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Article ; Online: Protocol to determine antibody affinity and concentration in complex solutions using microfluidic antibody affinity profiling.

Emmenegger, Marc / Worth, Roland / Fiedler, Sebastian / Devenish, Sean R A / Knowles, Tuomas P J / Aguzzi, Adriano

STAR protocols

2023 Volume 4, Issue 1, Page(s) 102095

Abstract: Conventional methods of measuring affinity are limited by artificial immobilization, large sample volumes, and homogeneous solutions. This protocol describes microfluidic antibody affinity profiling on complex human samples in solution to obtain a ... ...

Abstract	Conventional methods of measuring affinity are limited by artificial immobilization, large sample volumes, and homogeneous solutions. This protocol describes microfluidic antibody affinity profiling on complex human samples in solution to obtain a fingerprint reflecting both affinity and active concentration of the target protein. To illustrate the protocol, we analyze the antibody response in SARS-CoV-2 omicron-naïve samples against different SARS-CoV-2 variants of concern. However, the protocol and the technology are amenable to a broad spectrum of biomedical questions. For complete details on the use and execution of this protocol, please refer to Emmenegger et al. (2022),
MeSH term(s)	Humans ; Antibody Affinity ; COVID-19 ; Microfluidics ; SARS-CoV-2
Language	English
Publishing date	2023-02-13
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2023.102095
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Article ; Online: Author Correction: High content genome-wide siRNA screen to investigate the coordination of cell size and RNA production.

Müller, Micha / Avar, Merve / Heinzer, Daniel / Emmenegger, Marc / Aguzzi, Adriano / Pelkmans, Lucas / Berry, Scott

Scientific data

2021 Volume 8, Issue 1, Page(s) 313

Language	English
Publishing date	2021-12-02
Publishing country	England
Document type	Published Erratum
ZDB-ID	2775191-0
ISSN	2052-4463 ; 2052-4463
ISSN (online)	2052-4463
ISSN	2052-4463
DOI	10.1038/s41597-021-01096-2
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Article ; Online: High content genome-wide siRNA screen to investigate the coordination of cell size and RNA production.

Müller, Micha / Avar, Merve / Heinzer, Daniel / Emmenegger, Marc / Aguzzi, Adriano / Pelkmans, Lucas / Berry, Scott

Scientific data

2021 Volume 8, Issue 1, Page(s) 162

Abstract: Coordination of RNA abundance and production rate with cell size has been observed in diverse organisms and cell populations. However, how cells achieve such 'scaling' of transcription with size is unknown. Here we describe a genome-wide siRNA screen to ... ...

Abstract	Coordination of RNA abundance and production rate with cell size has been observed in diverse organisms and cell populations. However, how cells achieve such 'scaling' of transcription with size is unknown. Here we describe a genome-wide siRNA screen to identify regulators of global RNA production rates in HeLa cells. We quantify the single-cell RNA production rate using metabolic pulse-labelling of RNA and subsequent high-content imaging. Our quantitative, single-cell measurements of DNA, nascent RNA, proliferating cell nuclear antigen (PCNA), and total protein, as well as cell morphology and population-context, capture a detailed cellular phenotype. This allows us to account for changes in cell size and cell-cycle distribution (G1/S/G2) in perturbation conditions, which indirectly affect global RNA production. We also take advantage of the subcellular information to distinguish between nascent RNA localised in the nucleolus and nucleoplasm, to approximate ribosomal and non-ribosomal RNA contributions to perturbation phenotypes. Perturbations uncovered through this screen provide a resource for exploring the mechanisms of regulation of global RNA metabolism and its coordination with cellular states.
MeSH term(s)	Cell Cycle ; Cell Nucleolus/metabolism ; Cell Size ; Genome-Wide Association Study ; HeLa Cells ; Humans ; Proliferating Cell Nuclear Antigen/genetics ; Proliferating Cell Nuclear Antigen/metabolism ; RNA/biosynthesis ; RNA/genetics ; RNA, Small Interfering/genetics ; Single-Cell Analysis
Chemical Substances	PCNA protein, human ; Proliferating Cell Nuclear Antigen ; RNA, Small Interfering ; RNA (63231-63-0)
Language	English
Publishing date	2021-06-28
Publishing country	England
Document type	Dataset ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2775191-0
ISSN	2052-4463 ; 2052-4463
ISSN (online)	2052-4463
ISSN	2052-4463
DOI	10.1038/s41597-021-00944-5
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Article ; Online: Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity.

Reimann, Regina R / Puzio, Martina / Rosati, Antonella / Emmenegger, Marc / Schneider, Bernard L / Valdés, Pamela / Huang, Danzhi / Caflisch, Amedeo / Aguzzi, Adriano

Brain pathology (Zurich, Switzerland)

2022 Volume 33, Issue 2, Page(s) e13130

Abstract: The cellular prion protein ... ...

Abstract	The cellular prion protein PrP
MeSH term(s)	Mice ; Animals ; Prion Proteins/genetics ; Neurodegenerative Diseases ; Reverse Genetics ; Prions/genetics ; Antibodies ; Prion Diseases/genetics
Chemical Substances	Prion Proteins ; Prions ; Antibodies
Language	English
Publishing date	2022-11-03
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1051484-3
ISSN	1750-3639 ; 1015-6305
ISSN (online)	1750-3639
ISSN	1015-6305
DOI	10.1111/bpa.13130
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Article ; Online: Antiphospholipid antibodies are enriched post-acute COVID-19 but do not modulate the thrombotic risk.

Emmenegger, Marc / Emmenegger, Vishalini / Shambat, Srikanth Mairpady / Scheier, Thomas C / Gomez-Mejia, Alejandro / Chang, Chun-Chi / Wendel-Garcia, Pedro D / Buehler, Philipp K / Buettner, Thomas / Roggenbuck, Dirk / Brugger, Silvio D / Frauenknecht, Katrin B M

Clinical immunology (Orlando, Fla.)

2023 Volume 257, Page(s) 109845

Abstract: Background and objectives: COVID-19-associated coagulopathy, shown to increase the risk for the occurrence of thromboses and microthromboses, displays phenotypic features of the antiphospholipid syndrome (APS), a prototype antibody-mediated autoimmune ... ...

Abstract	Background and objectives: COVID-19-associated coagulopathy, shown to increase the risk for the occurrence of thromboses and microthromboses, displays phenotypic features of the antiphospholipid syndrome (APS), a prototype antibody-mediated autoimmune disease. Several groups have reported elevated levels of criteria and non-criteria antiphospholipid antibodies (aPL), assumed to cause APS, during acute or post-acute COVID-19. However, disease heterogeneity of COVID-19 is accompanied by heterogeneity in molecular signatures, including aberrant cytokine profiles and an increased occurrence of autoantibodies. Moreover, little is known about the association between autoantibodies and the clinical events. Here, we first aim to characterise the antiphospholipid antibody, anti-SARS-CoV-2 antibody, and the cytokine profiles in a diverse collective of COVID-19 patients (disease severity: asymptomatic to intensive care), using vaccinated individuals and influenza patients as comparisons. We then aim to assess whether the presence of aPL in COVID-19 is associated with an increased incidence of thrombotic events in COVID-19. Methods and results: We conducted anti-SARS-CoV-2 IgG and IgA microELISA and IgG, IgA, and IgM antiphospholipid line immunoassay (LIA) against 10 criteria and non-criteria antigens in 155 plasma samples of 124 individuals, and we measured 16 cytokines and chemokines in 112 plasma samples. We additionally employed clinical and demographic parameters to conduct multivariable regression analyses within multiple paradigms. In line with recent results, we find that IgM autoantibodies against annexin V (AnV), β2-glycoprotein I (β2GPI), and prothrombin (PT) are enriched upon infection with SARS-CoV-2. There was no evidence for seroconversion from IgM to IgG or IgA. PT, β2GPI, and AnV IgM as well as cardiolipin (CL) IgG antiphospholipid levels were significantly elevated in the COVID-19 but not in the influenza or control groups. They were associated predominantly with the strength of the anti-SARS-CoV-2 antibody titres and the major correlate for thromboses was SARS-CoV-2 disease severity. Conclusion: While we have recapitulated previous findings, we conclude that the presence of the aPL, most notably PT, β2GPI, AnV IgM, and CL IgG in COVID-19 are not associated with a higher incidence of thrombotic events.
MeSH term(s)	Humans ; Antibodies, Antiphospholipid ; Influenza, Human ; COVID-19/complications ; SARS-CoV-2 ; Antiphospholipid Syndrome ; Antibodies, Anticardiolipin ; Thrombosis ; beta 2-Glycoprotein I ; Immunoglobulin G ; Prothrombin ; Immunoglobulin A ; Immunoglobulin M ; Cytokines
Chemical Substances	Antibodies, Antiphospholipid ; Antibodies, Anticardiolipin ; beta 2-Glycoprotein I ; Immunoglobulin G ; Prothrombin (9001-26-7) ; Immunoglobulin A ; Immunoglobulin M ; Cytokines
Language	English
Publishing date	2023-11-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1459903-x
ISSN	1521-7035 ; 1521-6616
ISSN (online)	1521-7035
ISSN	1521-6616
DOI	10.1016/j.clim.2023.109845
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Zs.A 880: Show issues

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Article ; Online: Correction: Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins.

Emmenegger, Marc / Kumar, Sreedhar Saseendran / Emmenegger, Vishalini / Malinauskas, Tomas / Buettner, Thomas / Rose, Laura / Schierack, Peter / Sprinzl, Martin F / Sommer, Clemens J / Lackner, Karl J / Aguzzi, Adriano / Roggenbuck, Dirk / Frauenknecht, Katrin B M

PLoS pathogens

2022 Volume 18, Issue 2, Page(s) e1010289

Abstract: This corrects the article DOI: 10.1371/journal.ppat.1010118.]. ...

Abstract	[This corrects the article DOI: 10.1371/journal.ppat.1010118.].
Language	English
Publishing date	2022-02-02
Publishing country	United States
Document type	Published Erratum
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1010289
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Article ; Online: Genome-wide identification of microRNAs regulating the human prion protein.

Pease, Daniel / Scheckel, Claudia / Schaper, Elke / Eckhardt, Valeria / Emmenegger, Marc / Xenarios, Ioannis / Aguzzi, Adriano

Brain pathology (Zurich, Switzerland)

2018 Volume 29, Issue 2, Page(s) 232–244

Abstract: The cellular prion protein ( ... ...

Abstract	The cellular prion protein (PrP
MeSH term(s)	3' Untranslated Regions/genetics ; Alzheimer Disease ; Cell Line ; Genome-Wide Association Study/methods ; Humans ; MicroRNAs/genetics ; MicroRNAs/physiology ; PrPC Proteins/genetics ; PrPC Proteins/metabolism ; Prion Diseases/genetics ; Prion Proteins/genetics ; Prions/genetics ; Protein Binding/genetics
Chemical Substances	3' Untranslated Regions ; MIRN124 microRNA, human ; MicroRNAs ; PrPC Proteins ; Prion Proteins ; Prions
Language	English
Publishing date	2018-12-21
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1051484-3
ISSN	1750-3639 ; 1015-6305
ISSN (online)	1750-3639
ISSN	1015-6305
DOI	10.1111/bpa.12679
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ab Jg. 2022: Lesesaal (EG)

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Article ; Online: Serological fingerprints link antiviral activity of therapeutic antibodies to affinity and concentration.

Fiedler, Sebastian / Devenish, Sean R A / Morgunov, Alexey S / Ilsley, Alison / Ricci, Francesco / Emmenegger, Marc / Kosmoliaptsis, Vasilis / Theel, Elitza S / Mills, John R / Sholukh, Anton M / Aguzzi, Adriano / Iwasaki, Akiko / Lynn, Andrew K / Knowles, Tuomas P J

Scientific reports

2022 Volume 12, Issue 1, Page(s) 19791

Abstract: The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor ...

Abstract	The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor binding domain (RBD) of wild type (A), Delta (B.1.617.2), and Omicron BA.1 SARS-CoV-2 (B.1.1.529.1) spike using microfluidic diffusional sizing (MDS). Four therapeutic mAbs showed strongly reduced affinity to Omicron BA.1 RBD, whereas one (sotrovimab) was less impacted. These affinity reductions correlate with reduced antiviral activities suggesting that affinity could serve as a rapid indicator for activity before time-consuming virus neutralization assays are performed. We also compared the same mAbs to serological fingerprints (affinity and concentration) obtained by MDS of antibodies in sera of 65 convalescent individuals. The affinities of the therapeutic mAbs to wild type and Delta RBD were similar to the serum antibody response, indicating high antiviral activities. For Omicron BA.1 RBD, only sotrovimab retained affinities within the range of the serum antibody response, in agreement with high antiviral activity. These results suggest that serological fingerprints provide a route to evaluating affinity and antiviral activity of mAb drugs and could guide the development of new therapeutics.
MeSH term(s)	Humans ; Neutralization Tests ; Spike Glycoprotein, Coronavirus/chemistry ; Antibodies, Viral ; Viral Envelope Proteins ; Antiviral Agents/pharmacology ; Membrane Glycoproteins/chemistry ; SARS-CoV-2 ; Antibodies, Monoclonal ; COVID-19 Drug Treatment
Chemical Substances	sotrovimab (1MTK0BPN8V) ; Spike Glycoprotein, Coronavirus ; Antibodies, Viral ; Viral Envelope Proteins ; Antiviral Agents ; Membrane Glycoproteins ; Antibodies, Monoclonal ; spike protein, SARS-CoV-2
Language	English
Publishing date	2022-11-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-22214-z
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Article ; Online: Both COVID-19 infection and vaccination induce high-affinity cross-clade responses to SARS-CoV-2 variants.

Emmenegger, Marc / Fiedler, Sebastian / Brugger, Silvio D / Devenish, Sean R A / Morgunov, Alexey S / Ilsley, Alison / Ricci, Francesco / Malik, Anisa Y / Scheier, Thomas / Batkitar, Leyla / Madrigal, Lidia / Rossi, Marco / Meisl, Georg / Lynn, Andrew K / Saleh, Lanja / von Eckardstein, Arnold / Knowles, Tuomas P J / Aguzzi, Adriano

iScience

2022 Volume 25, Issue 8, Page(s) 104766

Abstract: The B.1.1.529 (omicron) variant has rapidly supplanted most other SARS-CoV-2 variants. Using microfluidics-based antibody affinity profiling (MAAP), we have characterized affinity and IgG concentration in the plasma of 39 individuals with multiple ... ...

Abstract	The B.1.1.529 (omicron) variant has rapidly supplanted most other SARS-CoV-2 variants. Using microfluidics-based antibody affinity profiling (MAAP), we have characterized affinity and IgG concentration in the plasma of 39 individuals with multiple trajectories of SARS-CoV-2 infection and/or vaccination. Antibody affinity was similar against the wild-type, delta, and omicron variants (
Language	English
Publishing date	2022-07-16
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2022.104766
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Article ; Online: Microfluidic Antibody Affinity Profiling Reveals the Role of Memory Reactivation and Cross-Reactivity in the Defense Against SARS-CoV-2.

Denninger, Viola / Xu, Catherine K / Meisl, Georg / Morgunov, Alexey S / Fiedler, Sebastian / Ilsley, Alison / Emmenegger, Marc / Malik, Anisa Y / Piziorska, Monika A / Schneider, Matthias M / Devenish, Sean R A / Kosmoliaptsis, Vasilis / Aguzzi, Adriano / Fiegler, Heike / Knowles, Tuomas P J

ACS infectious diseases

2022 Volume 8, Issue 4, Page(s) 790–799

Abstract: Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potentially beneficial and detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of ... ...

Abstract	Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potentially beneficial and detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be reactivated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Determining the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be disentangled by surface-based assays like enzyme-linked immunosorbent assays (ELISAs), which are routinely used to assess cross-reactivity. Here, we have used microfluidic antibody affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high-affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory reactivation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2.
MeSH term(s)	Antibodies, Viral ; Antibody Affinity ; COVID-19 ; Humans ; Microfluidics ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus
Chemical Substances	Antibodies, Viral ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2022-03-30
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2373-8227
ISSN (online)	2373-8227
DOI	10.1021/acsinfecdis.1c00486
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