Chemoenzymatic synthesis of an α-1,6-glucan-based conjugate vaccine against Helicobacter pylori.

In this study, we investigated the utility of glycoconjugates based on a linear α-1,6-glucan chain synthesized using a recombinant α-1,6-glucosyltransferase from the 26695 strain of Helicobacter pylori. Capillary electrophoresis-mass spectrometry analysis confirmed the main product to contain 9-10 sequentially added α-1,6-linked glucose residues. This was consistent with a length of α-1,6-glucan structure present in the outer core region of H. pylori lipopolysaccharide (LPS) from strains 26695 and 26695 HP0826::Kan. The synthetic α-1,6-glucan was conjugated to either bovine serum albumin or tetanus toxoid and immunological properties of resultant glycoconjugates investigated. The conjugates were immunogenic in rabbits and mice and induced strong and specific IgG responses against purified LPS from typeable and nontypeable α-1,6-glucan-positive H. pylori strains. Furthermore, the post-immune sera from rabbits that received the conjugates were bactericidal and cross-reacted with selected clarithromycin-resistant and clarithromycin-susceptible clinical isolates of H. pylori. This technology offers a novel approach to the design of a synthetic carbohydrate-based vaccine against H. pylori.

Assessment of stability of sulphated lactosyl archaeol archaeosomes for use as a vaccine adjuvant.

Archaeosomes, composed of sulphated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. In addition to efficacy, the stability of vaccine components including the adjuvant is an important parameter to consider when developing novel vaccine formulations. To properly evaluate the potential of SLA glycolipids to be used as vaccine adjuvants in a clinical setting, a comprehensive evaluation of their stability is required. Herein, we evaluated the long term stability of preformed empty SLA archaeosomes prior to admixing with antigen at 4 °C or 37 °C for up to 6 months. In addition, the stability of adjuvant and antigen was evaluated for up to 1 month following admixing. Multiple analytical parameters evaluating the molecular integrity of SLA and the liposomal profile were assessed. Following incubation at 4 °C or 37 °C, the SLA glycolipid did not show any pattern of degradation as determined by mass spectroscopy, nuclear magnetic resonance (NMR) and thin layer chromatography (TLC). In addition, SLA archaeosome vesicle characteristics, such as size, zeta potential, membrane fluidity and vesicular morphology, were largely consistent throughout the course of the study. Importantly, following storage for 6 months at both 4 °C and 37 °C, the adjuvant properties of empty SLA archaeosomes were unchanged, and following admixing with antigen, the immunogenicity of the vaccine formulations was also unchanged when stored at both 4 °C and 37 °C for up to 1 month. Overall this indicates that SLA archaeosomes are highly stable adjuvants that retain their activity over an extended period of time even when stored at high temperatures.

The potential of dextran-based glycoconjugates for development of Helicobacter pylori vaccine.

We have recently demonstrated that synthetic glycoconjugates based on delipidated lipopolysaccharide (LPS) of Helicobacter pylori and containing an α(1-6)-glucan chain induced broadly cross-reactive functional antibodies in immunized animals. To investigate the candidacy of α(1-6)-glucan as an alternative vaccine strategy we prepared glycoconjugates based on dextrans produced by lactic acid bacteria Leuconostoc mesenteroides B512F and consisting of linear α(1-6)-glucan chains with limited branching. Three dextrans with averaged molecular masses of 5,000 Da, 3,500 Da and 1,500 Da, respectively, were modified with a diamino group-containing linker and conjugated to a carrier protein, tetanus toxoid (TT) or diphtheria toxoid (DT), and their immunological properties investigated. The conjugates were immunogenic in both rabbits and mice and induced specific IgG responses against α(1-6)-glucan-expressing H. pylori LPS. Studies performed with post-immune sera of mice and rabbits immunized with dextran-based conjugates demonstrated cross-reactivity with LPS from typeable and non-typeable strains of H. pylori and selected mutants. The post-immune sera from rabbits that received the conjugates exhibited functional activity against α(1-6)-glucan-positive strains of H. pylori. These data provide evidence that dextran-based conjugates may offer a simplified approach to the development of carbohydrate-based vaccines against H. pylori.

Lipopolysaccharide glycotyping of clarithromycin-resistant and clarithromycin-susceptible Canadian isolates of Helicobacter pylori.

Lipopolysaccharide (LPS) of Helicobacter pylori exhibits several unique structures, such as Lewis (Le) antigens, α-1,6-glucan, and dd-heptan. To investigate the relationship between LPS structure and resistance to clarithromycin, 41 Canadian isolates of H. pylori were characterized by whole-cell ELISA (enzyme-linked immunosorbent assay), sugar analysis, immunoblotting, and indirect immunofluorescence. The expression of type 2 Lewis X and (or) Lewis Y antigens was detected in 22 of 23 (95.7%) clarithromycin-resistant and in 14 of 18 (77.7%) clarithromycin-susceptible H. pylori strains (P < 0.05), and 8 isolates co-expressed type 1 and type 2 Le antigens (8/41, 19.5%). A significantly higher frequency of α-1,6-glucan (P < 0.01) was detected in clarithromycin-resistant strains than in clarithromycin-susceptible strains (19/23 (82.6%) versus 11/18 (61.1%)). Sugar analysis of selected α-1,6-glucan-positive H. pylori strains confirmed that they frequently contained elevated amounts of dd-heptose. Clarithromycin-resistant isolates were also characterized by low expression levels or absence of CagA (17/23, 73.9%). Indirect immunofluorescence studies carried out on selected H. pylori strains with rabbit immune sera specific for α-1,6-glucan confirmed broad recognition of α-1,6-glucan epitope. The binding was not affected by LPS glycotype of H. pylori isolates examined nor by their CagA status or resistance to clarithromycin. These findings suggest α-1,6-glucan as a potential vaccine target, especially in an era of increasing clarithromycin resistance in H. pylori.

Sulfated Lactosyl Archaeol Archaeosome-Adjuvanted Vaccine Formulations Targeting Rabbit Hemorrhagic Disease Virus Are Immunogenic and Efficacious.

Vaccines play an important role in maintaining human and animal health worldwide. There is continued demand for effective and safe adjuvants capable of enhancing antigen-specific responses to a target pathogen. Rabbit hemorrhagic disease virus (RHDV) is a highly contagious calicivirus that often induces high mortality rates in rabbits. Herein, we evaluated the activity of an experimental sulfated lactosyl archaeol (SLA) archaeosome adjuvant when incorporated in subunit vaccine formulations targeting RHDV. The subunit antigens consisted of RHDV-CRM197 peptide conjugates or recombinant RHDV2 VP60. SLA was able to enhance antigen-specific antibody titers and cellular responses in mice and rabbits. Three weeks following immunization, antigen-specific antibody levels in rabbits vaccinated with RHDV2 VP60 + SLA were significantly higher than those immunized with antigen alone, with geomean titers of 7393 vs. 117. In addition, the SLA-adjuvanted VP60-based formulations were highly efficacious in a rabbit RHDV2 challenge model with up to 87.5% animals surviving the viral challenge. These findings demonstrate the potential utility of SLA adjuvants in veterinary applications and highlight its activity in different types of mammalian species.

Generation of a Liposomal Vaccine Adjuvant Based on Sulfated S-Lactosylarchaeol (SLA) Glycolipids.

Vaccine formulations utilize adjuvants to enhance the level and breadth of the immune response to a target antigen. Liposomes composed of sulfated S-lactosylarchaeol (SLA) glycolipids can induce strong humoral and cell-mediated antigen-specific immune responses to co-administered antigens in mice. This has been demonstrated with a variety of protein antigens, where the protein is either encapsulated within or simply admixed with the archaeal liposomes (archaeosomes). In this process, a dried film of SLA glycolipid is hydrated in water or antigen solution to generate a large multilamellar (ML) liposomal suspension which is then size reduced by sonication to form unilamellar vesicles (UL) with a narrower size distribution. Herein, we describe the generation of liposomes based on the archaeal-based lipid SLA for use as an adjuvant in vaccine formulations.

Evaluation of Adjuvant Activity and Bio-Distribution of Archaeosomes Prepared Using Microfluidic Technology.

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. They have classically been prepared using a thin-film hydration method with an average particle size of 100-200 nm. In this study, we developed methods to generate SLA archaeosomes at different sizes, i.e., 30 nm and 100 nm, via microfluidic mixing technology and evaluated their physicochemical characteristics, as well as adjuvant activity and in vivo biodistribution in mice. Archaeosomes, prepared using thin-film and microfluidic mixing techniques, had similar nanostructures and physicochemical characteristics, with both appearing stable during the course of this study when stored at 4 °C or 37 °C. They also demonstrated similar adjuvant activity when admixed with ovalbumin antigen and used to immunize mice, generating equivalent antigen-specific immune responses. Archaeosomes, labeled with CellVueTM NIR815, had an equivalent biodistribution with both sizes, namely the highest signal at the injection site at 24 h post injection, followed by liver, spleen and inguinal lymph node. The presence of SLA archaeosomes of either size helped to retain OVA antigen (OVA-Cy5.5) longer at the injection site than unadjuvanted OVA. Overall, archaeosomes of two sizes (30 nm and 100 nm) prepared using microfluidic mixing maintained similar physicochemical properties, adjuvant activity and biodistribution of antigen, in comparison to those compared by the conventional thin film hydration method. This suggests that microfluidics based approaches could be applied to generate consistently sized archaeosomes for use as a vaccine adjuvant.

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide.

BACKGROUND: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6-glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6-glucan and characterize their binding properties and functional activity. MATERIALS AND METHODS: BALB/c mice were injected intraperitoneally with 10(8) formalin-fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti-α1,6-glucan-producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole-cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild-type and mutant strains of H. pylori. RESULTS: The generated anti-α1,6-glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6-linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD450 ≥ 0.2). Bactericidal activity was observed against selective wild-type and mutant H. pylori strains exhibiting OD450 values of ≥ 0.45 in WCE. CONCLUSIONS: Anti-α1,6-glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS-based vaccine against H. pylori infections.

The Synergistic Effects of Sulfated Lactosyl Archaeol Archaeosomes When Combined with Different Adjuvants in a Murine Model.

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. SLA archaeosomes are a promising adjuvant candidate due to their ability to strongly stimulate both humoral and cytotoxic immune responses when simply admixed with an antigen. In the present study, we evaluated whether the adjuvant effects of SLA archaeosomes could be further enhanced when combined with other adjuvants. SLA archaeosomes were co-administered with five different Toll-like Receptor (TLR) agonists or the saponin QS-21 using ovalbumin as a model antigen in mice. Both humoral and cellular immune responses were greatly enhanced compared to either adjuvant alone when SLA archaeosomes were combined with either the TLR3 agonist poly(I:C) or the TLR9 agonist CpG. These results were also confirmed in a separate study using Hepatitis B surface antigen (HBsAg) and support the further evaluation of these adjuvant combinations.

Sulfated Lactosyl Archaeol Archaeosomes Synergize with Poly(I:C) to Enhance the Immunogenicity and Efficacy of a Synthetic Long Peptide-Based Vaccine in a Melanoma Tumor Model.

Cancer remains a leading cause of morbidity and mortality worldwide. While novel treatments have improved survival outcomes for some patients, new treatment modalities/platforms are needed to combat a wider variety of tumor types. Cancer vaccines harness the power of the immune system to generate targeted tumor-specific immune responses. Liposomes composed of glycolipids derived from archaea (i.e., archaeosomes) have been shown to be potent adjuvants, inducing robust, long-lasting humoral and cell-mediated immune responses to a variety of antigens. Herein, we evaluated the ability of archaeosomes composed of sulfated lactosyl archaeol (SLA), a semi-synthetic archaeal glycolipid, to enhance the immunogenicity of a synthetic long peptide-based vaccine formulation containing the dominant CD8+ T cell epitope, SIINFEKL, from the weakly immunogenic model antigen ovalbumin. One advantage of immunizing with long peptides is the ability to include multiple epitopes, for example, the long peptide antigen was also designed to include the immediately adjacent CD4+ epitope, TEWTSSNVMEER. SLA archaeosomes were tested alone or in combination with the toll-like receptor 3 (TLR3) agonist Poly(I:C). Overall, SLA archaeosomes synergized strongly with Poly(I:C) to induce robust antigen-specific CD8+ T cell responses, which were highly functional in an in vivo cytolytic assay. Furthermore, immunization with this vaccine formulation suppressed tumor growth and extended mouse survival in a mouse melanoma tumor model. Overall, the combination of SLA archaeosomes and Poly(I:C) appears to be a promising adjuvant system when used along with long peptide-based antigens targeting cancer.

Mechanistic insight into the induction of cellular immune responses by encapsulated and admixed archaeosome-based vaccine formulations.

Archaeosomes are liposomes formulated using total polar lipids (TPLs) or semi-synthetic glycolipids derived from archaea. Conventional archaeosomes with entrapped antigen exhibit robust adjuvant activity as demonstrated by increased antigen-specific humoral and cell-mediated responses and enhanced protective immunity in various murine infection and cancer models. However, antigen entrapment efficiency can vary greatly resulting in antigen loss during formulation and variable antigen:lipid ratios. In order to circumvent this, we recently developed an admixed archaeosome formulation composed of a single semi-synthetic archaeal lipid (SLA, sulfated lactosylarchaeol) which can induce similarly robust adjuvant activity as an encapsulated formulation. Herein, we evaluate and compare the mechanisms involved in the induction of early innate and antigen-specific responses by both admixed (Adm) and encapsulated (Enc) SLA archaeosomes. We demonstrate that both archaeosome formulations result in increased immune cell infiltration, enhanced antigen retention at injection site and increased antigen uptake by antigen-presenting cells and other immune cell types, including neutrophils and monocytes following intramuscular injection to mice using ovalbumin as a model antigen. In vitro studies demonstrate SLA in either formulation is preferentially taken up by macrophages. Although the encapsulated formulation was better able to induce antigen-specific CD8+ T cell activation by dendritic cells in vitro, both encapsulated and admixed formulations gave equivalently enhanced protection from tumor challenge when tested in vivo using a B16-OVA melanoma model. Despite some differences in the immunostimulatory profile relative to the SLA (Enc) formulation, SLA (Adm) induces strong in vivo immunogenicity and efficacy, while offering an ease of formulation.

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony.

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 microg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.

A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations.

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that can be used as adjuvants to induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigen. However, the entrapment efficiency of antigen within archaeosomes constituted using standard liposome forming methodology is often only 5-40%. In this study, we evaluated different formulation methods using a simple semi-synthetic archaeal lipid (SLA, sulfated lactosyl archaeol) and two different antigens, ovalbumin (OVA) and hepatitis B surface antigen (HBsAg). Antigen was entrapped within archaeosomes using the conventional thin film hydration-rehydration method with or without removal of non-entrapped antigen, or pre-formed empty archaeosomes were simply admixed with an antigen solution. Physicochemical characteristics were determined (size distribution, zeta potential, vesicle morphology and lamellarity), as well as location of antigen relative to bilayer using cryogenic transmission electron microscopy (TEM). We demonstrate that antigen (OVA or HBsAg) formulated with SLA lipid adjuvants using all the different methodologies resulted in a strong antigen-specific immune response. Nevertheless, the advantage of using a drug substance process that comprises of simply admixing antigen with pre-formed empty archaeosomes, represents a simple, efficient and antigenic dose-sparing formulation for adjuvanting and delivering vaccine antigens.

Analysis of Helicobacter pylori isolates from Chile: occurrence of selective type 1 Lewis b antigen expression in lipopolysaccharide.

Previous studies have shown that the LPS of Helicobacter pylori isolated from North American and European hosts predominantly expresses type 2 Lewis x (Le(x)) and Le(y) epitopes, whilst the LPS from Asian strains has the capacity to express type 1 Le(a) and Le(b) structures. The aim of this study was to evaluate the expression of Le antigens and the cytotoxin-associated antigen (CagA) by H. pylori isolates from Chile. A total of 38 isolates were screened. The expression of Le antigens and CagA was determined by whole-cell indirect ELISA, using commercially available monoclonal anti-Le and polyclonal anti-CagA antibodies. LPS profiles of H. pylori isolates were assessed by gel electrophoresis and Western blotting. Expression of Le(x) and/or Le(y) epitopes was confirmed in 32/38 isolates (84 %), whilst 9/38 isolates (24 %) expressed type 1 Le(b) blood group determinants, in addition to type 2 Le(x) and Le(y) structures. Six strains (16 %) were non-typeable. The majority of H. pylori strains examined were CagA-positive (83.3 %).

Effect of the HP0159 ORF mutation on the lipopolysaccharide structure and colonizing ability of Helicobacter pylori.

The outer core region of Helicobacter pylori lipopolysaccharide of the majority of isolates contains an alpha-1,6-glucan polymer synthesized by the product of the HP0159 ORF. Structural studies carried out on HP0159 lipopolysaccharide mutants by a combination of chemical methods, mass spectrometry and nuclear magnetic resonance spectroscopy confirmed that insertional inactivation of HP0159 gene in H. pylori strains 26695 and SS1 resulted in formation of a truncated lipopolysaccharide molecule characterized by the presence of a terminal dd-heptose residue in the side-chain outer core fragment and maintaining an inner core backbone structure compared with the wild-type Lewis antigen-expressing strains. Colonization studies with HP0159 mutants of two mouse-colonizing strains, SS1 and M6, confirmed their inability to successfully colonize the murine stomach.

Structural and immunological characterization of a glycoconjugate based on the delipidated lipopolysaccharide from a nontypeable Helicobacter pylori strain PJ1 containing an extended d-glycero-d-manno-heptan.

Structural characterization of the lipopolysaccharide (LPS) from a nontypeable Helicobacter pylori strain PJ1 and two corresponding mutants, PJ1 HP1283:cam and PJ1 HP1284:cam, was performed using a combination of NMR and mass spectrometric techniques. It resulted in the core structure that differed significantly from the one proposed previously. Overall architecture of PJ1 LPS was found to be consistent with a structural model described for several other H. pylori strains. It contained a polymer of d-glycero-d-manno-heptose (dd-Hep) as the O-chain component, linked to α-1,6-glucan through a dd-Hep oligosaccharide. H. pylori PJ1 HP1283:cam LPS was missing dd-heptan, terminating with an α-1,6-glucan chain containing 5-13 glucose residues. LPS of strain PJ1 HP1284:cam was missing dd-Hep from the core and had ß-GlcNAc attached directly to O-3 of the inner-core ld-Hep residue. To investigate the role of dd-heptan in protective immunity, delipidated LPS (dLPS) from strain PJ1 was conjugated to tetanus toxoid (TT) and immunological properties of the resultant glycoconjugate dLPS(PJ1)-TT determined. The dLPS(PJ1)-TT conjugate was immunogenic in mice and rabbits and induced specific and cross-reactive functional antibodies against homologous and heterologous strains of H. pylori. Whole cell indirect ELISA performed on a selected number of H. pylori isolates confirmed that the immune response correlated with the presence of α-1,6-glucan and was not augmented by the dd-Hep content of these strains.

Lipopolysaccharide structure of Helicobacter pylori serogroup O:3.

In this study, we describe a re-investigation of the lipopolysaccharide structure of Helicobacter pylori serogroup O:3. Application of NMR and MS approaches to the analysis of oligosaccharides obtained through degradation of LPS from H. pylori serogroup O:3 by various methods confirmed that its general architecture was identical to that of LPS from H. pylori strains 26695 and SS1 and followed a sequential linear assembly of the α-1,6-glucan, dd-heptan, and O-chain components. Additionally, MALDI-MS analysis demonstrated that a significant proportion of H. pylori serogroup O:3 LPS was terminated with α-1,6-glucan and was not further substituted by dd-heptan and the O-chain polysaccharide.

Colonization of gerbils with Helicobacter pylori O-chain-deficient mutant SS1 HP0826::Kan results in gastritis and is associated with de novo synthesis of extended homopolymers.

The O-chain polysaccharide of Helicobacter pylori is important for colonization and generation of chronic gastritis in mice. There are marked host differences in the development of H. pylori-induced gastric pathology in mice and gerbils. To investigate the role of the O-chain polysaccharide of H. pylori in colonization and gastritis in Mongolian gerbils, inoculation by oral gavage with H. pylori strain SS1 and its corresponding O-chain polysaccharide-deficient mutant SS1 HP0826::Kan was undertaken. Infection with both strains resulted in corpus atrophy, loss of parietal cells, and extensive mucous metaplasia at both 18 and 30 weeks postinfection. Contrary to previous results in splenocyte recipient severe combined immunodeficiency (SCID) mice, no difference was found in the grade of chronic gastritis, polymorphonuclear cell infiltration, atrophy, and mucous metaplasia in gerbils infected with the wild-type SS1 strain or SS1 HP0826::Kan strain. Examination of the effects of gerbil passage on LPS profiles of output SS1 HP0826::Kan isolates by SDS-PAGE, sugar, and methylation analyses revealed significant differences in LPS profiles of SS1 HP0826::Kan cells recovered from infected gerbils as compared to input bacteria. Specifically, the presence of a novel homopolymer of d-galactan, as well as an extended polymer of d-riban, was detected. These data provide evidence for the role of H. pylori LPS in bacterial adaption to promote colonization and pathology.

Simultaneous analysis of cardiolipin and lipid A from Helicobacter pylori by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cardiolipin (CL) is an anionic tetraacylphospholipid found in mammalian tissues, inner membrane of mitochondria and in the cytoplasmic membrane of Gram-positive and -negative bacteria. Lipid A is the principal structural component responsible for the range of biological activities of lipopolysaccharides. Here we report a MALDI-MS-based method for the sensitive simultaneous analysis of CL and lipid A from Helicobacter pylori cells. The sensitivity was demonstrated by the analysis of CL and lipid A from a single bacterial colony of in vitro grown H. pylori strain NCTC 11637 (ATCC 43504). We then characterized the CL and lipid A structures in H. pylori cells grown under three different conditions, on agar-horse blood plates, in liquid culture and ex vivo. The results revealed the presence of high amounts of myristic (C14:0) and 19-carbon cyclopropane (C19:0cyc) fatty acids. Alterations in CL structure were observed in H. pylori cells cultivated on plates as compared with the bacteria grown in broth culture. Furthermore, significant changes in lipid A acylation pattern were detected in H. pylori cells during formation of coccoids. In contrast, structural analysis of CL from ex vivo H. pylori cells recovered from the stomachs of infected Mongolian gerbils demonstrated only minor changes in acyl chain combination. This is the first report of simultaneous analysis of CL and lipid A from ex vivo cells of H. pylori.

Design and immunological properties of Helicobacter pylori glycoconjugates based on a truncated lipopolysaccharide lacking Lewis antigen and comprising an α-1,6-glucan chain.

To investigate the vaccine potential of H. pylori lipopolysaccharide (LPS), truncated LPS of H. pylori strain 26695 HP0826::Kan lacking O-chain polysaccharide and comprising an extended α-1,6-linked glucan chain was conjugated to tetanus toxoid (TT) or bovine serum albumin (BSA). Two approaches were used for delipidation or partial delipidation of H. pylori LPS: (1) mild hydrolysis resulting in delipidated LPS (dLPS) and (2) treatment with anhydrous hydrazine resulting in removal of O-linked fatty acids (LPS-OH). Both LPS-OH and dLPS were covalently linked through a 2-keto-3-deoxy-octulosonic acid (Kdo) residue to a diamino group-containing spacer, followed by conjugation to thiolated TT or BSA to give conjugates LPS-OH-TT, dLPS-BSA and dLPS-TT, respectively. The LPS-OH-TT, dLPS-BSA and dLPS-TT conjugates were immunogenic in both rabbits and mice, inducing strong and specific IgG responses against homologous and heterologous strains of H. pylori. Moreover, the rabbit post-immune sera showed cross-reactivity against clinical isolates of H. pylori in a whole-cell indirect ELISA, which was further confirmed by indirect immunofluorescent microscopy. A tenfold stronger IgG immune response to the immunizing antigen was generated in mice and rabbits that received dLPS-containing conjugate. The post-immune sera of rabbits immunized with LPS-OH-TT, dLPS-BSA or dLPS-TT displayed significant bactericidal activity against mutant and wild-type α-1,6-glucan-expressing strains and selected clinical isolates of H. pylori. Finally, partial protection against H. pylori challenge was demonstrated in mice vaccinated with dLPS-TT conjugate adjuvanted with cholera toxin. In summary, this study shows that glycoconjugates based on delipidated or partially delipidated LPS from H. pylori 26695 HP0826::Kan mutant induce broadly cross-reactive functional antibodies in immunized animals and should be considered for further vaccine development and testing.