Midtveisevaluering - Karl Erik Müller

Abstrakt

Leishmaniasis – diagnosis, virulence studies and vaccine development

Karl Erik Müller, Juliana Ide Aoki, Lucile Maria Floeter-Winter, Audun Helge Nerland

Leishmaniasis is one of the most neglected diseases in the world, affecting around 12 million people and over 350 million people are at risk of infection. Leishmania, a vector borne protozoan parasite, causes a spectrum of clinical manifestations in humans – cutaneous, mucosal and visceral. Where the visceral form is lethal if left untreated. No commercial vaccine is available and the current treatment options have unpleasant side effects, and there has also been an emergence of drug resistance. L-Arginine is an essential amino acid for Leishmania. The parasite relies on an external supply as no endogenous synthesis has been reported. The arginine transporter in Leishmania is arginine permease 3 (AAP3), whereas the arginine uptake in mammalian cells is mediated by the cationic amino acid transporter (CAT). The aim of this thesis has been to develop new assays for diagnosis using the AAP3 gene as a target. Further, to use AAP3 as a target in virulence studies comparing parasites propagated in vitro and in vivo. And lastly, to investigate AAP3 as a potential vaccine candidate.

In the first project we developed a real-time-PCR technique targeting AAP3 that was able to detect a wide range of species from different geographical locations (Eurasia, Africa and Americas). The assay has a high sensitivity and can be used for both quantitative and qualitative purposes. The assay, ran in duplex, with a host specific gene, proved also to be successful in measuring the parasite load in mice footpads.

In the second project we are developing a new diagnostic method based on high-resolution melting (HRM) analysis targeting the AAP3-gene. This technique has been applied to other targets, such as Hsp70. This technique can be used for rapid detection of Leishmania, and at the same time be able to identify the species causing leishmaniasis which then can be correlated to the main clinical forms (cutaneous, mucocutaneous and visceral).

In the third project we have compared L.major promastigotes repeatedly propagated in vitro for two years (weekly passages, i.e. >100), with L.major promastigotes that have been repeatedly propagated in vivo in mice (5 passages in mice). We found significant elevated levels of parasites in mice footpads of the in vivo propagated parasites. These parasites also has increased expression of GP63 and resistance to complement lysis. However, no significant difference in phagocytosis by neutrophils, monocytes or macrophages was found. Transcriptomic analysis revealed that for the in vivo versus the in vitro passages parasites, there were 25 up-regulated genes involved in 31 pathways, 79 down-regulated genes involved in 82 pathways, and 28 pathways were common to the up- and down-regulated genes.

In the fourth project we have used a DNA-vaccine approach to investigate if AAP3 can be a vaccine-candidate. Development of infection and parasite load in the footpad of mice will be evaluated alongside humoral and cellular immune responses to the vaccine before and after challenge with L.amazonensis. The first results demonstrate a 50% efficiency and tendency of lower parasite load in the vaccinated group.

To conclude, we have developed and are developing new diagnostic assays for leishmaniasis. Further, we have investigated the differences in virulence for in vitro and in vivo propagated parasites. Lastly, we are evaluating AAP3 as a potential vaccine target where initial results are promising.