Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection.

The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H. pylori urease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H. pylori gastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice against H. pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H. pylori infection.

The composition and function of M cell apical membranes: implications for microbial pathogenesis.

M cells, an epithelial cell phenotype that occurs only over organized mucosal lymphoid follicles, deliver samples of foreign material by transepithelial transport from the lumen to organized lymphoid tissues within the mucosa of the small and large intestines. The apical membranes of M cells in the intestine are designed to facilitate adherence and uptake of antigens and microorganisms, a prerequisite for immunological sampling. The molecular features of M cell apical surfaces that promote adherence and transport are crucial for understanding the strategies that pathogens use to exploit this pathway.

Serum antitoxin antibodies mediate systemic and mucosal protection from Clostridium difficile disease in hamsters.

Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis. The pathologic findings associated with this infection are believed to be caused by two large (approximately 300-kDa) exotoxins, toxins A and B. Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes. Using the hamster model of C. difficile infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid. We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not. Protection was associated with high levels of toxin-neutralizing antibodies in serum. The requirement for adjuvants for protection was assessed by using sequential i.m. and rectal or i.m. vaccination regimens. Unexpectedly, i.m. immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers. Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease. These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from C. difficile disease in humans.

Human intestinal M cells display the sialyl Lewis A antigen.

The biochemical features that distinguish human M cells from other intestinal epithelial cell types are important for understanding microbial pathogenesis and for targeting vaccines to the mucosal immune system. We applied a large panel of carbohydrate-specific monoclonal antibodies and lectins to Peyer's patch and cecum biopsy specimens from three normal individuals and a patient with inflammatory bowel disease. The results show that human M-cell glycosylation patterns are distinct from those of other species examined and that human M cells preferentially display the sialyl Lewis A antigen. This carbohydrate epitope is also present in a small subpopulation of enterocytes in the follicle-associated epithelium and in goblet cell mucins.

Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses.

We examined the roles of cell- and antibody-mediated immunity in urease vaccine-induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [beta2-microglobulin (-/-)] but not MHC class II knockout mice [I-Ab (-/-)]. In B cell knockout mice [microMT (-/-)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4(+) cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II-restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.

Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal administration.

Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

Adherence of Salmonella typhimurium to Caco-2 cells: identification of a glycoconjugate receptor.

The mechanism by which Salmonella species adhere to the epithelium of the intestine is not well understood. To identify components on intestinal epithelial cells that may be involved in the initial adherence of Salmonella typhimurium, we correlated patterns of adherence to well-differentiated Caco-2BBe cell monolayers with expression of brush border membrane components and lectin binding sites. This cloned cell line shows heterogeneous expression of sucrase-isomaltase and most lectin receptors. S. typhimurium adhered to a subpopulation of living or formaldehyde-fixed cells with a high multiplicity (up to 150 bacteria per cell). Bacterial binding to selected cells was not correlated with expression of the brush border hydrolases dipeptidyl-peptidase IV and sucrase-isomaltase or with binding of 10 of the 12 lectins tested. However, binding was correlated with the presence of binding sites for peanut agglutinin (PNA) [specific for Gal beta (1-3) GalNAc] and soybean agglutinin (specific for terminal GalNAc). Preincubation of live and fixed Caco-2BBe monolayers with PNA inhibited bacterial binding, while preincubation with soybean agglutinin did not. Electron microscopic analysis demonstrated that the initial adherence of S. typhimurium to Caco-2 cells in vitro involved peripheral components of the glycocalyx on apical microvilli. These results suggest that a Gal beta (1-3)GalNAc epitope recognized by PNA and located in the glycocalyx is involved in the early recognition events between S. typhimurium and Caco-2 cells and that differences in glycosylation patterns among individual epithelial cells may be a determinant in cell-selective adherence of S. typhimurium.

Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines.

We have used a panel of lectins and antibodies to describe the composition of complex carbohydrates associated with M cells in various regions of the intestinal tract of adult BALB/c mice. The fucose-specific lectin Ulex europaeus agglutinin type I (UEA I) is a marker of M cells in the small intestine and recognized M cells at an early stage of differentiation. Subpopulations of M cells in a single follicle-associated epithelium (FAE) could be distinguished by different fucose-specific probes. Certain lectins revealed that M cells have basal processes that extend into the underlying lymphoid tissue. Colonic and rectal M cells display glycosylation patterns distinct from M cells of Peyer's patches and are characterized by terminal galactose. UEA I selectively adhered to Peyer's patch M cells in mucosal explants and in ligated intestinal loops in vivo. The lectin was taken up into endocytic vesicles and transported to the intra-epithelial pocket and other domains of the basolateral membrane. Thus M cell-specific glycoconjugates could serve as "receptors" for targeting of lectin-antigen conjugates to the mucosal immune system.

Transitions between in situ and isolated chromatin.

We show that the mechanism by which chromatin displaying higher-order structure is usually isolated from nuclei involves a transition to an extended nucleosomal arrangement. After being released from nuclei, chromatin must refold in order to produce the typical chromatin fibers observed in solution. For starfish sperm chromatin with a long nucleosome repeat (222 bp), isolated fibers are significantly wider than those in the nucleus, indicating that the refolding process does not regenerate the native higher-order structure. We also propose that for typical eukaryotic nuclei, the concept that the native state of the (inactive) bulk of the genome is a chromatin fiber with defined architecture be reconsidered.

Ultrastructural preservation of nuclei and chromatin: improvement with low-temperature methods.

The ultrastructure of chromatin has been examined in nuclei prepared by a variety of low-temperature methods. Embedding glutaraldehyde (GA)-fixed nuclei in Lowicryl K4M or K11M following dehydration by the progressive lowering of temperature (PLT) method, or in K11M following spray freezing and freeze substitution (FS), produces chromatin fibres that have, in situ, a diameter close to the in vivo state, and show internal structural details consistent with patterns of nucleosome packing previously observed only in preparations of isolated fibres. This is a temperature-dependent effect; fibres conventionally dehydrated and embedded in Lowicryl at 0 degrees C or in conventional epoxy resin at 60 degrees C have lower and less uniform diameters, and lack internal structural details. Of the techniques used, spray freezing followed by FS resulted in the most notable improvement over conventional methods. Inclusion of GA during FS of rapidly frozen, unfixed nuclei in methanol does not result in cross-linking of nuclear proteins. In acetone, however, cross-linking by GA occurs at -45 degrees C, or at lower temperatures if the water content of the acetone-based FS media is kept deliberately high. Substitution regimes employing GA alone or in combination with uranyl acetate and/or osmium tetroxide do not result in fibre morphologies comparable to either prefixed or unfixed nuclei substituted in additive-free substitution media. Whole fibroblasts show excellent preservation of nuclei and the nuclear/cytoplasmic interface after spray freezing followed by FS and low-temperature embedding.

High-frequency mutations in a plasmid-encoded gas vesicle gene in Halobacterium halobium.

Gas vesicle-deficient mutants of Halobacterium halobium arise spontaneously at high frequency (about 1%). The mutants are readily detected, forming translucent colonies on agar plates in contrast to opaque wild-type colonies. To investigate the mechanism of this mutation, we recently cloned a plasmid-encoded gas vesicle protein gene, gvpA, from H. halobium. In the wild-type NRC-1 strain the gvpA gene is encoded by a multicopy plasmid of approximately 150 kilobase pairs (kb). We have now characterized 18 gas vesicle-deficient mutants and 4 revertants by phenotypic and Southern hybridization analyses. Our results indicate that the mutants fall into three major classes. Class I mutants are partially gas vesicle-deficient (Vac(delta-)) and unstable, giving rise to completely gas vesicle-deficient (Vac(-)) derivatives and Vac(+) revertants at frequencies of 1-5%. The restriction map of the gvpA gene region in class I mutants is unchanged but the gene copy number is reduced compared to the Vac(+) strains. Class II mutants can be either Vac(delta-) or completely Vac(-) but are relatively stable. They contain insertion sequences within or upstream of the gvpA gene. A Vac(-) class II mutant, R1, contains the 1.3-kb insertion sequence, ISH3, within the gvpA gene, whereas four Vac(delta-) class II mutants contain other insertion sequences upstream of the gene. Class III mutants are stable Vac(-) derivatives of either the wild-type or class I mutants and have no detectable copies of the gvpA gene. Based on these results, we discuss the mechanisms of gas vesicle mutations in H. halobium.