Charakterisierung und Identifizierung von immundominanten Bereichen der L3-Chitinase von Onchocerca volvulus
Chemotherapeutic treatment of filarial infections has rendered difficult and still insufficient. Therefore, the identification of potentially protective molecules which can be used for vaccine development is desirable. The chitinase of larvae stage three of Onchocerca volvulus constitutes a promising antigen. Immunological characterization of this protein and the identification of immunodominat regions was performed in this study. The complete sequence (OvL3-chitinase) and the C-terminal end responsible for the binding of chitin (OvL3-CBD) were cloned in an expression vector, overexpressed in E. coli and subsequently purified. Recombinant chitinase was enzymatically active. The OvL3-chitinase and the OvL3-CBD were used for immunization studies using the rodent filaria A. viteae in the animal model M. unguiculatus. No decreased number of adult worms was observed after immunization with OvL3-CBD in the present of Alum as adjuvans, while chitinase together with STP resulted in the reduction the worm burden to 40 % (first trial) and to 17,7 % (second trial). Additional immunization studies using BALB/c-mice were performed with OvL3-chitinase in the absence or the presence of two different adjuvans. Spleen cells isolated from mice immunized with chitinase in the absence of adjuvans were devoid of proliferative capacity after in vitro antigenic restimulation. Antigen-specific IgG1 antibodies were the only subtype detectable in sera from mice. These results suggested that a Th2 response was induced after immunization with chitinase under these conditions. Including STP or Alum in the immunization protocol a polarization of the obtained immune response was found. Immunization with chitinase together with Alum elicited strong IgG1 response followed by slightly increase of IgG2a and IgG2b. Co-administration of chitinase and STP evoked increased IgG2a and IgG2b antibodies in addition to the observed IgG1 response. Good proliferative responses were observed for spleen cells from mice immunized with chitinase in the present of both adjuvans after in vitro restimulation with chitinase. Furthermore stronger T-cell reactivity was found in the group immunized with chitinase/STP. Also chitinase was expressed in Salmonella and oral immunization of mice with this construct enforced the T-cell reactivity, however antigen specific antibodies were undetectable. To further characterize T cell reactivity against OvL3-chitinase T cell epitopes using the Rothbard and Taylor algorithm (1988), Humphreys prediction and the MHC-II-binding motifs from Rammmensee (1995) were identified. All predicted epitopes were synthesized and tested in T-cell proliferation assays. Additional synthetic L3-chitinase-derived overlapping peptides were also used in T-cell proliferation assays. The sensitivity and specificity of the algorithms was evaluated by comparing the predicted epitopes with the epitopes determined by overlapping peptides. Using this approach, prediction based on MHC-II-binding motifs showed the highest sensitivity (0,47) and specificity (0,7). A further increase of the predictive power was obtained by a combining MHC-II-binding motifs and Rothbard and Taylor algorithm, resulting in increased sensitivity (0,67) but lower specificity (0,33). Five immunodominant regions were identified by all algorithm and four of them were confirmed as T-cell epitopes in T-cell assays. PBMCs isolated from patients affected with Onchocerca volvulus from Cameroon were also tested for their reactivity against OvL3-chitinase and the OvL3 CBD in T-cell proliferation assays. After in vitro restimulation with OvL3-chitinase and CBD only marginal T-cell response was observed accompanied by release of Th2-like cytokines such as IL-10 when stimulated with chitinase and IL-4, IL-5 and IL-10 when stimulated with CBD. Strong antibody responses of the IgG4 isotype were detected in the serum of 77% of the patients against chitinase and only in 20% of the patients against CBD.
School:Humboldt-Universität zu Berlin
Source Type:Master's Thesis
Date of Publication:05/08/2000