The mucosal surfaces are often the first barriers for infectious agents. Consequently, mucosal immunity is probably one of the most efficient host defense mechanism aimed at by new generation vaccines. The objectives of this project are the development and assessment of new technologies to induce mucosal immune responses, using the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) as the model antigen. Several live and non-living vectors are used to study optimal antigen presentation to the mucosal respiratory and gastro-intestinal surfaces. Immunized animals are then studied for their quantitative and qualitative (antibody isotypes, cytokines) immune responses in conjunction with the protective efficacy against parasite infection in terms of reduction of worm burden, worm fecundity, and egg viability of the schistosome. The live vectors include non-colonizing (Lactococcus lactis), as well as colonizing microorganisms (Bordetella pertussis). Non-living vehicles include liposomes, and biodegradable microparticles.
Role of IgA: (Partner A) The IgG and IgA antibody responses to 28GSTs has been monitored in two vaccination trials involving: -1- Calves immunized with Sb28GST and exposed to Schistosoma matteei and -2- in patas monkeys vaccinated with Sh28GST and challenge with S. haematobium. In both models, the IgA dependent inhibition of GST activity was associated with protective effects: impairment of egg hatching in naturally infected calves and a reduction of urinary egg excretion in monkeys. In parallel, the induction of specific secretory IgA has been obtained in the milk of pigs immunized with Sm28GST. The purification of the different IgA molecular forms and the analysis of their fonctional properties are in progress.
Expression of Sm28GST in L. lactis: (Partner B) A set of four lactococcal strains have been prepared which express either whole Sm28GST, or whole Sm28GST fused to the Cter of TTFC, or two copies of the GST immunodominant region (115-131) fused to TTFC, or two copies of the GST Cter epitope (190-211) fused to TTFC. Immune reponse has been evaluated for each of these expressor strains in mice using either sc, intranasal, or oral route. Whereas the antibody responses were disappointed for the epitope expressor constructs whatever the route of administration used, constructs including the whole Sm28GST induced significant specific immune responses predominantly consisted of IgG1, IgG2a, and included IgA and IgM. Specific IgA were detected in faecal pellets in mice receiving the Sm28GST constructs by nasal or oral route. Protective experiments showed the efficacy of the live vaccine construct.
Expression of Sm28GST in B. pertussis: (Partner C) We determined the FHA secretion domain by site-directed mutagenesis, and confirmed the specificity of the accessory protein FhaC in the secretory machinery of FHA. Construction of a recombinant strain of B. pertussis expressing the parasite antigen has shown the feasibility and the vaccine efficacy of the chosen live vector. The next major achievements has been the construction of genetically attenuated B. pertussis strains and secretion of FHA-Sm28GST hybrid proteins in these strains. FHA-Sm28GST hybrid protein was well secreted and presented at the bacterial surface by a strain deleted to its pertussis toxin gene. Infection in mice resulted in colonisation of the lungs but in absence of pathological effect. Results indicated that nasal administration reulted in the production of significant amounts of specific antibodies and was effective against a parasite challenge in mice.
Microspheres: (Partner D) A multiple emulsion-evaporation solvent technique and spray-drying are the two methods which have been optimalized to produce calibrated microparticles (1-10 mm) containing the Sm28GST from several biodegradable polymers (PLA, PLAGA & PCL) selected on basis of their huydrophobicity. Immune reponse has been evaluated for each of these preparations. The systemic administration of these microparticulate vectors provoked a very significant specific immune response characterized by high serum levels of IgG1, IgG2a and IgG2b. Four oral administrations of these carriers also induced a strong serum immune response which could be delay when PCL was used as carrier. A significant secretory specific IgA production at the level of the respiratory tract was also elicited when Sm28 GST loaded - PLAGA RG505 microparticles were administered.
Liposomes: (Partner E) We developed new formulations of liposomes adapted to mucosal routes and defined the physico-chemical parameters of the lipid-protein relationships. After evaluation of the stability and the avalaibility of different liposome-protein complexes in vivo following oral or nasal administration, we evaluated the immune responses. Oral administration induced a systemic and mucosal immune response with a strong efficacy in terms of protection against parasite infection. The nasal administration in mice indicated a strong efficiency of our constructions in terms of secretory and systemic antibody production with a Th2 like profile, defined by the isotypic and the cytokine profile. Nasal administration of the liposomal constructions in non-human primates induced a prolonged immune response with an significant production of specific IgA in serum. Furthermore, new formulations bearing bacterial adhesins potentially increased the efficacy of the nasal immunisation.
Funding SchemeCSC - Cost-sharing contracts
CB2 1QP Cambridge