Oral Immunization Of Mice Research Articles

PLG microparticles stabilised using enteric coating polymers as oral vaccine delivery systems

Novel poly(dl-lactide-co-glycolide) microparticles for oral vaccine delivery were formulated using the enteric polymers Eudragit ® L100-55 and carboxymethylethylcellulose (CMEC) as stabilisers. To serve as a control, microparticles were also produced using the conventional PVA surfactant. In all three cases the antigen, ovalbumin (OVA)-loaded microparticles produced were less than 5 μm in diameter and had a spherical, smooth rounded appearance. The presence of surfactants at the microparticle surface was demonstrated by the surface analysis techniques, XPS and SSIMS. Incubation of microparticles with solutions of pepsin or trypsin led to the removal of a proportion of the antigen associated with all three systems. However, in three CMEC-stabilised microparticle formulations and one of three Eudragit formulations, a high percentage of the associated antigen was protected from removal by a solution of pepsin at pH 1.2 compared with the PVA-stabilised microparticles. In addition, with certain CMEC and Eudragit formulations a degree of protection was also afforded to the associated OVA against removal by trypsin at pH 7.4. Following the incubation of microparticles in simulated gastric fluid a higher percentage of intact antigenic OVA was detected in microparticles stabilised using CMEC than in the PVA - and Eudragit - stabilised formulations. Oral immunisation of mice with OVA-loaded microparticles stabilised using either of the three surfactants led to the induction of specific serum IgG and salivary IgA antibodies. Significantly higher levels of specific salivary IgA antibody to OVA were measured in mice immunised with the CMEC-stabilised microparticles than with the other two formulations. This novel approach in PLG microparticle formulation may have potential in increasing the efficacy of microparticulate systems for the oral administration of vaccines.

The alternative sigma factor, sigmaE, is critically important for the virulence of Salmonella typhimurium.

In Escherichia coli, extracytoplasmic stress is partially controlled by the alternative sigma factor, RpoE (sigmaE). In response to environmental stress or alteration in the protein content of the cell envelope, sigmaE upregulates the expression of a number of genes, including htrA. It has been shown that htrA is required for intramacrophage survival and virulence in Salmonella typhimurium. To investigate whether sigmaE-regulated genes other than htrA are involved in salmonella virulence, we inactivated the rpoE gene of S. typhimurium SL1344 by allelic exchange and compared the phenotype of the mutant (GVB311) in vitro and in vivo with its parent and an isogenic htrA mutant (BRD915). Unlike E. coli, sigmaE is not required for the growth and survival of S. typhimurium at high temperatures. However, GVB311 did display a defect in its ability to utilize carbon sources other than glucose. GVB311 was more sensitive to hydrogen peroxide, superoxide, and antimicrobial peptides than SL1344 and BRD915. Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant was defective in its ability to survive and proliferate in both cell lines. The effect of the rpoE mutation on the intracellular behavior of S. typhimurium was greater than that of the htrA mutation. Both GVB311 and BRD915 were highly attenuated in mice. Neither strain was able to kill mice via the oral route, and the 50% lethal dose (LD50) for both strains via the intravenous (i.v.) route was very high. The i.v. LD50s for SL1344, BRD915, and GVB311 were <10, 5.5 x 10(5), and 1.24 x 10(7) CFU, respectively. Growth in murine tissues after oral and i.v. inoculation was impaired for both the htrA and rpoE mutant, with the latter mutant being more severely affected. Neither mutant was able to translocate successfully from the Peyer's patches to other organs after oral infection or to proliferate in the liver and spleen after i.v. inoculation. However, the htrA mutant efficiently colonized the livers and spleens of mice infected i.v., but the rpoE mutant did not. Previous studies have shown that salmonella htrA mutants are excellent live vaccines. In contrast, oral immunization of mice with GVB311 was unable to protect any of the mice from oral challenge with SL1344. Furthermore, i.v. immunization with a large dose ( approximately 10(6) CFU) of GVB311 protected less than half of the orally challenged mice. Thus, our results indicate that genes in the sigmaE regulon other than htrA play a critical role in the virulence and immunogenicity of S. typhimurium.

Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B

Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma. The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice. UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain. Evaluation of protection against H. pylori was performed in Balb c mice by oral immunization with a single dose of the vaccine strain. Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H. pylori wild type strain and, six weeks later, mice were sacrificed to determine H. pylori infection by detection of urease activity from the antral region of the mouse stomachs. In several independent experiments, we observed 100% infection with H. pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S. typhimurium expressing recombinant UreA and UreB. Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays. These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H. pylori colonization. Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H. pylori infection. In addition, this model will aid to elucidate the effective protection mechanisms against H. pylori in the gastric mucosa.

Detoxification of the Helicobacter pylori cytotoxin.

Treatment of the Helicobacter pylori vacuolating cytotoxin with very low concentrations of formaldehyde resulted in abrogation of toxic activity in both a HeLa cell vacuolation assay and an in vivo assay of gastric epithelial damage. Detoxification had only a minimal effect on the integrity of the oligomeric or monomeric structure. The toxoid retained the ability to bind to target cells and to induce high-titer neutralizing antibodies after immunization of rabbits. Furthermore, oral immunization of mice with the toxoid resulted in protection against infective challenge with mouse-adapted strains of H. pylori. The sensitivity of the toxin to formaldehyde treatment suggests that a few lysine residues in the protein may be essential for toxic activity and that VacA detoxified in this manner may be a potential candidate for inclusion in a vaccine against H. pylori infection and disease.

Gastritis in urease-immunized mice after Helicobacter felis challenge may be due to residual bacteria

BACKGROUND & AIMS: Oral immunization with recombinant Helicobacter pylori urease (rUre) coadministered with a mucosal adjuvant protects mice against challenge with Helicobacter felis. In this study, the duration of protection and gastritis after challenge were characterized at sequential time intervals up to 1 year.METHODS: Outbred Swiss- Webster mice were orally immunized with rUre plus adjuvant and examined for the presence of H. felis infection and leukocyte infiltration into the gastric mucosa.RESULTS: When defined by gastric urease activity, 70%-95% of rUre-immunized mice were protected for between 2 and 57 weeks. Challenge with H. felis increased the inflammatory response in the gastric mucosa of rUre-immunized mice, which also had elevated CD4+ and CD8+ T cells. The CD8+ cells represented a population of gastric intraepithelial cells, which expressed the mucosal alpha E-integrin. Epithelial changes consisting of parietal cell loss and hyperplasia of the epithelium occurred in approximately 20% of the mice. Antimicrobial triple therapy significantly decreased the degree of gastritis and epithelial alteration in the stomach.CONCLUSIONS: These results indicate that oral immunization of mice with rUre produces a long- lasting inhibition of H. felis infection but that residual bacteria may produce a persistent lymphocytic infiltration under these experimental conditions.(Gastroenterology 1997 Oct;113(4):1118-28)

Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.

Oral immunization with recombinant Mycobacterium bovis BCG simian immunodeficiency virus nef induces local and systemic cytotoxic T-lymphocyte responses in mice.

Recombinant live Mycobacterium bovis BCG vectors (rBCG) induce strong cellular and humoral immune responses against various antigens after either systemic or oral immunization of mice. Cytotoxic T-lymphocyte (CTL) responses may contribute to the control of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infections whose portal of entry is the gastrointestinal or genital mucosa. In this study, we immunized BALB/c mice with a recombinant BCG SIV nef and observed its behavior in oropharyngeal and target organ lymphoid tissues. The cellular immune responses, particularly the intestinal intraepithelial and systemic CTL responses, were investigated. The results showed that rBCG SIV nef translocated the oropharyngeal mucosa and intestinal epithelium. It diffused to and persisted in target lymphoid organs. Specific SIV Nef peptide proliferative responses and cytokine production were observed. Strong systemic and mucosal CTL responses were induced. In particular, we demonstrated direct specific anti-Nef CTL in intestinal intraepithelial CD8beta+ T cells. These findings provide evidence that orally administered rBCG SIV nef may contribute to local defenses against viral invasion. Therefore, rBCG SIV nef could be a candidate vaccine to protect against SIV infection and may be used to develop an oral rBCG HIV nef vaccine.

Production and Characterization of Murine IgA Monoclonal Antibodies to the Surface Antigens of Rhesus Rotavirus

Rotavirus is the single most important cause of severe diarrhea in humans and is diffuse in most animal species worldwide, and an understanding of the antigenic properties of the virus is essential to the design of rational vaccine strategies. To better understand the localization of viral epitopes involved in antibody-mediated neutralization of virus infectivity, we have orally immunized mice with live rhesus rotavirus (RRV) and generated a panel of hybridoma cell clones secreting IgA class monoclonal antibodies. A total of 12 neutralizing IgA MAbs to VP4 and VP7 proteins were studied for their epitope specificity and topographical relationships by hemagglutination-inhibition assays, neutralization assays, and competitive-binding assays with previously mapped MAbs. In addition, neutralization-escape virus mutants were selected and gene segments for each variant were cloned and sequenced. Two IgA MAbs were found to be directed to the antigenic region A of the VP7 protein at amino acid 94, and 10 MAbs were directed at the VP8 trypsin cleavage fragment of VP4. Five of the VP4-specific MAbs identified the same neutralization epitope on the RRV VP8 protein, not previously associated with RRV neutralization. All neutralization-escape variants selected by this antibody group contained mutations at amino acids 132–135 of VP4. One IgA MAb selected for a mutation at amino acid 190 of VP4, and the corresponding viral mutant failed to agglutinate erythrocytes. This MAb mapped to an epitope recognized by 2 additional IgA MAbs. These results suggest that oral immunization of mice with RRV elicits an IgA immune response which is predominantly directed toward antigenic determinants on the VP8 portion of VP4. As a consequence, the route of immunization may alter immunodominant neutralization responses elicited to rotavirus.

Oral immunization of mice against candidiasis.

Oral immunization with live Candida albicans evoked antibody- and cell-mediated immune responses in gnotobiotic C.B-17 and BALB/c mice. No deaths or systemic candidiasis of endogenous origin were evident in these C. albicans-colonized (pure culture) mice. Histologic examination showed minimal to no infection of the stomach, esophagus, or tongue by C. albicans. Not only were orally immunized mice more resistant to systemic candidiasis (intravenous challenge) than were germfree (nonimmunized) controls, but immunity could be transferred to susceptible mice with immune spleen cells. Oral immunization elicited a Th1-type response in spleen cells and a Th2 response in Peyer's patch lymphocytes. The alimentary tracts of these orally immunized mice remained chronically colonized with C. albicans in spite of the presence of both antibody- and cell-mediated immune responses to C. albicans.

Oral immunization with the B subunit of the heat-labile enterotoxin of Escherichia coli induces early Th1 and late Th2 cytokine expression in Peyer's patches.

The B subunit of heat-labile toxin (LT-B) from enterotoxigenic Escherichia coli has been shown to be a powerful mucosal immunogen. Oral immunization of mice with LT-B revealed that BALB/c (H-2d) and C57BL/6 (H-2b) mice gave high serum IgG and mucosal IgA responses specific for LT-B. However, ALY (H-2b) mice lacking intestinal Peyer's patches (PP) did not respond to oral LT-B with either serum or mucosal antibodies. These results indicate that PP lymphocytes supported both systemic and mucosal immune responses when the antigen was administered orally. Reverse transcription polymerase chain reaction analyses revealed that PP CD4 T cells expressed early Th1-type (interferon-gamma and interleukin [IL]-2) and late Th2-type (IL-4, -5, and -6) cytokine mRNA. These results suggest that the systemic and mucosal antibody responses induced by LT-B are regulated by a cooperation of PP Th1 and Th2 cells.

Outer membrane proteins of Pseudomonas aeruginosa as vaccine candidates

We tested the ability of the recombinant outer membrane proteins of Pseudomonas aeruginosa to serve as a protective vaccine against this Gram-negative pathogen in the presence of two main pathophysiological events leading to P. aeruginosa sepsis: (i) systemic infection during immunosuppression; and (ii) bacterial translocation. A hybrid vaccine was cloned which combined the protective epitopes of outer membrane protein F (OprF) and outer membrane protein I (OprI). This vaccine proved to be highly protective against an intraperitoneal challenge with P. aeruginosa in immunosuppressed mice. Oral immunization of mice with recombinant OprI expressing Salmonella dublin, induced s-IgA antibodies in the gut mucosa against OprI. These provided protection against translocation of P. aeruginosa in an immunosuppressed mouse model. To test whether OprI is effective in man, recombinant OprI was purified and used for the immunization of human volunteers. Immunization was tolerated well, and no side effects were observed. Antibody titers against OprI were measured in 90% of the volunteers after immunization.

Optimization of live oral Salmonella-HIV-l vaccine vectors for the induction of HIV-specific mucosal and systemic immune responses

Recent evidence suggests that live oral Salmonella-HIV vaccine vectors have the potential to elicit HIV-specific T cell-mediated immunity in both the mucosal and systemic compartments. We are using the mouse-typhoid model to identify Salmonella::HIV vaccine vector constructs that elicit HIV-specific mucosal and systemic immune responses. Oral immunization of mice with a Salmonella strain that expresses recombinant gp120 (rgp120) in the cytoplasm of the vector elicits a modest gpl20-specific T cell proliferation response in the spleen. However, such Salmonella constructs did not stimulate the development of gpl20-specific serum IgG or cytotoxic T lymphocytes (CTLs). Interestingly, the majority of cytoplasmi-cally-expressed rgp120 forms inclusion bodies in Salmonella. We believe that in this form rgpl20 is highly susceptible to protease degradation by the vector. As such, cytoplasmic rgp120 may not persist in the host after vaccination, resulting in the modest immunogenicity of rgp120 in these constructs. To circumvent this problem we constructed Salmonella strains that express rgp120 on the surface of the vector. Preliminary data suggest that surface-expressed rgp120 is significantly more immunogenic in both the mucosal and systemic compartments than cytoplasmic rgp120. These results, therefore, support the proposal that Salmonella vectors will be a safe and inexpensive means for delivery of HIV antigens to, and the dictation of HIV-specific T cells in, the mucosal and systemic compartments.

Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR.

An asd-stabilized plasmid carrying enterotoxigenic Escherichia coli cfaABCE genes was constructed and called pJGX15C-asd+. Expression of colonization factor antigen I (CFA/I) by this plasmid occurs independently of the cfaABCE positive regulator cfaR in attenuated Salmonella delta aro delta asd strain H683 and nonpathogenic laboratory E. coli asd strain chi 6212. Oral immunization of mice with nonpathogenic E. coli chi 6212 (pJGX15C-asd+) does not elicit significant serum or mucosal responses against CFA/I. In contrast, oral immunization with a single dose of attenuated S. typhimurium H683(pJGX15C-asd+) elicits a 10(5)-fold increase in CFA/I-specific serum immunoglobulin G and significant elevation of CFA/I-specific immunoglobulin A-secreting B cells in the lamina propria, mesenteric lymph nodes, and spleen. Thus, only the Salmonella-CFA/I construct effectively delivered CFA/I to the inductive sites of the gut-associated and systemic lymphoid tissues.

BCG-induced mucosal immune responses

The induction of mucosal immune responses is particularly important for protection against diseases for which entry and pathogenesis are clearly related to the mucosal system, such as salmonellosis, AIDS or tuberculosis. We investigated the immune responses in guinea-pigs vaccinated by BCG via the respiratory compared to the intradermal route. The results demonstrate that the aerogenic BCG induced a better activation of broncho-alveolar macrophages and a substantially improved protective effect against a virulent challenge with Mycobacterium tuberculosis. We also use a DNA recombinant BCG expressing LacZ gene to investigate the influence of various routes of administration on the immunogenicity of the β-galactosidase, a foreign antigen expressed by the LacZ-BCG recombinant. Thus, lymph-node proliferative responses, delayed type hypersensitivity and antibody responses specific for β-galactosidase can be produced in guinea-pigs immunized orally, respiratorily and intradermally. The respiratory and especially the oral route of administration produced higher mucosal and systemic immune responses compared with the intradermal route of immunization. Moreover, the oral immunization of mice with recombinant BCG induced IgA responses which can be detected both in sera and in intestinal secretions. In conclusion, BCG recombinants may be of potential use as an adjuvant vaccine.

Oral immunization of mice and swine with an attenuated Salmonella choleraesuis [delta cya-12 delta(crp-cdt)19] mutant containing a recombinant plasmid

Salmonella choleraesuis chi 3781, an attenuated [delta cya-12 delta(crp-cdt)19] mutant, was electroporated with the plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Recombinant S. choleraesuis chi 3781 stably maintained the pBA31-R7 plasmid and continued to express the cloned protein following recovery of the organism from orally inoculated animals. Unlike previous studies using S. typhimurium chi 4064(pBA31-R7), S. choleraesuis chi 3781(pBA31-R7) was able to colonize both the gut mucosa and deep tissues of both BALB/cByJ mice and crossbred swine. Orally inoculated mice developed serum antibodies to both the cloned 31-kDa protein (rBCSP31) and to S. choleraesuis chi 3781 endotoxin. These mice also developed a local intestinal antibody response to Salmonella endotoxin but not to rBCSP31. Similarly, mice inoculated with recombinant S. choleraesuis chi 3781 did not develop a delayed-type hypersensitivity (DTH) footpad response following injection with rBCSP31; however, these mice did respond to S. choleraesuis chi 3781 soluble antigen. Conversely, orally inoculated swine did not develop significant serum or intestinal antibody responses to cloned protein or Salmonella endotoxin, but DTH responses to both cloned protein and S. choleraesuis chi 3781 soluble antigen were strongly positive. The cell-mediated nature of these DTH responses was confirmed by histological examination. Results suggest that S. choleraesuis chi 3781 may be a suitable choice for further studies of vaccine efficacy in swine, especially for diseases which require cell-mediated immunity for resolution.

Biodegradable microparticles for oral immunization

Ovalbumin (OVA) was entrapped in poly(lactide-co-glycolide) microparticles and administered to mice. Following intraperitoneal immunization, the microparticles induced both proliferative T-cell responses and cytotoxic T-cell responses in spleen cells. Following oral immunization, the mean salivary IgA antibody response to microparticles was significantly greater than the response to soluble OVA (p < 0.0001). Serum IgG antibody levels were also significantly greater in the group administered microparticles (p < 0.001). Cholera toxin B subunit was also entrapped in microparticles. Following oral immunization in mice, specific antibody-secreting cells were detected both in the spleens and in the mesenteric lymph nodes.

Specific lung mucosal and systemic immune responses after oral immunization of mice with Salmonella typhimurium aroA, Salmonella typhi Ty21a, and invasive Escherichia coli expressing recombinant pertussis toxin S1 subunit.

Pertussis toxin (PT) is considered an essential protective component for incorporation into new generation vaccines against Bordetella pertussis, the causative agent of whooping cough. Traditionally, antipertussis vaccination has employed an intramuscular route. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunization with live vaccine carrier strains of Salmonella spp. or Escherichia coli. Recombinant S1 subunit of pertussis toxin was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, in the human typhoid vaccine strain Salmonella typhi Ty21a, and in E. coli CAG629 containing the Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness of epithelial cells. Expression of recombinant PT S1 subunit (rPT-S1) did not affect in vitro invasiveness of the tested strains, which retained the ability to adhere to and invade the embryonic human intestinal cell line HI-407. Following oral immunization of mice with the live vaccine strains expressing rPT-S1, immunoglobulin G (IgG), IgA, and IgM responses were monitored. IgG specific to PT was detected in serum samples of mice, while IgG and IgA specific to PT were detected in lung washes after oral immunization with living Salmonella spp. or E. coli (pWR110) expressing rPT-S1. Utilization of live oral vaccines expressing B. pertussis antigens, which stimulate both a systemic and lung mucosal response, may provide an attractive alternative to purified component vaccines against whooping cough.

Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody

Studies were performed to determine whether active immunity against murine rotavirus (EDIM) infection of mice correlated with titers of neutralizing antibody to the challenge virus. Neonatal mice administered either murine or heterologous rotaviruses all developed diarrhea and high titers of serum rotavirus IgG. However, only mice given EDIM, the murine EB, or simian SA11-FEM strains were protected against EDIM infection when challenged 60 days later. Other serotype 3 strains (RRV, SA11-SEM), as well as strains belonging to serotypes 5 and 6 (OSU, NCDV, WC3), were not protective. Serum neutralizing antibody titers to EDIM were almost undetectable after rotavirus infection with any strain and could not, therefore, be correlated with protection. Likewise, intestinal neutralizing antibody titers were extremely low 21 days after EDIM infection, and by 60 days after inoculation, EDIM-infected mice had no greater intestinal neutralizing antibody titers than uninoculated controls. Mice inoculated with SA11-FEM as neonates had much higher serum rotavirus IgG responses than mice inoculated as adults, and only those infected with this virus as neonates were protected. Thus, although immunity to EDIM did not correlate with the presence of neutralizing antibody to EDIM, it did correlate with the overall magnitude of the immune response after inoculation with SA11-FEM.

Amplification of the secretory IgA response to Toxoplasma gondii using cholera toxin

Our study demonstrates that cholera toxin (CT) markedly enhances the intestinal anti-T. gondii antibody response following oral immunisation of mice with a T. gondii sonicate (TSo) and CT. The antibodies induced were mostly IgA and secretory IgA but a small quantity of IgG was also produced. In contrast, no intestinal anti-T. gondii IgM antibodies were detected. Anti-CT IgA antibodies were also present in intestinal secretions but in much lower quantities than the T. gondii-specific IgA. No anti-CT IgG nor IgM antibodies were detected. Western blot analysis showed that CT induced not only an increase of the intensity of the intestinal IgA antibody response to the 30-kDa band but also induced intestinal IgA antibodies against other major T. gondii proteins (p22, and the 28-kDa antigen) as recognised by specific monoclonal antibodies. The amplification of the anti-T. gondii secretory IgA response by means of an appropriate adjuvant may be one major step leading towards an orally induced immune protection against toxoplasmosis.

Amplification of the secretory IgA response toToxoplasma gondiiusing cholera toxin

Our study demonstrates that cholera toxin (CT) markedly enhances the intestinal anti-T. gondii antibody response following oral immunisation of mice with a T. gondii sonicate (TSo) and CT. The antibodies induced were mostly IgA and secretory IgA but a small quantity of IgG was also produced. In contrast, no intestinal anti-T. gondii IgM antibodies were detected. Anti-CT IgA antibodies were also present in intestinal secretions but in much lower quantities than the T. gondii-specific IgA. No anti-CT IgG nor IgM antibodies were detected. Western blot analysis showed that CT induced not only an increase of the intensity of the intestinal IgA antibody response to the 30-kDa band but also induced intestinal IgA antibodies against other major T. gondii proteins (p22, and the 28-kDa antigen) as recognised by specific monoclonal antibodies. The amplification of the anti-T. gondii secretory IgA response by means of an appropriate adjuvant may be one major step leading towards an orally induced immune protection against toxoplasmosis.