Mucosal immunization - cluster project | MUCIMM | Project | Results | FP5 | CORDIS

The antigens pET clones obtained from Siena were introduced into a range of E. coli host strains. Under conditions, which facilitated expression of the cloned antigens, the plasmid vectors segregated heavily from the bacterial hosts unless antibiotic selection was maintained. To assess expression in Salmonella strains, NMB 0992 was cloned with an intact signal sequence into a medium copy number plasmid, under the control of the dps promoter (induced during the stationary phase in vitro and within macrophages). The new plasmid was then transformed into E. coli and Salmonella hosts. Once again, the plasmid was lost in vitro in the absence of antibiotic. Western blotting confirmed expression of NMB 0992 in a number of host strains. So, expression of meningococcal antigens in Salmonella appears to present a number of technical challenges in terms of maintaining stable expression of antigen in the absence of antibiotic selection. This would clearly compromise in vivo immunization experiments. More stable expression might be achieved by expression of antigens from the Salmonella chromosome. These data led us to concentrate subsequent efforts on immunization studies with purified meningococcal proteins and mucosal adjuvants. Three immunization experiments were carried out in BALB/c mice, immunized intranasally, using purified meningococcal antigens and a range of mucosal adjuvants (CT, LT, LTR72, LTK63). Qualitative differences were apparent between the immune responses induced following immunization with the three meningococcal antigens selected for study. All induced clear cellular immune responses. However, in contrast with NMB 0992 and NMB 1994, NMB 1985 was a very poor inducer of either local or systemic antibody responses. These results are in close agreement with those obtained by Kingston Mills at the Dublin group. One of the criteria used for selection of the antigens was that they had all been shown to induce bactericidal antibodies. In our studies however, bactericidal antibody production was only observed following immunization with NMB 1994. This may reflect differences between the protocol used for initial screening of proteins carried out in CHIRON (i.p. inoculation in presence of FCA) and the mucosal delivery employed in the experiments described above. LTR72 produced responses which in general were quantitatively similar to, or greater than, those induced by LT. Even at a ten-fold higher dose than that used for LT or LTR72, LTK63 was a far less potent adjuvant. In conclusion, our studies reveals that, although technical problems linked to instable expression of antigens in Salmonella, the all three meningococcal antigens selected for the study induce clear cellular immune responses after mucosal immunization in mice, strengthen the idea that mucosal immunization can be an effective alternative for vaccine delivery. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

We have studied the immunization of healthy volunteers with novel oral attenuated Salmonella typhi and S. typhimurium vaccine strains harbouring defined combined aroC and type III secretion (ssaV) mutations. In particular, the attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 Delta aroC Delta ssaV, designated ZH9) and S. enterica serovar Typhimurium (TML Delta aroC Delta ssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

In a previous study, we found that oral vaccination induces strong B cell responses in the stomach of Helicobacter pylori infected but not of uninfected individuals. In this study, we have evaluated the possibility of inducing gastric immune responses in H pylori infected volunteers by intestinal and gastric immunisation. H pylori infected subjects were given two doses of an inactivated cholera vaccine, either intestinally via an endoscope approximately 30 cm distal to the pylorus sphincter or intragastrically as small droplets applied directly onto the stomach mucosa. Uninfected individuals received the vaccine by standard oral procedure. Vaccine specific antibody secreting cells in antral and duodenal biopsies were detected by the enzyme linked immunospot assay technique before and seven days after the second immunisation. Results show that intestinal immunisations resulted in induction of vaccine specific gastric IgA secreting cells in five of eight volunteers. This immunisation schedule also gave rise to specific duodenal antibody secreting cells in seven of eight individuals. Local gastric immunisation resulted in the induction of specific B cells in the gastric mucosa of four of eight volunteers. Gastric antigen application also resulted in B cell responses in the duodenum in all volunteers. Uninfected volunteers receiving the vaccine perorally responded in the duodenum but not in the stomach. We conclude that H pylori infection increases the ability of the gastric mucosa to serve as an expression site for intestinally induced B cell responses. These findings are of importance when designing a therapeutic H pylori vaccine, and based on our results such a vaccine can be delivered along the whole upper gastrointestinal tract. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

The Gram positive commensal bacterium Streptococcus gordonii was employed as a live delivery system for mucosal immunization. The genetic system that allows the expression of foreign proteins on the bacterial surface is based on the integration of the heterologous gene into the bacterial chromosome downstream of a strong resident promoter. The streptococcal M6 surface protein is employed as a fusion partner to allow the surface expression of heterologous proteins. By using this system it is also possible to simultaneously express two different foreign proteins on the same bacterial surface. This vector is therefore able to co-express a vaccine antigen with an adjuvant molecule. Recombinant strains of S. gordonii developed within the MUCIMM project expressed the meningococcal antigens NadA (NMB1984) and Orf40 (NMB 0992) as single heterologous proteins or co-expressed NadA with the B subunit of LT (LTB). Meningococcal antigens had previously been identified by Chiron as novel and potential vaccine candidates. The expression of the proteins, tested by flow cytometric analysis, was very efficient. Recombinant bacterial strains were employed in an immunization experiment of mice by the intranasal route. BALB/c mice were inoculated at 0, 3, and 6 weeks with 109 CFU of different bacterial strains. The recombinant strains GP1349 and GP1369 were administered alone or mixed with the soluble mucosal adjuvant LTR72, a genetically modified LT-derived molecule (developed by Chiron). This adjuvant was chosen as it was shown to be an effective mucosal adjuvant in previous studies. Recombinant bacteria administered by the mucosal route were extremely efficient in inducing serum antibody response both against NadA and Orf40. The mean value (calculated among 7 mice) of serum NadA-specific IgG concentration was 21.3 mg/ml while anti Orf40 was 6.3 mg/ml. When recombinant bacteria were intranasally administered with the LTR72 mucosal adjuvant, a further increase in serum antigen-specific IgG was detected (320 mg/ml of anti-NadA and 25 mg/ml of anti-Orf40 serum IgG). A significant serum bactericidal activity against N. meningitidis 2996, measured in collaboration with partner CR3, was detected in most mice immunized with recombinant bacteria expressing NadA. A correlation between antigen-specific antibodies titre and bactericidal titre was observed in 57% of mice immunized with GP1349 (4 out of 7 mice) and in 85% of mice immunized with GP1349 mixed with LTR72 (6 out of 7 mice). NadA-specific immune response against was also observed at the local level in nasal washes (NW) and bronchoalveolar lavages (BAL). Antigen-specific IgA production was statistically significant (P = 0.015 and P = 0.045 in NW and BAL respectively) compared to mice immunized with wild type bacteria (with or without LTR72). Local IgA response against Orf40, assessed in both NW and BAL, was not significant. Intranasal immunization was also performed with recombinant bacteria co-expressing NadA and LTB on the bacterial surface (GP1351). The lower result observed compared to mice immunized with GP1349 (both with and without LTR72), is probably due to the lower expression efficiency of the NadA vaccine antigen on the bacterial vector. The expected results were all reached. We have indeed constructed recombinant S. gordonii strains efficiently expressing the meningococcal vaccine candidates NadA and Orf40. Immunization experiments in mice have shown the immunogenicity of the meningococcal antigens expressed on the bacterial surface and delivered by the mucosal route. A high antibody response was detected both locally and systemically. A significant bactericidal activity against N. meningitidis 2996 was assayed by partner CR3 in sera of mice immunized with bacteria expressing NadA and a correlation between NadA-specific IgG titre and bactericidal titre was assayed in a high percentage of animals. As a potential vaccine candidate carried by recombinant S. gordonii, NadA has shown to be more immunogenic (both at the systemic and at the local level) than Orf40. The use of the soluble mucosal adjuvant LTR72 was able to increase the serum antibody response against NadA, even if a high immune response was induced also by recombinant bacteria alone, specially at the mucosal level. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

Preclinical and clinical activities carried out within the Mucimm project have provided the necessary proof of principle that mucosal vaccines are feasible in humans. This successful experience and fruitful model of coordination in the field of mucosal immunity and vaccine development has created the basis to build an Integarted Project within the 6th framework programme focused on the development of mucosally delivered vaccines against HIV and TB which will induce local immunity able to neutralise the pathogens at their port of entry and systemic immunity able to prevent systemic spread of the infection. The possible development of mucosal vaccines against malaria will also be investigated. The Project consists of two major parallel interlinked tracks: - Phase I trials of mucosal immunization using antigens and adjuvants validated in previous preclinical and clinical studies; - Development of new candidate vaccines for mucosal immunization and their comparative selection in animal models. Antigens such as Gag, V2-deleted gp120, Tat of HIV-1, Ag85B/ESAT-6 hybrid molecule of TB, the C-terminal fragment of CS protein from P. berghei and P. falciparum, and adjuvants such as LTK63 and HSP70, will be used in combination for phase I trials to be performed first in Europe and than in Africa. Development of new vaccine candidates will not include antigen discovery but would rather be based on the novel use of adjuvants and delivery systems in combination with promising antigens. Animal models of increasing relevance for human vaccines will be used to compare new candidates and to select those, which should proceed to phase I trials. The final expected result of this Project is to provide candidate mucosal vaccines, with proved safety and immunogenicity in humans, that could subsequently enter the path towards licensure through phase II and III clinical trials. The European Developing Countries Clinical Trials Partnership (EDCTP), which is expected to be soon operative, could be instrumental in exploiting the results obtained in this project by testing mucosal vaccines against these devastating diseases in developing countries. MUVAPRED consortium is composed of 23 partners from 10 counties including one participant form Eastern Europe (Czech Republic) and one from Developing Countries (Guinea) under the coordination of Chiron srl. These participating groups have been carefully chosen for their technical expertise and scientific excellence. So, the most excellent scientists in the field of vaccines, mucosal immunology, tuberculosis, HIV/AIDS, malaria and clinical trials will take part to this project. Thus, this project will offer the opportunity to evaluate at the same time different potential candidates and formulations for mucosal vaccination against HIV, TB and malaria bringing together technologies, molecules, and know-how that otherwise would have had little opportunities to be evaluated in the same experimental (preclinical and/or clinical) context. The unique proposed approach of a centralised comparative testing of the vaccine candidates in animal models of increasing relevance for humans, will assure that new promising vaccine candidates will be efficiently selected. More importantly, a centralised structure will be used to perform the clinical studies with mucosal vaccines in Europe, leading to the creation of a worldwide centre of excellence for clinical studies of mucosal vaccines. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

We have characterised immune response to oral BCG in healthy human volunteers. As part of an integrated program to develop recombinant BCG for oral use (partly funded by MUCIMM) the immune response to a non-recombinant oral BCG Moreau (RdJ) preparation in healthy human volunteers who have previously been intradermally immunised with BCG Glaxo has being evaluated. Fifteen healthy volunteers have to date taken the vaccine. We have shown that this vaccine is well tolerated by healthy British adults when delivered in a single dose. We have used T cell proliferation assays, cytokine assays from the supernatants of stimulated PBMCs, cytokine Elispot responses, and serology, to many different mycobacterial antigens to investigate the immune response. The T cell proliferation assays have shown that the oral BCG Moreau (RdJ) elicited a T cell response in the majority of our volunteers (12/15). With the cytokine response to PPD stimulated PBMCs, we have shown a Th1 response with an increase in IFN-g in the supernatant. Similarly a cytokine Elispot assay has shown a statistically significant increase in the number of cell secreting IFN-g to PPD post vaccination. Serological and skin test cytokine responses have also been measured but not yet analysed. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

In this study we have compared different routes of vaccination for eliciting antibody responses in the human stomach and duodenum. H. pylori infected individuals were immunized either nasally or rectally with a model antigen (cholera toxin B subunit) and immune responses after these routes were compared with responses after oral or intrajejunal vaccination. Specific antibody levels in serum as well as specific antibodies and antibody-secreting cells in biopsies from antrum and duodenum were determined by ELISA and ELISPOT-methods. In contrast to oral vaccination, neither nasal nor rectal vaccination induce significant increases in specific ASC either in antrum or duodenum. Furthermore, when analysing the antibody levels in saponin extracted biopsies, intrajejunal vaccination was superior to both nasal and rectal vaccination in inducing antigen-specific IgA. We conclude that oral vaccination is the optimal route for induction of antigen-specific responses in the stomach and duodenum in humans while nasal or rectal vaccination are relatively ineffective for this purpose. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

The works performed under this part of the project concerned with mechanisms explaining mucosal adjuvanticity of toxin-based immunomodulators. These studies focused on the bacterial toxins to probe the role of B cells and DC as APC in mucosal immune responses. The scientific basis of this work was the notion that toxin-based adjuvants affect the functional status of mucosal APC and hence, will determine whether active immunity or tolerance to co-administered antigens will be induced. For the proposed studies we have used six categories of immunomodulatory molecules: - The enzymatically active holotoxins, - The mutated enzymatically inactive holotoxins (e.g. LTK63), - The partially active mutant holotoxins (e.g. LTR72), - The B cell targeted enzymatically active CTA1-DD, - The enzymatically inactive mutants of this fusion protein and finally - The B-subunits. The central hypothesis entertained in the project was that the vectors which activate B cells or DC will allow co-administered antigen to prime local and systemic immune responses. Conversely those vectors that bind, but do not activate them fully, will enhance the induction of systemic tolerance that normally occurs after mucosal administration of antigen. Hence we have investigated the ability of adjuvant treated APC to stimulate antigen-specific T cells and have explored the effects in vivo. We compared the effects of the intact holotoxins and CTA1-DD with those of the enzymatically inactive mutant holotoxins, such as LTK63, or the B-subunits, to understand to what extent one or several distinctive immunomodulatory pathways exist for these mucosal adjuvants. Using the CTA1-DD fusion protein and its inactive mutants we tested the hypothesis that enzymatic activity in general augments APC function and results in active immune responses, whereas enzymatically inactive molecules may suppress/tolerize immune responses. We have also evaluated the effect of another enzyme, Pertussis toxin S1, which replaced the CTA1 in the fusion protein together with DD, S1-DD. This construct was explored for adjuvant effects and the distinctive and separate enzymatic activity of S1 on Gia was compared with that of CTA1-DD, acting on Gsa. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

Recombinant BCG strains expressing the model antigen chicken ova and the N. meningitidis ORF961 (nadA or NMB1994) encoding gene have been successfully constructed. These strains express the gene from an episomal vector and both Moreau and Pasteur rBCG strains have been obtained and grown as vaccine preparations. Expression cassettes have also been integrated into the BCG chromosome by means of a vector derived from mycobacteriophage Ms6. The integrative rBCG-nadA strain is available in the BCG Pasteur background. The integrative rBCG-nadA strain in the BCG Moreau background is ongoing. The two replicative rBCG-nadA strains (Moreau and Pasteur) and the integrative rBCG-nadA strain (Pasteur) have been sent to Dr David Lewis to perform immunization studies in mice by the oral and intraperitoneal routes. In a first experiment, BALB/c mice were immunized i.p. with 2x109 CFUs of rBCG-Pasteur-(pGIP3) and rBCG-Moreau (pGIP3). Animals were bled on day 63 and day 85 following immunization. Sera were sent to Chiron laboratories to evaluate anti-NadA antibodies produced and to test their bactericidal activity. Data indicate that rBCG Pasteur-nadA is more immunogenic than the BCG-Moreau-nadA strain. Anti-NadA titers were quite low but this could be due to the fact that the mice received only one dose of rBCG. Since bactericidal activity seems to correlate with high anti-NadA titers it is then not surprising that none of the sera tested were bactericidal. Another experiment where mice will receive a second dose of rBCG-nadA is needed. In order to dissect the immune response induced by rBCG, the model antigen ova has been expressed in BCG. Several rBCG-ova strains have been constructed in order to target the heterologous antigen to the cytosol, the cell wall or to secrete the antigen. The constructs (both in replicative and integrative vectors) have been transformed into BCG Pasteur and Moreau background. Work is in progress to localize OVA produced in all strains. Antigen presentation experiments have been performed using CD4 T-cell hybridomas specific for the OVA I-Ad restricted epitope as well as OT2 OVA-TCR transgenic T cells. Bone-marrow derived dendritic cells (BMDC) infected by all rBCG-ova strains do present the epitope to CD4 T-cells in the two systems. However, presentation using rBCG-ova is less efficient than presentation using Salmonella typhimurium ova strains despite comparable in vitro levels of expression. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

As a conclusion of the MUCIMM project, we have organised an Euroconference/Workshop entitled Novel Strategies of Mucosal Immunisation through Exploitation of Mechanisms of Innate Immunity in Pathogen-Host Interaction held in Siena on November 6-9, 2002. This event, supported by the EU Commission aimed at implementing European efforts towards novel vaccination strategies, has represented the most important appointment of all participants of the 5FP involved with mucosal vaccine projects and has paved the way to the 6FP. The purpose of the meeting has been multiple: - To gather the co-ordinators of the best EU-funded projects on vaccine strategies, mucosal immunisation, and innate immunity, in a Euroconference aimed at establishing the state of the art of the European research in the field. - To hold a Workshop on "Innate Immunity and Pathogen-Host Interaction", which included a number of overviews from top scientists and opinion leaders, plus a series of contributions from younger investigators. The Workshop aimed at providing both participating European researchers and the EU Commission with the newest trends and directions of research in the area of mechanisms of innate immunity and their exploitation for the design of new vaccination strategies. - To discuss, in a concluding Round Table, the future trends of mucosal immunisation strategies within the European health policies. - To implement participation of younger investigators through encouraging and training measures during the scientific sessions of the meeting. Participation awards has been available.A special issue of the journal Vaccine has been prepared by participants based on the work presented at the meeting and aimed to describe the state of art of European research in mucosal immunology and vaccinology. This has been published in June 2003. Thus, all journal subscribers worldwide have received the information about the actions of the EU Commission in the fields. Furthermore, copies of that issue of Vaccine has been distributed to all participants at the Global Vaccine Research Forum held in Seoul, South Korea on June 30th - July 2nd 2003 organized by GAVI and WHO. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

The whole-genome sequence of N. meningitidis serogroup B strain MC58 was used to identify novel vaccine candidates. Putative novel antigens were expressed in E. coli as His-tag or GST fusions, purified, and used to immunize mice. Analysis of sera allowed for the identification of proteins that are surface-exposed and able to induce a bactericidal antibody response. Among the novel identified antigens considerable as vaccine candidates against MenB, three proteins were selected as model antigens for these studies: Orf1 (NMB1985), Orf40 (NMB0992), 961 (NMB1994). These antigens have been be used to evaluate the efficacy of different live delivery systems and the potentiality of mucosal vaccination against meningococcus. All the three proteins are: - Surface-exposed in MenB; - Well expressed in E. coli as fusion proteins; - Able to induce a bactericidal antibody response (an important property because it correlates with protection in humans); - Homologous to known bacterial virulence factors; - Surface-localized in E. coli using E. coli as a model system for heterologous bacterial surface-expression (native-expression). Orf1 is a protein of 1457 aa homologous to Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae. In MenB, as well in E. coli expressing the full-length gene, it is exported to the outer membrane, processed and secreted in culture supernatant. Orf40 (591 aa) is homologous to Hsf and Hia adhesins of Haemophilus influenzae. In MenB it is found on the outer membrane as a protein of about 200 kDa (oligomers). In E. coli expressing the full-length gene, Orf40 is surface-localized, expressed as monomer and possibly forms also multimers. 961 (405 aa) is homologous to YadA of enterophatogenic Yersinia, a non-pilus associated adhesin and to UspA2 of Moraxella catarrhalis, involved in serum resistance. The sequence homology is limited to the carboxyl terminal region, while an overall similarity can be observed at the level of the secondary structure. In fact, 961, YadA and UspA2, have a carboxyl terminal membrane anchor formed by four amphipatic beta-strands and internal alpha-helical regions which probably form coiled-coils. In MenB and in E. coli expressing the native form, 961 are localized in the outer membrane and forms very stable oligomers. These genes were expressed in E. coli under the control of T7 promoter. Orf1 and Orf40 were produced and purified as cytoplasmicinsoluble His-tag fusions, while 961 was produced as different forms (GST/His-fusions, untagged protein, domains, cytoplasmic proteins, secreted forms), most of which are able to elicit a bactericidal activity. The three MenB antigens, as well the novel mucosal adjuvants derivatives of LT, LTK63 and LTR72, have been provided, as genes and as purified recombinant proteins, to different consortium partners (Imperial College of Sciences, Institute Pasteur and University of Siena) for expression in Salmonella, BCG and Streptococcus gordonii. More information on the MUCIMM project can be found at http://www.altaweb.it/mucimm/.

We have studied the role of DC in mucosal immunity. In particular, we have established a method for DC and epithelial co-culture in vitro and we have studied the effects of bacterial vectors on DC using both in vitro and in vivo approaches. Penetration of pathogens expressing invasion genes in the gut mucosa is believed to occur mainly through specialized epithelial cells, called M cells, which are located in Peyers patches (PP). However, S. typhimurium deficient in invasion genes encoded by Salmonella Pathogenicity Island 1 (SPI1), are still able to reach the spleen following oral administration, suggesting the existence of an alternative route for bacterial invasion, independent of M-cells. We have been able to show a new mechanism for bacterial uptake in the mucosa tissues, which is mediated by dendritic cells (DC). DC open the tight junctions between epithelial cells, send dendrites outside of the epithelium and directly sample bacteria. Moreover, as DC expresses tight junction proteins, such as occludin, claudin 1 and Zonula occludens 1, the integrity of the epithelial barrier is preserved. With this knowledge we should be able to design bacterial vectors as suitable delivery systems at the mucosal sites. Since new evidence suggests that dendritic cells (DC) orchestrate the two types of mucosal immune responses, immunity and tolerance, we wanted to evaluate the mechanism involved in this regulation. Thus, our aim was to study mucosal dendritic cells and their interaction with the mucosal adjuvant molecule, cholera toxin (CT), and its derivative forms. By the use of antigen conjugated to CT we managed to induce DC maturation in vitro, with up-regulation of different co-stimulatory surface markers. In contrast, using the enzymatically inactive binding moiety of CT, the CTB adjuvant, and the immature form of the DC was preserved. These phenotypically distinct DC stages go along with our in vivo findings, showing that only CT conjugated to the OVA antigen triggered an immune response, while CTB-OVA induced tolerance. The different responses were induced despite equal uptake of antigen coupled to each carrier molecule, emphasizing the importance of enzymatic activity comprised by the holotoxin. Conjugates traced in vivo were found accumulated at the periphery of the B cell follicles, both after CT-OVA as well as CTB-OVA administration, where the main part of OVA carrying cells were CD11c+ i.e. DC. We continued to use these conjugates to target DC at mucosal membranes, herewith controlling the induction of mucosal responses. Hence, by oral feeding of OVA coupled to CT or its B-subunit we were able to target mucosal DC and manipulate their differential stages. Antigen bound to CT induced strong local recruitment of DC to the GALT, in line with high production of specific IgA in the gut mucosa. The influx of DC was most evident in the Peyer´s patches (PP) with a huge amount of cells entering the subepithelial dome. This infiltration into the PP was also found after administering CTB-OVA, although poor gut IgA immunity was detected. Interestingly, both CT- and CTB-OVA conjugates triggered influx of DC to the draining mesenteric lymph node, suggesting migration of immature- as well as mature DC to the lymph nodes. To find out which genes are differentially regulated by CT- and CTB stimuli we have now performed a global gene analysis to analyse the mechanisms involved in tolerance and immunity. Preliminary results are that more than 400 genes in CT treated DC are induced, whereas only 25 are induced using the CTB molecule. These genes will now be further analysed to define their role in vivo. Thus, through the use of distinct immunomodulatory adjuvants, CT and CTB, and their interaction with DC, we are now able to monitor the immune responses in favour of active immunity using the enzymatically active CT-OVA conjugate or tolerance by the use of CTB-OVA conjugate. In addition, studies expanding mucosal DC with flt3L injection have recently been initiated to thoroughly characterize the DC in the gut mucosa and unravel the cellular features that distinguish mucosal IgA response from oral tolerance. More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

Mucosal immunization - cluster project

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