Entwicklung einer verbesserten Serodiagnostik und Untersuchung der Eignung eines Proteasom-Inhibitors zur Behandlung der Viszeralen Leishmaniose
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Date
2023-03-09
Authors
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Philipps-Universität Marburg
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Abstract
A reliable VL diagnosis is essential for effective disease control. However, current diagnostic
methods have limitations because they are not sufficiently sensitive and/or specific and may
not identify all cases of VL from different endemic areas or may cross-react with other
infections. The aim of this study was to develop a kinesin-based antigen suitable for highly
sensitive and/or specific strain-independent VL serodiagnosis.
The kinesin proteins of Leishmania are dominant B-cell antigens in patients with VL, i.e.
infected individuals form IgG antibodies against the kinesin. However, the sequence and
structure of kinesins (number of repeats) varies greatly between different isolates. This
explains why rapid serological diagnosis of VL is unreliable, as commercially available tests are
based on rK39 (L. infantum, Brazil, 6.4 repeats) and rK16 (L. donovani, India, 4.0 repeats). In
this work, the recombinant kinesin proteins of different VL strains were generated, which
consisted of an increasing number of repeats. The reactivity of these proteins was studied
using sera from VL patients and it was shown that the number of TR affects the binding affinity
between antigens and antibodies due to the thermodynamic binding kinetics.
The recombinant protein, rKLi8.3, from L. infantum was produced and its performance was
compared with other kinesin-derived antigens (rK39 and rKLO8). The rKLi8.3 antigen was used
for the production of an ELISA and lateral flow POC device that were subsequently tested on
a panel of sera from East-Africa, India and South America. In addition, rKLi8.3 was tested on
dogs suffering from leishmaniasis and showed that rKLi8.3 can also be best used for CVL
diagnostics. In conclusion, the POC test based on rKLi8.3 has a reliable and improved
diagnostic performance that performed equally well in all endemic areas.
Currently available treatments for VL are limited to pentavalent antimony agents, Amp B,
paromomycin, and miltefosine. Treatment with these drugs is lengthy, poorly tolerated, and
sometimes has severe side effects. Therefore, effective, safe, and easily administered drugs
are needed for VL. Recently, a selective proteasome inhibitor (GNF-6702) for parasites of the
Kinetoplastida (T. brucei, T. cruzi, and L. donovani) was described and successfully tested in
animal models.
Additionally, the inhibitor GNF-6702 was tested for its efficacy against different pathogen
strains of VL. Sequential studies of the proteasomal β4- and β5-subunits performed in advance
indicated that the inhibitor might also be effective against the kinetoplastid pathogens of KL,
sleeping sickness, and Chagas disease. The data provided genetic and biochemical validation
of the effectiveness of the inhibitor on the visceral leishmanial proteasome, regardless of their
morphological form (promastigote, axenic, or intracellular amastigote). At the same time, it
showed minimal effects in human cells. Thus, the inhibitor GNF-6702 is a promising
pharmacological candidate for the treatment of VL. Interestingly, the immunoproteasome
inhibitor ONX-0914 showed no effect in promastigote Leishmania, while it was able to
significantly affect the viability of amastigote Leishmania. This suggests a structural alteration
of the proteasome during the transition from promastigote to amastigote stage, which has
never been described before. Further studies in this direction may uncover a new drug target
in the amastigote proteasome.
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Dates
Created: 2022Issued: 2023-03-09Updated: 2023-03-09
Faculty
Medizin
Language
ger
Data types
DoctoralThesis
Keywords
Kinesin antigenPOC diagnosticsverbesserte Sero-DiagnostikPOC-DiagnostikKinesin antigenimproved sero-diagnosticsVisceral leishmaniasisLateral Flow AssayViszerale Leishmanioselateral flow assay
DDC-Numbers
610
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Rouzbeh Mahdavi: Entwicklung einer verbesserten Serodiagnostik und Untersuchung der Eignung eines Proteasom-Inhibitors zur Behandlung der Viszeralen Leishmaniose. : Philipps-Universität Marburg 2023-03-09. DOI: https://doi.org/10.17192/z2023.0135.