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Zusammenfassung Masterarbeit Anna Bollinger

SARS-CoV-2 Multiplex Serology of Dried Blood Spot samples and expression of SARS-CoV-2 spike protein in protozoan cell culture

In early 2020, the infectious disease COVID-19 (coronavirus disease 2019) caused
by SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2) emerged
worldwide. Since then, the research on SARS-CoV-2 has become a global priority in
science. In this context, Immunoassays detecting antibodies against specific SARSCoV-2 antigens are used to determine prior exposure and to estimate the current
seroprevalence. The department of Infection and Cancer Epidemiology at the
German Cancer Research Center (DKFZ) uses Multiplex Serology to measure these antibodies in serum samples. To assess reliable seroepidemiological data with this high-throughput platform, a large number of samples is required.
Dried Blood Spot (DBS) cards offer an easy way to collect blood samples on a large scale. The independent preparation of DBS samples by the patients is a major
contribution to generate seroepidemiological data on SARS-CoV-2. Therefore, the
first objective of this thesis was to validate antibody responses to selected SARSCoV-2 antigens of DBS-derived samples against serum samples using Multiplex
Serology. For this purpose, samples from 150 post-infected individuals were used.
Results indicate that antibody responses from DBS are well comparable to those of
serum samples. Their respective quantitative readout, median fluorescence intensity (MFI), correlates very well with a median Pearson’s r of 0.91. A special focus is directed to the receptor-binding-domain of SARS-CoV-2 spike protein subunit 1 (S1RBD) and the SARS-CoV-2 nucleocapsid protein (N). Both antigens performed especially well with MFI values of up to 20,000 and a perfect concordance (Cohen’s kapa of 1.00).
Altogether, results indicate that DBS samples can be used in the future as a simple,
rapid and reliable alternative to serum samples to assess SARS-CoV-2 epidemiology.To study SARS-CoV-2 antibody responses in a large scale, the antigens used in Multiplex Serology also need to be easily available in a large scale and are therefore mainly expressed in Escherichia coli (E. coli). The SARS-CoV-2 spike protein subunit 1 (S1) and S1RBD are highly glycosylated proteins and lose their seroreactivity when expressed in a prokaryotic system. Therefore, the second objective of the thesis was to test an alternative expression system based on Leishmania tarentolae (L. tarentolae) for an in-house expression of the glycosylated proteins S1 and S1RBD.
The protozoan expression system was successfully established. It was, however, not yet possible to express functional S1 and S1RBD due to limitations. Most likely, the Glutathione-S-Transferase (GST)-tag, which was fused to the proteins, prohibited successful protein expression. Since an in-house expression of the glycosylated S1 and S1RBD would still provide significant advantages, the recombinant proteins were re-designed using different affinity tags and will be characterized in future experiments.

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