in vivo models used for the study of Helicobacter pylori infection have relied heavily on the use of gastric cancer cell lines and rodents. Major challenges faced by these methods have been the inability to study cancer initiation in already cancerous cell lines, and the difficulty in translating results obtained in animal models due to genetic differences. These challenges have prevented a thorough understanding of the pathogenesis of disease and slowed the development of cancer therapies and a suitable vaccine against the pathogen. In recent years, the development of gastric organoids has provided great advantages over the traditional in vivo and in vitro models due to their similarities to the human stomach in vivo, their ease of use, and the capacity for long-term culture. This review discusses the advantages and limitations of existing in vivo and in vitro models of H. pylori infection, and how gastric organoids have been applied to study H. pylori pathogenesis, with a focus on how the pathogen interacts with the gastric epithelium, inflammatory processes, epithelial repair, and cancer initiation. The potential applications of organoids to address more complex questions on the role of hormones, vaccine-induced immunity are also discussed.

Homeostasis and Cancer Initiation: Organoids as Models to Study the Initiation of Gastric Cancer.

Gastric cancer represents a significant disease burden worldwide. The factors that initiate cancer are not well understood. Chronic inflammation such as that triggered by H. pylori infection is the most significant cause of gastric cancer. In recent years, organoid cultures developed from human and animal adult stem cells have facilitated great advances in our understanding of gastric homeostasis. Organoid models are now being exploited to investigate the role of host genetics and bacterial factors on proliferation and DNA damage in gastric stem cells. The impact of a chronic inflammatory state on gastric stem cells and the stroma has been less well addressed. This review discusses what we have learned from the use of organoid models to investigate cancer initiation, and highlights questions on the contribution of the microbiota, chronic inflammatory milieu, and stromal cells that can now be addressed by more complex coculture models.

Solid Lipid Nanoparticle Carrier Platform Containing Synthetic TLR4 Agonist Mediates Non-Viral DNA Vaccine Delivery.

There is a growing demand for better delivery systems to improve the stability and efficacy of DNA vaccines. Here we report the synthesis of a non-viral DNA vaccine delivery system using a novel adjuvanted solid lipid nanoparticle (SLN-A) platform as a carrier for a DNA vaccine candidate encoding the Urease alpha (UreA) antigen from Helicobacter pylori. Cationic SLN-A particles containing monophosphoryl lipid A (adjuvant) were synthesised by a modified solvent-emulsification method and were investigated for their morphology, zeta potential and in vitro transfection capacity. Particles were found to bind plasmid DNA to form lipoplexes, which were characterised by electron microscopy, dynamic light scattering and fluorescence microscopy. Cellular uptake studies confirmed particle uptake within 3 h, and intracellular localisation within endosomal compartments. In vitro studies further confirmed the ability of SLN-A particles to stimulate expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) in human macrophage-like Tohoku Hospital Pediatrics-1 (THP-1) cells. Lipoplexes were found to be biocompatible and could be efficiently transfected in murine immune cells for expression of recombinant H. pylori antigen Urease A, demonstrating their potential as a DNA vaccine delivery system.

Treatment of Staphylococcus aureus skin infection in vivo using rifampicin loaded lipid nanoparticles.

We have previously reported on a novel nanoparticle formulation that was effective at killing Staphylococcus aureus in vitro. Here, we report for the first time, the antibacterial effects of a lipidic nano-carrier containing rifampicin (NanoRIF) which can be used to successfully treat Methicillin-Resistant S. aureus (MRSA) infection at a reduced antibiotic dosage compared to the free drug in a skin wound model in mice. The formulation used contains the lipid monoolein, a cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) and the antibiotic. We have shown that rifampicin-loaded nanoparticles are more effective at treating infection in the skin wound model than the antibiotic alone. Cryo-TEM was used to capture for the first time, interactions of the formed nanoparticles with the cell wall of an individual bacterium. Our data strongly indicate enhanced binding of these charged nanoparticles with the negatively charged bacterial membrane. The efficacy we have now observed in vivo is of significant importance for the continued development of nanomedicine-based strategies to combat antibiotic resistant bacterial skin infections.

The frequency of circulating integrin α4ß7+ cells correlates with protection against Helicobacter pylori infection in immunized mice.

BACKGROUND: Chronic Helicobacter pylori infection is the cause of peptic ulcers in a subpopulation of individuals and a risk factor for the development of gastric cancer. A vaccine against H pylori infection can prevent the acquisition of the infection and protect against reinfections. Clinical trials to date evaluating the efficacy of H pylori vaccines in human challenge models have shown moderate to poor protection with difficulties in predicting efficacy. Thus, while further studies are needed to design an effective vaccine, we also need to find relevant correlates for vaccine efficacy. OBJECTIVE: To find immune correlates to vaccine efficacy, the frequencies of neutrophils, eosinophils and inflammatory monocytes and CD4+ T-cell memory and mucosa homing integrin α4ß7+ cells were assessed by flow cytometry in the blood of mice after vaccination. MATERIALS AND METHODS: H pylori antigens and cholera toxin or the multiple mutant CT (mmCT) were administered via the sublingual (SL) and intragastric route (IG). The vaccinated mice were infected with H pylori strain SS1 bacteria, and colonization in the stomach and immune responses were evaluated. RESULTS: The H pylori vaccine was effective in reducing bacterial load in the stomach of mice and enhancing immune responses compared to unvaccinated infection controls. In the blood of mice after SL or IG route of vaccination, we observed changes in frequencies of innate and adaptive immune cell subsets compared to infection controls. Remarkably, the frequency of circulating mucosal homing α4ß7+ CD4+ T cells after vaccination correlated with low bacterial load in the stomach of individual mice irrespective of the immunization route. CONCLUSIONS: Our study shows that the innate and adaptive immune cell subsets can be measured in the blood after vaccination and that increased frequency of α4ß7+ CD4+ in the blood after immunization could be used as a predictive marker for the efficacy of vaccine against H pylori infection.

Immunity and Vaccine Development Against Helicobacter pylori.

Helicobacter pylori is a highly-adapted gastrointestinal pathogen of humans and the immunology of this chronic infection is extremely complex. Despite the availability of antibiotic therapy, the global incidence of H. pylori infection remains high, particularly in low to middle-income nations. Failure of therapy and the spread of antibiotic resistance among the bacteria are significant problems and provide impetus for the development of new therapies and vaccines to treat or prevent gastric ulcer, and gastric carcinoma. The expansion of knowledge on gastric conventional and regulatory T cell responses, and the role of TH17 in chronic gastritis from studies in mouse models and patients have provided valuable insights into how gastritis is initiated and maintained. The development of human challenge models for testing candidate vaccines has meant a unique opportunity to study acute infection, but the field of vaccine development has not progressed as rapidly as anticipated. One clear lesson learned from previous studies is that we need a better understanding of the immune suppressive mechanisms in vivo to be able to design vaccine strategies. There is still an urgent need to identify practical surrogate markers of protection that could be deployed in future field vaccine trials. Important developments in our understanding of the chronic inflammatory response, progress and problems arising from human studies, and an outlook for the future of clinical vaccine trials will be discussed.

Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly-N-acetylglucosamine.

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly-N-acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N-acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms.

Induction of mucosal immune responses against Helicobacter pylori infection after sublingual and intragastric route of immunization.

There is a current lack of effective mucosal vaccines against major gastroenteric pathogens and particularly against Helicobacter pylori, which causes a chronic infection that can lead to peptic ulcers and gastric cancer in a subpopulation of infected individuals. Mucosal CD4+ T-cell responses have been shown to be essential for vaccine-induced protection against H. pylori infection. The current study addresses the influence of the adjuvant and site of mucosal immunization on early CD4+ T-cell priming to H. pylori antigens. The vaccine formulation consisted of H. pylori lysate antigens and mucosal adjuvants, cholera toxin (CT) or a detoxified double-mutant heat-labile enterotoxin from Escherichia coli (dmLT), which were administered by either the sublingual or intragastric route. We report that in vitro, adjuvants CT and dmLT induce up-regulation of pro-inflammatory gene expression in purified dendritic cells and enhance the H. pylori-specific CD4+ T-cell response including interleukin-17A (IL-17A), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) secretion. In vivo, sublingual immunization led to an increased frequency of IL-17A+ , IFN-γ+ and TNF-α+ secreting CD4+ T cells in the cervical lymph nodes compared with in the mesenteric lymph nodes after intragastric immunization. Subsequently, IL-17A+ cells were visualized in the stomach of sublingually immunized and challenged mice. In summary, our results suggest that addition of an adjuvant to the vaccine clearly activated dendritic cells, which in turn, enhanced CD4+ T-cell cytokines IL-17A, IFN-γ and TNF-α responses, particularly in the cervical lymph nodes after sublingual vaccination.

Excitability and Synaptic Transmission in the Enteric Nervous System: Does Diet Play a Role?

Changes in diet are a challenge to the gastrointestinal tract which needs to alter its processing mechanisms to continue to process nutrients and maintain health. In particular, the enteric nervous system (ENS) needs to adapt its motor and secretory programs to deal with changes in nutrient type and load in order to optimise nutrient absorption.The nerve circuits in the gut are complex, and the numbers and types of neurons make recordings of specific cell types difficult, time-consuming, and prone to sampling errors. Nonetheless, traditional research methods like intracellular electrophysiological approaches have provided the basis for our understanding of the ENS circuitry. In particular, animal models of intestinal inflammation have shown us that we can document changes to neuronal excitability and synaptic transmission.Recent studies examining diet-induced changes to ENS programming have opted to use fast imaging techniques to reveal changes in neuron function. Advances in imaging techniques using voltage- or calcium-sensitive dyes to record neuronal activity promise to overcome many limitations inherent to electrophysiological approaches. Imaging techniques allow access to a wide range of ENS phenotypes and to the changes they undergo during dietary challenges. These sorts of studies have shown that dietary variation or obesity can change how the ENS processes information-in effect reprogramming the ENS. In this review, the data gathered from intracellular recordings will be compared with measurements made using imaging techniques in an effort to determine if the lessons learnt from inflammatory changes are relevant to the understanding of diet-induced reprogramming.