In vitro measurements of protein–protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum

Fiedler, Sebastian; Piziorska, Monika A.; Denninger, Viola; Morgunov, Alexey S.; Ilsley, Alison; Malik, Anisa Y; Schneider, Matthias M.; Devenish, Sean R. A.; Meisl, Georg; Aguzzi, Adriano; Fiegler, Heike; Knowles, Tuomas P. J.

doi:10.1101/2020.12.20.422820

Cited by 3 publications

(5 citation statements)

References 24 publications

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“…According to this analysis,

, which, after dividing by the ACE2 molecular mass, equals to

. This dissociation constant is in the range of

values reported by others (1.2–120 nM) for this interaction [ 10 , 38 , 39 , 40 , 41 ]. To calculate the

using ELISA, one has to perform a modified ELISA that also includes an indirect ELISA to find the free concentration of ligand [ 42 , 43 ].…”

Section: Discussionsupporting

confidence: 74%

See 1 more Smart Citation

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Roth

Danielli

2021

Sensors

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Inhibitor screening is an important tool for drug development, especially during the COVID-19 pandemic. The most used in vitro inhibitor screening tool is an enzyme-linked immunosorbent assay (ELISA). However, ELISA-based inhibitor screening is time consuming and has a limited dynamic range. Using fluorescently and magnetically modulated biosensors (MMB), we developed a rapid and sensitive inhibitor screening tool. This study demonstrates its performance by screening small molecules and neutralizing antibodies as potential inhibitors of the interaction between the spike protein 1 (S1) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the angiotensin-converting enzyme 2 (ACE2) receptor. The MMB-based assay is highly sensitive, has minimal non-specific binding, and is much faster than the commonly used ELISA (2 h vs. 7–24 h). We anticipate that our method will lead to a remarkable advance in screening for new drug candidates.

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“…According to this analysis,

, which, after dividing by the ACE2 molecular mass, equals to

. This dissociation constant is in the range of

values reported by others (1.2–120 nM) for this interaction [ 10 , 38 , 39 , 40 , 41 ]. To calculate the

using ELISA, one has to perform a modified ELISA that also includes an indirect ELISA to find the free concentration of ligand [ 42 , 43 ].…”

Section: Discussionsupporting

confidence: 74%

“…To calculate the

using MMB, we fitted the MMB data to one site-total binding model. This model was selected based on a 1:1 binding of S1 to the ACE2 receptor [ 10 , 38 , 39 , 40 ]. The model considers the total binding, including specific and non-specific binding.…”

Section: Discussionmentioning

confidence: 99%

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Roth

Danielli

2021

Sensors

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“…To further investigate, we then assessed cross-reactivity directly via a competition assay based on the principle described in Schneider et al 16 and Fiedler et al 17 ( Figure 3A ). For this approach, a quantity of each serum corresponding to 20 nM antibody binding sites was mixed with 10 nM labeled HCoV-NL63 RBD, to allow at least 80% of the labeled antigen to be bound by antibodies.…”

Section: Resultsmentioning

confidence: 99%

“…Here, we have used microfluidic antibody-affinity profiling (MAAP) 15–17 to quantify the cross-reactivity of antibodies against SARS-CoV-2 S1, S2 and the receptor binding domain (RBD) in nine convalescent COVID-19 patient samples and three pre-pandemic controls, and compared our results to standard ELISA assays. While MAAP matched the ELISA data qualitatively, crucially it enabled the simultaneous and independent determination of antibody affinities and concentrations to reveal interindividual variation of antibody responses to SARS-CoV-2 not detectable by ELISA.…”

Section: Introductionmentioning

confidence: 99%

Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2

Denninger¹,

Meisl

et al. 2021

Preprint

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Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potential beneficial as well as 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 re-activated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Understanding 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 deconvoluted 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 re-activation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS CoV-2.

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“…We then compared these results to the inhibitory effect of antibodies directed against the RBD of the spike protein using our microfluidics-based methodology 37 . We first incubated S1 and fluorescently labelled ACE2; upon complex formation, we incubated this complex with samples of seropositive individuals ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Microfluidic Affinity Profiling reveals a Broad Range of Target Affinities for Anti-SARS-CoV-2 Antibodies in Plasma of COVID-19 Survivors

Schneider

Emmenegger

et al. 2020

Preprint

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The clinical outcome of SARS-CoV-2 infections can range from asymptomatic to lethal, and is thought to be crucially shaped by the quality of the immune response which includes antibody titres and affinity for their targets. Using Microfluidic Antibody Affinity Profiling (MAAP), we determined the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 23 of whom were confirmed to be SARS-CoV-2-positive by PCR testing. We found that dissociation constants (Kd) of anti-RBD antibodies spanned more than two orders of magnitude from 80 pM to 25 nM, despite having similar antibody concentrations. Individual patients showed progressively higher antibody concentrations but constant Kd values, suggesting that affinities did not mature over time. 33 sera showed affinities higher than that of the CoV2 spike for its ACE2 receptor. Accordingly, addition of seropositive plasma to pre-formed spike-ACE2 receptor complexes led to their dissociation. Finally, we observed that the RBD of HKU1, OC43, and SARS-CoV coronaviruses, but not unrelated control proteins, were able to compete substantially with the RBD of SARS-CoV-2 in solution. Therefore, the affinity of total plasma immunoglobulins to SARS-CoV-2 is an indicator of the quality of the immune response to SARS-CoV-2, and may help select the most efficacious samples for therapeutic plasmapheresis.

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In vitro measurements of protein–protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum

Cited by 3 publications

References 24 publications

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2

Microfluidic Affinity Profiling reveals a Broad Range of Target Affinities for Anti-SARS-CoV-2 Antibodies in Plasma of COVID-19 Survivors

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