Intranasal Cry1Ac protoxin is an effective mucosal and systemic carrier and adjuvant of Streptococcus pneumoniae polysaccharides in mice.

Streptococcus pneumoniae is a major respiratory pathogen in infants, children and the elderly. Available parenteral anti-pneumococcal vaccines based on type-specific capsular polysaccharides (CPSs) are useful in adults but do not elicit protective immunity in infants and young children. To enhance their immunogenicity, pneumococcal CPSs conjugated to proteins are being developed. Mucosal vaccines may induce mucosal and systemic immune responses, but their development has been hampered by the lack of effective, inexpensive innocuous mucosal adjuvants or immunogenic vaccine carriers. We have demonstrated that the recombinant Cry1Ac protoxin from Bacillus thuringiensis is highly immunogenic and has mucosal and systemic adjuvant effects on proteins coadministered in mice. In this work, we evaluated Cry1Ac as a carrier and adjuvant of S. pneumoniae CPS for the induction of mucosal and systemic antibody responses after intranasal and intraperitoneal immunization in mice. Our results demonstrate that intranasal application of pneumococcal polysaccharides either coadministered or conjugated with Cry1Ac induces higher systemic and mucosal specific antibody responses than those elicited by pneumococcal polysaccharides alone. Adjuvant effects of Cry1Ac on polysaccharides may be appropriate for vaccine design.

Differences between the large and small intestine in the immunodominant amoebic proteins recognized by IgG and IgA antibodies in BALB/c mice.

We have previously reported that there are differences in the number of predominant amoebic antigens recognized by serum and small intestinal antibodies induced after local and systemic immunization with glutarldehyde-fixed Entamoeba histolytica trophozoites (GFT) in BALB/c mice, by an immunoblot analysis. Moreover, by enzyme-linked immunosorbent assay (ELISA) analysis, we found differences in the antiamoebic antibody isotype patterns elicited at the large and small intestines. To further characterize the antiamoebic immune response induced in BALB/c mice, after local (oral and rectal) and systemic (intraperitoneal and intramuscular) immunization with GFT, we performed an immunoblot analysis of the amoebic proteins predominantly recognized by immunoglobulins (Ig)G, IgA and IgM in the serum and in the small and large intestines. The present work shows differences between the large and small intestine in the IgG- and IgA-antibody recognition pattern of amoebic proteins, thus confirming and extending our previous findings supporting the compartmentalization of the intestinal immune response. Furthermore, our reported observation that there are differences in the amoebic proteins predominantly recognized by antibodies of different isotypes was extended to the intestines, as some proteins with relative molecular weights of 24-25, 66, 140 kDa are strongly recognized by IgG but not by other antibody isotypes.

Immunodulation of rat serum and mucosal antibody responses to Entamoeba histolytica trophozoites by beta-1,3-glucan and cholera toxin.

Systemic and mucosal and immune responses can be manipulated with immunomodulators. Here we show the modulatory effects of cholera toxin (CT) and beta-1,3-glucan (GLU) on the rat antiamebic serum and fecal antibody responses to one or four intraperitoneal (IP) or intragastric (IG) doses of glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). One IP dose of GFT maximized serum IgM and IgG antiamebic antibodies on days 4 and 9, respectively; CT coadministration increased IgM antibodies, whereas IgG titers increased with CT or GLU; coproantibodies were undetectable after GFT alone or coadministered with GLU, whereas CT coadministration maximized fecal IgA antibodies on day 6. One IG dose of GFT alone increased serum IgM and IgG antibodies 2.5 times and no further increases were detected using GLU, whereas CT doubled serum IgG antibodies; GFT did not affect the coproantibody responses, whereas GLU coadministration maximized IgG coproantibody levels on day 6 and CT increased IgG and IgA coproantibody levels on the same day. On the other hand, four IG doses of GFT alone or with GLU induced tolerance, whereas GFT alone via the IP route increased serum antibodies slightly and GLU coadministration increased serum IgG antibody titers 300-fold. CT coadministration by both routes increased IgA coproantibodies, and simultaneous CT+GLU coadministration induced lower responses than either CT or GLU. Different antiamebic immune responses might therefore be attained through the use of different immunization routes and immunomodulators to induce protective immunity against intestinal or extraintestinal amebiasis.

Intranasal, rectal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis induces compartmentalized serum, intestinal, vaginal and pulmonary immune responses in Balb/c mice.

Recently we discovered that the Cry1Ac protoxin of Bacillus thuringiensis administered to Balb/c mice intraperitoneally (i.p.) or intragastrically is a systemic and intestinal immunogen as potent as cholera toxin. To further characterize the mucosal immunogenicity of Cry1Ac we additionally tried the intranasal (i.n.) and rectal routes and used enzyme-linked immunoassays to determine anti-Cry1Ac antibody responses in the serum as well as in vaginal and tracheobronchial washes and in the fluids of the large and the small intestine. Immunization by the i.p., i.n. and rectal routes induced IgM, IgG and IgA antibodies in all the mucosal surfaces analyzed, but the magnitude and predominant isotype of each response depended on the route used and the mucosal site analyzed. These data extend our findings on the striking mucosal immunogencity of Cry1Ac and provide additional evidence on the compartmentalization of the mucosal immune system.

Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine.

Bacillus thuringiensis (Bt), considered a safe insecticide, produces insecticidal proteins named Cry during sporulation, which possess exceptional immunological properties. In this work using an immunohistochemical test we demonstrated that Cry1Ac protoxin (pCry1Ac) binds to the mucosal surface of the mouse small intestine. Ligand blot assay allowed us to detect, under denaturing conditions, six pCry1Ac-binding polypeptides present in brush border membrane vesicles isolated from the small intestine. Moreover, this protein induced in situ temporal changes in the electrophysiological properties of the mouse jejunum. The data obtained indicate a possible interaction in vivo of Cry proteins with the animal bowel which could induce changes in the physiological status of the intestine.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice.

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins

Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice.

The spore-forming soil bacterium Bacillus thuringiensis produces parasporal inclusion bodies composed by delta-endotoxins also known as Cry proteins, whose resistance to proteolysis, stability in highly alkaline pH and innocuity to vertebrates make them an interesting candidate to carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of Cry1Ac protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the protoxin were administered by intraperitoneal or intragastric route and anti-Cry1Ac antibodies of the major isotypes were determined in serum and intestinal fluids. The two forms of Cry1Ac protoxin administered by intraperitoneal route induced a high systemic antibody response, however, only soluble Cry1Ac induced a mucosal response via intragastric. Serum antibody levels were higher than those induced by cholera toxin. Systemic immune responses were attained with doses of soluble Cry1Ac ranging from 0.1 to 100 microg by both routes, and the maximal effect was obtained with the highest doses. High anti-Cry1Ac IgG antibody levels were detected in the large and small intestine fluids from mice receiving the antigen via i.p. These data indicate that Cry1Ac is a potent systemic and mucosal immunogen.

Bacillus thuringiensis Cry1Ac protoxin is a potent systemic and mucosal adjuvant.

Recently we demonstrated that recombinant Cry1Ac protoxin from Bacillus thuringiensis is a potent systemic and mucosal immunogen. In this study we compared the adjuvant effects of Cry1Ac and cholera toxin (CT) for the hepatitis B surface antigen (HBsAg) and bovine serum albumin (BSA). The antibody responses of intestinal secretions and serum were determined by ELISA in Balb/c mice immunized through the intragastric (IG) or intraperitoneal (IP) routes. When HBsAg was administered via IG, the anti-HBsAg intestinal response was not enhanced by either Cry1Ac or CT, whereas via IP Cry1Ac increased the anti-HBsAg intestinal immunoglobulin (Ig)G response and CT increased the intestinal IgA and IgM responses. Serum anti-BSA antibodies increased when BSA was co-administered with CT or Cry1Ac by both routes. Cholera toxin and Cry1Ac co-administered via IP increased the IgG anti-BSA response in fluid of the large intestine and CT also increased the IgA and IgM responses slightly. When co-administered via IP, CT and Cry1Ac did not affect the IgG anti-BSA response of the small intestine significantly. We conclude that Cry1Ac is a mucosal and systemic adjuvant as potent as CT which enhances mostly serum and intestinal IgG antibody responses, especially at the large intestine, and its effects depend on the route and antigen used. These features make Cry1Ac of potential use as carrier and/or adjuvant in mucosal and parenteral vaccines.

Different antiamebic antibody isotype patterns in the large and small intestine after local and systemic immunization of mice with glutaraldehyde fixed Entamoeba histolytica trophozoites.

We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.

In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli.

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.

A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites.

We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine orange, and by in situ hybridization to trophozoites with a rDNA probe. The EhkO was found in the axenically grown clones A, L6 (strain HMI:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV). Bacteria present in MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments. Autoradiography of metabolically [3H]thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract and examined by light microscopy, exhibited [3H]thymidine incorporation into this structure. DNA was purified from the EhkO and enriched nuclear fractions and analyzed by transmission electron microscopy. The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas nuclear DNA was present mainly as linear molecules and some circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected.

Intragastric immunization of rats with Entamoeba histolytica trophozoites induces cecal mucosal IgE, eosinophilic infiltration, and type I hypersensitivity.

We have investigated the role of IgE in the local immunity of intestinal amebiasis, a parasitic infection known to induce specific antibody-forming cells (AFC) and IgA antibodies in rodents and humans. We found that intragastric immunization of rats with glutaraldehyde-fixed Entamoeba histolytica trophozoites significantly increased antiameba AFC in the Peyer's patches and spleen and that the lamina propria of the cecum from immunized animals was infiltrated by eosinophils armed with IgE antibodies. Morphometric analysis showed that IgE-containing cells and eosinophils were nearly three times more abundant in the cecum of immunized rats. Antigenic challenge with amebal lysates provoked an increase in the short-circuit current and in the transepithelial potential difference in Ussing-chambered cecum preparations from immunized rats. Although eosinophilia and the increase of IgE are common consequences of infection by parasitic worms, our results indicate that local immunity in intestinal amebiasis also involves IgE deposition, eosinophil infiltration, and type I hypersensitivity, which may explain some symptoms of amebic dysentery such as colic, abdominal tension, tenesmus, and bloody stools.

In vivo and in vitro experimental intestinal amebiasis in Mongolian gerbils (Meriones unguiculatus).

One of the main drawbacks of experimental amebiasis is the lack of an adequate animal model for invasive intestinal lesions. Mongolian gerbils are useful because both intestinal and hepatic amebiasis can be produced experimentally with Entamoeba histolytica trophozoites. In this paper we show results obtained with in vivo and in vitro models of intestinal amebiasis in gerbils. We inoculated gerbils intracecally with monoxenic cultures of a highly virulent E. histolytica HM1:IMSS substrain. In the in vivo model an increase in mucus production was observed during the first 6 h of interaction. Microulcerative mucosal lesions appeared at 24-72 h postinoculation. Inflammatory infiltrate and edema of the lamina propria were associated with superficial foci of necrosis. At 96 h the cecal mucosa had an almost normal appearance and live amebas were no longer detected. In the in vitro model, early damage was detected in cecal strips mounted in Ussing chambers as a rapid fall in potential difference, short-circuit current, and transepithelial resistance that correlated with the extent of the microscopic lesions produced. The latter consisted of cellular edema and the appearance of small, translucent vacuoles at the base of epithelial cells. Further damage led to loss of intercellular junctions, destruction of interglandular epithelial cells, and edema of the lamina propria. The present results demonstrate that the gerbil is useful as an experimental model for the analysis of early stages of invasive intestinal amebiasis both in vivo and in vitro.