Cyclic peptides as biological tools : Applications for diagnosis and therapeutics in rheumatoid arthritis and COVID-19

Abstract: The overall aims of the projects included in this thesis were to design, synthesize and evaluate cyclic peptide scaffolds grafted with biological epitopes for their ability to bind antibodies in rheumatoid arthritis and COVID-19 sera. One of the main outcomes of this thesis was the observation that scaffold peptides grafted with target epitopes efficiently neutralized anti-citrullinated protein autoantibodies (ACPA) from rheumatoid arthritis patients. Another main outcome was that grafted cyclic scaffold peptides demonstrated potential for use in COVID-19 diagnostics.In paper I and II, peptides grafted with epitopes from reported ACPA target proteins were designed, synthesized and evaluated for ACPA-neutralizing activity. Cyclic scaffold peptides and linear peptides were compared. In paper I, we demonstrate that the peptides based on the stable scaffold sunflower trypsin inhibitor-1 (SFTI-1) can be used to neutralize ACPA. The peptides s[Cit-Fil](306-323), s[Cit-Vim](60-75) and s[Cit-Vim](2-17) were particularly efficient, and blocked 80-93% with IC50-values ranging from 5.6 -82 µM. Moreover we show that compared to their linear counterparts, these peptides demonstrated enhanced stability in human serum.In paper II, scaffold peptides from the PDP-family (PawS-derived peptides) with different structural properties were evaluated for ACPA-neutralizing activity. By grafting the same ACPA target epitope on three PDP-peptides, their impact on antibody binding could be compared to the linear epitope and to an SFTI-grafted epitope. No substantial difference in antibody binding was contributed by the scaffolds, but a reduction in background was observed for the PDP-peptides when compared to the SFTI-1-peptide. The peptides demonstrated enhanced serum stability in vitro.In Paper III, grafted SFTI-1-scaffold peptides were applied to COVID-19 antibody detection. SFTI-1 was grafted with immunodominant epitopes from SARS-CoV-2 virus spike protein. Epitopes in linear form were synthesized for comparison. A SARS-CoV-2 ELISA was developed and a cohort of positive and negative samples were tested for anti-SARS-CoV-2 reactivity. The peptide (S2_1146-1161_c) stood out as a promising antigen and recognized positive and negative sera with similar capacity as both a lateral flow test and a commercial SARS-CoV-2 ELISA. In paper IV, cyclic scaffold peptides grafted with epitopes from SARS-CoV-2 proved useful in an antibody proximity extension assay, both at detecting and distinguishing COVID-19 positive and negative sera.