Christof Berberich

The humoral immune response to the kinetoplastid membrane protein-11 in patients with American leishmaniasis and Chagas disease: prevalence of IgG subclasses and mapping of epitopes.

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 > > IgG3 > IgG2 > IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.

Attenuated Toxoplasma gondii ts-4 mutants engineered to express the Leishmania antigen KMP-11 elicit a specific immune response in BALB/c mice

In order to test recombinant Toxoplasma as adjuvant and live vaccine carrier in the infectious disease model of murine experimental leishmaniasis, we engineered the attenuated, temperature-sensitive Toxoplasma gondii strain ts-4 to express the heterologous Leishmania antigen kinetoplastid membrane protein-11 (KMP-11). Transgenic ts-4 clones were obtained which express KMP-11 as cytoplasmatic protein or target it to the secretory pathway of the tachyzoites. Immunization of BALB/c mice with these stably transformed parasites elicited proliferative responses to both T. gondii antigen and recombinant KMP-11. When challenged with Leishmania major, we observed significant protection in animals that had been vaccinated with the KMP-11-expressing ts-4 mutants. The adjuvant attenuated only the onset of the Leishmania infection, but animals were ultimately not able to control the disease. Thus, our findings demonstrate that recombinant Toxoplasma has the potential to serve as an efficient vaccine carrier for cutaneous leishmaniasis. Furthermore, they establish a protective role for the antigen KMP-11 when given in such a vaccine formulation.

Molecular and antigenic characterization of the Leishmania (Viannia) panamensis kinetoplastid membrane protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire encoding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was clone, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. the L. (V) panamensis ORF was subsequently clone in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We proposed that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

The method used to sample ulcers influences the diagnosis of cutaneous leishmaniasis

Before beginning treatment for cutaneous leishmaniasis, parasitological confirmation of the disease is required. The most commonly used diagnostic procedures are microscopy and culture of samples taken from the active edge of the lesion. In this study, we compared the sensitivity of previous diagnostic procedures with the polymerase chain reaction (PCR), using smears taken from the edge of the lesion and its centre. The sensitivity was greater with smears taken from the centre of the lesion, both for microscopical examination (85%) and for PCR (81%), compared to those obtained from the edge of the lesion (69% and 58% respectively). When PCR was carried out on biopsy material from the edge of the lesion the sensitivity was 63%.

The gene encoding the metacyclogenesis-associated transcript Mat-1 is conserved in the genus Leishmania and shows a tendency to form dimers upon protein expression.

Abstract The Leishmania infantum Mat-1 gene – recently described in L. major as a highly stage-specific, metacyclogenesis-associated transcript – has been cloned. The 420-bp Mat-1 coding region is conserved with respect to the L. major gene (82% sequence homology). Analysis of the predicted amino-acid sequence reveals structural motifs showing homology with the class of leucine-zipper transcription factors. Southern-blot hybridization analysis suggests that Mat-1 is a low-copy-number gene, probably consisting of two gene copies. The recombinant Mat-1 protein expressed in fusion with the Escherichia coli maltose-binding protein shows a tendency to form dimers in the presence of the leucine-rich C-terminal domain. Bacteria expressing the Mat-1 open reading frame are highly growth-attenuated and tend to delete or modify the insert, which suggests that expression of Mat-1 is toxic for the bacteria.