Phage display-based discovery of cyclic peptides against the broad spectrum bacterial anti-virulence target CsrA

Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broadacting virulence modulators

Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes

Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization

Generation of sequence defined antibodies from universal libraries by phage display has been established over the past three decades as a robust method to cope with the increasing market demand in therapy, diagnostics and research. For applications requiring the bivalent antigen binding and an Fc part for detection, phage display generated single chain Fv (scFv) antibody fragments can rapidly be genetically fused to the Fc moiety of an IgG for the production in eukaryotic cells of antibodies with IgG-like properties. In contrast to conversion of scFv into IgG format, the conversion to scFv-Fc requires only a single cloning step, and provides significantly higher yields in transient cell culture production than IgG. ScFv-Fcs can be effective as neutralizing antibodies in vivo against a panel of pathogens and toxins. However, different scFv fragments are more heterologous in respect of stability than Fab fragments. While some scFv fragments can be made extremely stable, this may change due to few mutations, and is not predictable from the sequence of a newly selected antibody. To mitigate the necessity to assess the stability for every scFv-Fc antibody, we developed a generic lyophilization protocol to improve their shelf life. We compared long-term stability and binding activity of phage display-derived antibodies in the scFv-Fc and IgG format, either stored in liquid or lyophilized state. Conversion of scFv-Fcs into the full IgG format reduced protein degradation and aggregation, but in some cases compromised binding activity. Comparably to IgG conversion, lyophilization of scFv-Fc resulted in the preservation of the antibodies' initial properties after storage, without any drop in affinity for any of the tested antibody clones

DataSheet_1_Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.pdf

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.</p

Human serum from SARS-CoV-2-vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant

Abstract Background: The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omi‑ cron variant was discovered and immediately classifed as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. Methods: All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale ther‑ mophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutraliza‑ tion efcacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vacci‑ nated persons was analyzed. Results: Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were signif‑ cantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. Conclusion: These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicro

Human serum from SARS-CoV-2-vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant

Abstract Background: The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omi‑ cron variant was discovered and immediately classifed as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. Methods: All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale ther‑ mophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutraliza‑ tion efcacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vacci‑ nated persons was analyzed. Results: Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were signif‑ cantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. Conclusion: These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicro

Human serum from SARS-CoV-2-vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant

Abstract Background: The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omi‑ cron variant was discovered and immediately classifed as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. Methods: All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale ther‑ mophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutraliza‑ tion efcacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vacci‑ nated persons was analyzed. Results: Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were signif‑ cantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. Conclusion: These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicro