Rusch, Sebastian Marco. Recombinant expression of "Plasmodium falciparum" erythrocyte membrane protein 1 fragments and serological studies. 2008, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_8157
responsible for all clinical symptoms of malaria. Both children and adults that are
infected with P. falciparum can either have symptoms of variable severity or be
asymptomatic. However, it is mostly young children who suffer from severe
symptoms ranging from severe anaemia to cerebral malaria, and it is mostly adults
from endemic areas who experience comparatively mild episodes with headache
and sometimes fever.
The observed morbidity is largely associated with sequestration of parasitized
erythrocytes (iRBCs) on endothelial cells of host blood capillaries. This
cytoadherence prevents late stage iRBCs from being cleared by the spleen. Instead,
iRBCs bind to various host cell receptors such as CD36, ICAM, or CSA leading to
obstruction of blood vessels, impaired oxygen delivery in affected host organs and
immunological reactions of the affected tissues.
The key mediator of sequestration found is the P. falciparum Erythrocyte Membrane
Protein 1 (PfEMP1). This large parasite derived protein is exported from the parasite
and trafficked through various membranes and through the host cell cytosol until
becoming inserted into the erythrocyte cell membrane. It is located at the interface
between parasite and host immune system, and undergoes antigenic variation.
PfEMP1 is encoded by approximately 60 var genes per haploid genome, and is
expressed in a mutually exclusive manner, i.e. only one gene is expressed at any
one time. As one of its sophisticated immune evasion strategies, the parasite can
switch to another PfEMP1 variant and thus becomes no more recognizable by the
host immune system.
It is believed that protection against severe malaria is the result of the development
of immune responses against various variants of PfEMP1. However, immunity to
malaria is never sterile but instead only reduces parasite density and morbidity. We
have based our work on the hypothesis that not all variants of PfEMP1 are equally
pathogenic i.e. have the same affinity to host cell receptors. We believe that only a
certain subset of PfEMP1 variants is able to confer solid cytoadherence, and
consequently is responsible for severe malaria. Possessing an antibody repertoire
against these specific variants therefore will protect from severe episodes.
In this work we have chosen a multiple approach to generate molecular tools and to
test this hypothesis. Firstly, we elaborated on the generation of pan-specific noncross
reactive PfEMP1 antibodies using both recombinantly expressed domains
both from the molecule’s head structure (NTS domain) and synthetic peptides
corresponding to the semi-conserved intracellular part of PfEMP1 (ATS peptides).
By means of various molecular methods, however, we found that none of the
generated sera recognized full length endogenous PfEMP1 exclusively.
Secondly, we attempted expression of large fragments of PfEMP1 in E.coli to test
the recognition of sera from different malaria cases. At the same time we wanted to
exploit the possibility to express random fragments of PfEMP1 in a bacterial library
to similarly test these sera on. Insuperable obstacles with large recombinant protein
expression forced us to divert our approach towards smaller domains.
For this we isolated var mRNA from samples from several individuals presenting
either with asymptomatic infections or experiencing severe malaria episodes. 14 var
DBL domains were recombinantly expressed in E. coli and used to measure
antibody titers in sera from 100 semi-immune Papua New Guinean adults. The
frequency of recognition (FoR) for these antigens of was assessed and compared
between FoR of DBL domains deriving from severe cases and from asymptomatic
samples. We found that DBL domains deriving from severe cases were significantly
more often recognized by sera from semi immune Papua New Guinean adults than
DBL domains derived from asymptomatic samples. This is indicative for semiimmune
adults not suffering from clinical malaria because being better protected
against parasites expressing “severe” DBL domains of PfEMP1.
We also tested 34 sera from children with asymptomatic infections collected during
a longitudinal study in Tanzania. We selected sera that were collected at two time
points 6 months apart to assess the development and dynamics of antibodies
against those DBL domains. FoR increased significantly over time in these children
but only for DBL domains deriving from severe cases. As these children did not
suffer from clinical episodes between the two sampling dates, these results also
indicate that acquisition of antibodies against “severe” DBL domains is faster and
can confer protection.
In summary, our findings support the notion that development of antibodies against
PfEMP1 variants (in this case against DBL domains) is associated with protection
against severe disease and thus contributes as an important factor to the acquired
clinical immunity to severe malaria. These findings raise hope in the feasibility of a
putative protective vaccine against the major virulence factor PfEMP1.
|Committee Members:||Lingelbach, Klaus and Voss, Till|
|Faculties and Departments:||09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Epidemiology and Public Health (EPH) > Chronic Disease Epidemiology > Genetic Epidemiology of Non-Communicable Diseases (Probst-Hensch)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||141|
|Last Modified:||30 Jun 2016 10:41|
|Deposited On:||13 Feb 2009 16:20|
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