Identification and characterization of "Plasmodium falciparum" and "Mycobacterium ulcerans" antigens as potential vaccine components and targets for serological test and molecular typing methods

Abstract

The increase of Plasmodium falciparum’s drug resistance and the resurgence of Mycobacterium ulcerans disease through environmental changes aggravate health problems caused by these pathogens. Buruli ulcer, caused by M. ulcerans, is after tuberculosis and leprosy the third most common mycobacterial human infection and the most poorly understood of these three diseases. It is characterized by chronic, necrotizing ulceration of subcutaneous tissues and the overlying skin. M. ulcerans is a slow-growing mycobacterium which multiplies extracellularly in Buruli ulcer lesions. There is a broad antigenic overlap between mycobacterial species, which complicates the analysis of adaptive immune responses and hampers the development of specific sero-diagnostic tests for M. ulcerans in areas where BCG vaccination has been implemented and tuberculosis is endemic. In an effort to identify immunodominant antigens of M. ulcerans, we have generated panels of monoclonal antibodies from mice immunized with this pathogen. Cross-reactivity studies with other mycobacterial species performed by Western blot and immunofluorescence assays have identified immunodominant epitopes with a limited cross-species distribution (18kDa and the 34-37kDa proteins). In contrast, the majority of antigens were spread widely amongst different mycobacterial species. One set of non-crossreactive monoclonal antibodies recognized an 18kDa protein of M. ulcerans that is associated with the cell-wall fraction, and expressed in Buruli ulcer lesions. The target protein was identified by massspectroscopy as the M. ulcerans orthologue of the M. leprae 18kDa small heat shock protein, which has no orthologues in the genomes of M. bovis and M. tuberculosis. Human anti-18kDa small heat shock protein antibodies were found in the serum of all Buruli ulcer patients tested, but not in sera from Europeans volunteers and only rarely in sera from Africans living in Buruli ulcer non-endemic regions. Reactivity of sera from a large proportion of people living in a Buruli ulcer endemic area and in contact with Buruli ulcer patients indicated that an 18kDa small heat shock protein-based serological test is suitable to detect exposure to M. ulcerans. Since M. ulcerans shows only very limited genetic diversity, standard multi-locus sequence typing of housekeeping genes is not a suitable tool for molecular epidemiological analysis of Buruli ulcer. Among the monoclonal antibodies exhibiting broad inter-species cross-reactivity, one group recognized the M. ulcerans orthologue of mycobacterial laminin-binding protein. DNA sequence analysis demonstrated that the corresponding hupB gene from M. ulcerans isolates of diverse geographical origin exhibited considerable diversity based both on insertional/deletional polymorphism and on single base exchanges. Dominance of non-conservative exchanges was indicative of a diversifying selection pressure. Sequences analysis of a set of such variable genes may develop into a new tool for genetic fingerprinting of isolates. There is great need to identify new malaria vaccine and drug targets. Monoclonal antibodies were used to characterize a novel conserved protein of P. falciparum designated D13. Western blot analysis demonstrated that D13 is stage-specifically expressed during schizogony in asexual blood stages of the parasite. It has a functionally essential role in parasite biology, since anti-D13 monoclonal antibodies have parasite growth inhibitory activity. The D13 protein may represent a suitable target for a malaria vaccine design. Immunofluorescence analysis with monoclonal antibodies specific for glyceraldehydes-3- phosphate dehydrogenase (pfGAPDH) and pfAldolase showed that pfGAPDH and pfAldolase colocalise in early stages of both liver and asexual blood stage parasite development. However, during schizogony, unlike pfAldolase, pfGAPDH was enriched in the apical region of the parasites. In addition, Western blot analyses demonstrate that pfGAPDH is in both the membrane-containing pellet and supernatant fractions. These results have provided evidence that pfGAPDH exerts non-glycolytic function(s) in P. falciparum; including possibly a role in vesicular transport and biogenesis of apical organelles. This data together with the limited amino acid sequence identity with human GAPDH suggest that the pfGAPDH could be a promising safe target for drug treatment.