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

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.