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.

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.

Binding and transepithelial transport of immunoglobulins by intestinal M cells: demonstration using monoclonal IgA antibodies against enteric viral proteins.

M cells of intestinal epithelia overlying lymphoid follicles endocytose luminal macromolecules and microorganisms and deliver them to underlying lymphoid tissue. The effect of luminal secretory IgA antibodies on adherence and transepithelial transport of antigens and microorganisms by M cells is unknown. We have studied the interaction of monoclonal IgA antibodies directed against specific enteric viruses, or the hapten trinitrophenyl (TNP), with M cells. To produce monospecific IgA antibodies against mouse mammary tumor virus (MMTV) and reovirus type 1, Peyer's patch cells from mucosally immunized mice were fused with myeloma cells, generating hybridomas that secreted virus-specific IgA antibodies in monomeric and polymeric forms. One of two anti-MMTV IgA antibodies specifically bound the viral surface glycoprotein gp52, and 3 of 10 antireovirus IgA antibodies immunoprecipitated sigma 3 and mu lc surface proteins. 35S-labeled IgA antibodies injected intravenously into rats were recovered in bile as higher molecular weight species, suggesting that secretory component had been added on passage through the liver. Radiolabeled or colloidal gold-conjugated mouse IgA was injected into mouse, rat, and rabbit intestinal loops containing Peyer's patches. Light microscopic autoradiography and EM showed that all IgA antibodies (antivirus or anti-TNP) bound to M cell luminal membranes and were transported in vesicles across M cells. IgA-gold binding was inhibited by excess unlabeled IgA, indicating that binding was specific. IgG-gold also adhered to M cells and excess unlabeled IgG inhibited IgA-gold binding; thus binding was not isotype-specific. Immune complexes consisting of monoclonal anti-TNP IgA and TNP-ferritin adhered selectively to M cell membranes, while TNP-ferritin alone did not. These results suggest that selective adherence of luminal antibody to M cells may facilitate delivery of virus-antibody complexes to mucosal lymphoid tissue, enhancing subsequent secretory immune responses or facilitating viral invasion.

Transepithelial transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS.

This study was designed to determine whether human immunodeficiency virus type 1 (HIV-1) might enter the host by penetrating epithelial barriers through antigen-transporting M cells in lymphoid follicle-associated epithelia. Interaction of HIV-1 with epithelial cells was examined using mucosal explants from Peyer's patches of mice and rabbits. HIV-1 adhered to the luminal membranes of M cells of both species, and was endocytosed and delivered to intraepithelial spaces containing lymphocytes and macrophages. These observations suggest that M cells, which are numerous in the human rectal mucosa, may efficiently deliver HIV-1 to target cells in mucosal lymphoid tissue, and that such transport may contribute to sexual transmission of AIDS.

The therapeutic potential of monoclonal antibodies against respiratory syncytial virus.

Attempts to develop a vaccine against respiratory syncytial virus (RSV), the major cause of lower respiratory tract disease in infants and young children, have been unsuccessful. Passive immunisation with antibody to RSV has been found to be an effective alternative method for prophylaxis. The product currently in use for RSV passive immunisation, a preparation of purified human IgG containing virus-neutralising activity, requires monthly iv. infusions. Monoclonal antibodies (mAbs) are currently under development as an alternative means of treatment that would require lower doses. The first such mAb was recently approved for RSV prophylaxis in the USA. The mucosal delivery of antibodies is also effective and a mAb nose drop treatment for immunoprophylaxis is under development. The potential of passive immunisation for the treatment of existing RSV infections is not clear. Antibody treatment following infection clearly suppresses viral replication but it may not reduce disease once inflammatory processes have been initiated.

Intranasal antibody prophylaxis for protection against viral disease.

For more than a century, antibody has been used for passive parenteral immunization against viral and bacterial pathogens. This approach has been successful for prevention of viral respiratory infection and has led to testing of intranasal or aerosol delivery of antibody to passively immunize the respiratory tract mucosal surface. Mucosal delivery may be advantageous because it allows the antibody to neutralize the virus particles before they initiate infection and because it concentrates the antibody where viral replication takes place. Animal studies have shown the feasibility of passive intranasal immunization against a number of respiratory tract viruses. Development of nasal antibody treatments for humans is under way, and early clinical studies have confirmed that this approach is safe and can be used to prevent respiratory tract disease. Polyclonal human immunoglobulin from pooled plasma preparations can be used to provide broad protection against a number of different pathogens, while monoclonal antibodies or their fragments can be used to target specific viruses.

Cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus.

BACKGROUND: HNK20 is a murine IgA which is currently being investigated in clinical trials against respiratory syncytial virus (RSV) infections in infants and young children. OBJECTIVE: To produce a single chain antibody fragment (scFv) from HNK20 hybridoma cells and assess its functional activities in vitro and in vivo (mouse model). STUDY DESIGN: The V regions of heavy and light chains were cloned and linked by a sequence encoding for (Gly4 Ser)3 and expressed in Escherichia coli. RESULTS: Over 100 mg/l of the HNK20-scFv was produced in shake flasks after induction with isopropyl (beta-D-thiogalactopyranoside (IPTG). ScFv was purified under native conditions on a Ni2+ affinity column and migrated as a single band of 34 kDa on sodium dodecyl sulfate (SDS)-gels. ScFv demonstrated similar affinity as its parent IgA molecule, neutralized RSV in vitro and significantly reduced RSV titers in lungs of mice when administered intranasally shortly before or a day after RSV challenge. CONCLUSION: It is possible that this scFv or its derivatives, when applied by intranasal or pulmonary route, will be useful for treatment of RSV infections in infants and young children.

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.

Intranasal monoclonal immunoglobulin A against respiratory syncytial virus protects against upper and lower respiratory tract infections in mice.

The role of secretory antibody in protection against respiratory syncytial virus (RSV) infection was examined by using monoclonal immunoglobulin A (IgA) antibody for intranasal passive immunization of mice. Eight anti-RSV IgA hybridomas were produced by fusing myeloma cells with lung lymphocytes from RSV-immunized mice. Five IgA antibodies recognized RSV strains of both the A and the B subgroups, and two of these neutralized virus in a plaque reduction assay. Monoclonal IgA antibody HNK20, which bound to F glycoprotein, was most effective, reducing plaques by 50% at a concentration of 0.1 microgram/ml for both subgroup A and subgroup B strains. HNK20 also neutralized all of eight clinical isolates of RSV tested. When delivered intranasally to mice 24 h prior to RSV challenge, HNK20 reduced virus titers in the lungs by nearly 100-fold. Maximal protection occurred at a dose of 0.5 mg/kg of body weight. Significant protection against lung infection was seen when the interval between antibody treatment and challenge was as long as 72 h. HNK20 also decreased virus titers in the nose approximately 10-fold when given 1 h, but not 24 h, before challenge. When mice were treated with HNK20 intranasally 3 days after challenge, viral titers were reduced in the lungs but not the nose. The results indicate that topical application of relatively small amounts of monoclonal IgA can protect against both upper and lower respiratory tract infections caused by RSV.

Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection.

Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses. Mice were immunized i.n. with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant. To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia. Repeated daily i.n. administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces. Once weekly i.n. immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease. When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection.

A role for calcium-activated calmodulin in murine nonspecific cell-mediated cytotoxicity.

The role of Ca++ in mouse nonspecific cell-mediated cytotoxicity was studied using the calcium ionophore A23187, inhibitors of calmodulin activity, and agents which modulate cyclic AMP concentration. A23187 markedly enhanced spleen lymphocyte cytotoxicity against SV3T3 target cells, suggesting that Ca++ influx enhances cytolytic activity. This conclusion was supported by experiments in which verapamil, a Ca++ channel blocker, inhibited normal and ionophore-induced cytotoxicity. A23187 also enhanced cytolysis of YAC-1 cells in a 16-hr 51Cr release assay, but had little effect in the more typical 4-hr assay. Lysis of BHK, a cell line resistant to murine natural cytotoxicity, could not be induced by A23187. However, hamster effector cells, which can lyse xenogeneic target cells, showed increased against either SV3T3 or BHK. This indicates that the effect of the ionophore was not due to nonspecific release of toxic products from the effector cells. Both normal and ionophore-enhanced lysis were also inhibited by the phenothiazines chlorpromazine and trifluoperazine, which block the activity of calmodulin. A more specific calmodulin activity inhibitor, W13, was also shown to profoundly inhibit cytotoxicity induced by A23187. These results suggest that Ca++ acts as a stimulus-response coupler in cell-mediated cytotoxicity, and that calmodulin mediates the effects of the Ca++. Furthermore, cyclic AMP appears to modulate the action of Ca++ since agents which increase cAMP levels reduce the effects of A23187.

Membrane domains and macromolecular transport in intestinal epithelial cells.

Epithelial cell plasma membranes are organized as biochemically and functionally distinct domains and subdomains. We have explored the composition and maintenance of specific membrane regions during endocytosis and transepithelial vesicular transport in specialized cells of the intestinal epithelium. A unique membrane glycoprotein has been identified in apical endocytic subdomains and endosomal tubules of absorptive cells in suckling rat ileum. This endocytic system is involved in sorting and transepithelial transport of peptide growth factors as well as delivery of milk macromolecules to lysosomes. In M cells of follicle-associated epithelium, we have shown that membrane-bound proteins are efficiently transported to a specialized subdomain of basolateral membrane. The apical membranes of these cells bear immunoglobulin binding sites and transport monoclonal IgA antibodies to the basolateral side, where they may interact with cells of the mucosal immune system.

New model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin A from hybridoma tumors protects against Vibrio cholerae infection.

Secretory immunoglobulin A (sIgA) plays a role in defense against Vibrio cholerae and other microorganisms that infect mucosal surfaces, but it is not established whether sIgA alone can prevent disease. We report here a strategy for identifying the antigen specificities of monoclonal sIgA antibodies that are capable of providing such protection. IgA hybridomas were generated from Peyer's patch lymphocytes after oral immunization with V. cholerae Ogawa 395. A clone was selected that produced dimeric monoclonal IgA antibodies directed against an Ogawa-specific lipopolysaccharide carbohydrate antigen exposed on the bacterial surface. Hybridoma cells were used to produce subcutaneous "backpack" tumors in syngeneic mice, resulting in secretion of monoclonal sIgA onto mucosal surfaces. Neonatal mice bearing anti-lipopolysaccharide hybridoma backpack tumors were specifically protected against oral challenge with 100 50% lethal doses of virulent Ogawa 395 organisms. Thus, the IgA hybridoma backpack tumor method identifies protective epitopes in the mucosal system and demonstrates that a single monoclonal sIgA can be sufficient to protect against intestinal disease.

Oral administration of polymeric immunoglobulin A prevents colonization with Vibrio cholerae in neonatal mice.

A simple animal model was used to demonstrate passive protection by immunoglobulin A (IgA) against a mucosal pathogen, Vibrio cholerae. Oral administration of a monoclonal IgA directed against a lipopolysaccharide component of the vibrio protected neonatal mice against oral challenge, as measured by reduced intestinal colonization. A single dose of 0.1 microgram of polymeric monoclonal IgA given 1 h prior to challenge reduced the number of recoverable vibrios by at least 100-fold. An additional dose 3 h before challenge or 1 h after challenge did not enhance protection. A 10-fold-higher concentration of monomeric IgA was required to achieve the same level of protection as that conferred by polymeric IgA. Polymeric IgA digested with trypsin or human duodenal aspirates to lower-molecular-weight fragments retained most of its ability to protect mice against challenge.

Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori.

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively). The mucosal prime-parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.

Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates.

Chimeric yellow fever (YF)-dengue (DEN) viruses (ChimeriVax-DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290-7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [ approximately 7 to 8 log(10), plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log(10) PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax-DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.