Webinar Measuring What Really Matters: Safe, Rapid and Variant-Specific Affinity-Based Virus-Neutralization Assays Published on February 9th, 2021 The emergence of more transmissible SARS-CoV-2 mutants and associated concerns about the effects of these mutations on the efficacy of vaccines has highlighted the vital importance of understanding the humoral immune response to SARS-CoV-2. But despite the advances in developing rapid tests to detect active infections, immunoassays have made only limited progress towards establishing a functional understanding of antibody-mediated immunity after infection or vaccination. A major obstacle in obtaining this information has been the lack of tools that quantitively profile the molecular interactions via which neutralizing antibodies interfere with the binding of SARS-CoV-2 to host cells. In our webinar we present a new type of rapid assay that is based on quantifying these protein interactions and provides functional insights equivalent to the gold standard cell-based neutralization assay by measuring the affinity, concentration, and neutralization potential of antibodies against the SARS-CoV-2 spike protein directly in serum. The webinar aims to cover: Why quantitative information on protein interactions is key to provide actionable insights on the potency of the immune response to SARS-CoV-2 What are the drawbacks of the “gold standard” cell-based virus neutralization tests and why there is a need for a safe, rapid and easy-to use test How our technology is uniquely placed to rapidly assess the immune response to SARS-CoV-2 directly in serum How our novel affinity-based virus-neutralization assays could support the development of new vaccines and therapeutic antibodies, the investigation of new processes for tracking of functional immunity and vaccine escape and research into improvements in donor selection for convalescent plasma therapy You can find more information on the work we are doing on COVID-19 here. About the Speaker Sebastian Fiedler (PhD) Lead Application Scientist – Life Sciences Sebastian Fiedler is a Lead Application Scientist – Life Sciences, at Fluidic Analytics in Cambridge (UK). Before joining Fluidic Analytics, he helped developing antibody therapeutics at the CRUK–AstraZeneca Antibody Alliance Laboratory, studied GPCR–lipid interactions during his postdoc at the University of Toronto and, during his PhD at the University of Kaiserslautern and at the Leibniz Institute of Molecular Pharmacology in Berlin he investigated how membrane proteins gain and retain their structures. Find out more about Sebastian here. Transcript Introduction Haris: Welcome and thank you all for attending today’s webinar on how Fluidic Analytics’ can provide entirely new ways to quantify the functional immune response to SARS-COV-2. Before we begin just a couple of housekeeping notes. After the presentation there will be a Q&A with Sebastian, but all attendees are encouraged to submit questions through the chat box during the webinar. We are recording this webinar and it will be available to stream on our website so that you can revisit it at your convenience. So, without further ado, it is my pleasure to introduce to you our speaker today, Dr Sebastian Fiedler, who is the Lead Application Scientist for Life Sciences at Fluidic Analytics. Sebastian was part of the team that developed this affinity-based approach to measure and quantify neutralizing antibodies and I know he has a lot of new information he’s keen to share. So, I will now hand it over to Sebastian, take it away. Sebastian: Thank you very much Haris for that kind introduction. I would also like to welcome our listeners and thank everyone for joining us for our webinar on “Measuring what really matters”. Today, I will describe the development of safe, rapid and variant-specific affinity-based virus-neutralization assays to profile the functional immunity to SARS-CoV-2 directly in serum. In light of the ongoing pandemic, it has never been more important to understand the immune response on a functional level. Even though vaccines are now increasingly available, new variants of SARS-CoV-2 have appeared all over the world, and the key question remains as to what level and for how long natural and vaccine-induced immunities will last. In today’s webinar, we will show you the latest developments and present an entirely new affinity-based approach of quantifying the neutralization potential of anti-SARS-CoV-2 antibodies in convalescent serum. Background As most of you will be aware, the ever-increasing emergence of new mutant variants of SARS-CoV-2, together with concerns about the effects of these mutations on the efficacy of vaccines and therapeutics has underscored the necessity of understanding the antibody response to SARS-CoV-2. The ability to directly and rapidly quantify the interactions between antibodies and virus proteins is crucially important to understand how functional immunity varies over time, and in response to these new mutations. To date, however, a major impediment to obtaining functional, actionable insights into the immune response to this virus has been the lack of tools that quantitively profile the molecular interactions whereby neutralizing antibodies interfere with the binding of SARS-CoV-2 to host cells. I am very excited to present today our most recent data on the development of safe, rapid and variant specific affinity-based virus-neutralization assays using the quantitative analysis of the interactions between the SARS-CoV-2 spike protein and the human ACE2 host cell receptor. Cell-Based Neutralization Assays Currently, testing an individual’s functional immune status is routinely performed using a cell culture-based virus neutralization assay, in which live cells get infected with viruses or pseudoviruses in the presence of different dilutions of patient serum or plasma. The highest plasma dilution that still prevents viral infection and growth, is considered the neutralizing antibody titer. This approach is well established, provides results at high levels of sensitivity, selectivity, and accuracy, and provides extremely valuable information on people’s immune response which is why it is also deemed as the Gold Standard. However, this type of test provides a titre-only result. The information obtained about the immune status of an individual is qualitative rather than quantitative as antibody concentrations and affinities cannot be independently reported. This, however, can have important consequences. Should an antibody titer change over time it will not be clear whether the titre change is driven by a change of the antibody concentration, or by a change in the affinity that the antibodies have for the viral antigens. Other drawbacks of this test, in addition to the safety regulations required when working with live viruses, are that this type of assay takes several days from sample to result and has poor reproducibility across labs. It is therefore not surprising that cell-based virus-neutralization assays got quickly overwhelmed by the speed and scale posed by the COVID-19 pandemic. Fluidity One-W Serum So, what if we could do something differently here? And had an assay that could quantify protein-protein interactions directly in serum or plasma to determine functional immunity? We at Fluidic Analytics have developed the Fluidity One-W Serum, a microfluidic instrument that enables quantitative analyses of protein interactions directly in solution and in their native environment. With the Fluidity One-W Serum, we can determine the size, concentration and affinity of proteins and protein complexes. As the analysis is based on the measurement of robust equilibrium binding in solution, protein interactions, like for instance antigen/antibody interactions, can be monitored directly in complex backgrounds such as serum without the need to analyze complex binding kinetics. Also, the limitations often seen with surface-based technologies, such as false-positive results or other artefacts due to non-specific binding, are negligible with our technology. Microfluidic Diffusional Sizing (MDS) The underlying technology of our Fluidity One-W Serum instrument is microfluidic diffusional sizing, which takes place on a microfluidic chip that is inserted into the instrument, as seen in the top right corner of the screen. For the sizing experiment, a stream of fluorescently labeled protein is introduced into the diffusion chamber alongside an auxiliary stream. The streams flow in parallel with no active, convective mixing. So, the only way protein can migrate from one stream to the other is by diffusion. Small peptides and proteins diffuse rapidly, large proteins and aggregates slowly. At the end of the diffusion chamber, the streams are split. The quantity of protein in each stream is determined by the fluorescence from the label. The ratio of fluorescence between the two streams gives us the hydrodynamic radius, which is used to determine the size, concentration and affinity of proteins and their interaction partners. As only the labeled species (this can either be a protein or protein complex) gets detected, this approach is ideally suited for the quantification of protein-protein interactions not only in buffer, but more importantly, in complex solutions like for instance serum. It’s also worth mentioning that as the technology is based on microfluidics, only small amounts of serum are required per data point, making this technology highly accessible for even small sample volumes. Qualitative Affinity-Based Virus-Neutralization Assay So, driven by the need for a new type of functional virus neutralization assay, we have developed a rapid and safe, affinity-based receptor competition assay to replace the current Gold Standard cell-based tests. Our assay is based on an ACE2/spike/antibody competition experiment in which we compare the measured sizes of unbound ACE2 with the size of the ACE2/spike complex in the presence and in the absence of antibodies. A neutralizing antibody will compete with ACE2 for spike binding, reduce the fraction of ACE2 that is bound to spike and lead to a reduction of size to the level of unbound ACE2. By contrast, a non-neutralizing antibody will leave the ACE2/spike complex intact and have no impact on the measured size. This assay is safe and experimentally easy to perform as it does not require the handling of viruses. It gives clear and easy to interpret yes/no results, and as it relies solely on the affinity and concentration of the neutralizing antibodies in a patient’s blood, the results obtained with our assay are reproducible between different labs. Most importantly, our assay is significantly quicker than a cell-based virus-neutralization test with a turnaround time of less than 2 hours compared to ~5 days. Qualitative Assay Performance Compared to Gold Standard Cell-Based Assay on COVID-19 Patients We have used this approach in collaboration with the University Hospital of Zurich in Switzerland to test 37 individuals using both our qualitative, affinity-based neutralization assay as well as the gold standard cell-based virus neutralization assay. Comparing the results with the cell-based assay, with this patient cohort we obtained 94% sensitivity, 83% specificity and an accuracy of 93%, with an AUC of 0.88. These results clearly show that our assay performs at the level of the current Gold Standard assay, but can be performed in a much shorter time scale Rapid Qualitative Virus Neutralization Assay Although we observed high sensitivity and specificity values with the assay, the test was reliant on an upfront knowledge of the concentration of anti-spike antibodies in patient serum. To obtain the correct antibody concentration, independent experiments with various serum dilution steps will have to be carried out prior to performing the assay. To overcome these limitations and to reduce the turnaround time even further, we optimized the assay by utilizing minimally diluted serum. This way, we make use of the full amount of antibodies that is present in a sample. Crucially, the assay can now be used without any prior knowledge of the concentration of anti-spike antibodies and has a reduced turnaround time of less than 1 hour. As we can see on the graph on the right-hand side, the optimized assay gives similar results as seen on the previous slide. Neutralizing antibodies lead to sizes at the level of unbound ACE2, while non-neutralizing antibodies result in sizes that are similar to the ACE2/spike complex or larger. Quantitative Affinity-Based Virus-Neutralization Assay Our qualitative affinity-based virus neutralization assay provides clear information about the presence or absence of virus-neutralizing antibodies within an hour. However, for vaccine or antibody therapy development, or for the selection of suitable donors for convalescent plasma therapy, this information is simply not sufficient, and additional knowledge about the potency of the immune response is vital. A major obstacle in obtaining this information, however, has been the lack of tools that quantitively profile the molecular interactions via which neutralizing antibodies interfere with the binding of SARS-CoV-2 to host cells. We have used the unique capabilities of the Fluidity One-W Serum platform to measure these interactions directly in serum and developed an affinity-based receptor competition assay that not only detects the presence of neutralizing antibodies but also provides a true quantitative measure of their neutralizing potency. To this end, we employed the same principles as outlined in the qualitative assay by mixing labelled ACE2 and spike protein with patient serum. This approach leads to a decrease in the measured size when antibodies bind to spike and outcompete the ACE2 receptor. However, this time, we add different concentrations of patient serum to the ACE2/spike mixture to generate an inhibition curve and determine the Kd values as well as the concentration of neutralizing antibodies in the patient serum. Titrated ACE2/Spike Interactions Quantified—Proof of Concept To demonstrate the principle of the assay, we analyzed recombinant proteins added to pre-pandemic, COVID-19 negative human serum at a concentration of 90%. We first measured the affinities of ACE2 to the SARS-CoV-2 spike S1 domain as well as the affinity of the S1 domain to a monoclonal neutralizing antibody. This served to obtain the KD values of the two independent interactions that underly the antibody-mediated inhibition. For the ACE2/spike binding, we obtained a KD value of 7 nM, and for the antibody/spike binding the KD was 5.1 nM. We then predicted the inhibition curve of the antibody by using the two KD values of ACE2/spike binding and S1/antibody binding, as shown by the red circles in the third panel. If the antibody targets the receptor binding site of S1 and competes with ACE2 for binding, the experimental data should follow the predicted curve, which it indeed does, as shown by the black circles. All three curves can then be analyzed globally to check if the two KD values measured for the two individual interactions indeed define the shape of the inhibition curve. In the case of the neutralizing antibody chosen here, this generated very similar KD values (8.4 nM for ACE2/spike and 5.1 nM for antibody/spike binding) as obtained for the individual analyses shown in the two first graphs on the left-hand slide. This clearly demonstrates that the antibody chosen for this experiment has a competitive mode of action and that its neutralization potency can be predicted by the binding energies (in other words, the KD values) of the underlying binary interactions. The neutralization potency of this monoclonal antibody can then be calculated by determining the ratio of the KD value for spike binding to ACE2 and the KD value for spike binding to antibody. In our case this value equals 1.6. I will explain how this KD ratio can be interpreted on the next slide. As mentioned before, the two competing reactions in antibody-mediated inhibition are the binding of viral spike protein to ACE2 and the binding of antibodies to the spike protein. In order to effectively compete with the ACE2 binding, the KD value of the antibody binding to spike should be lower than the KD value of ACE2 binding to spike. By calculating the ratio of the two KD values, serum samples can be ranked based on their neutralization potency. In this hypothetical example, the first antibody binds 10x more spike protein than ACE2 receptor and is therefore, quite potent. In contrast, antibody three binds even less spike protein than the ACE2 receptor and is therefore considerably less potent. Quantification of ACE2/Spike Interactions in COVID-19 Patient Serum To show that this also holds true in patients, we performed the same experiments using two convalescent serum samples. The panels on the left-hand side for each of the two patient samples show binding curves in which three different concentrations of labeled S1 were titrated with convalescent serum. The panels on the right-hand side show competition assays in which complexes composed of labeled ACE2 and unlabeled spike S1 were titrated with convalescent serum. Both sets of data were analyzed globally using KD values of antibody/spike binding and concentration of antibodies as shared fitting parameters. The KD value for spike binding to ACE2 was known to us from the previous experiments and was used as a constant parameter. We can see that both samples contain anti-S1 antibodies with KD values in the sub nanomolar range; 0.8 nM for patient A and <0.5 nM for patient B. However, patient A had approximately five times more antibodies than patient B with a concentration of 55 nM vs 9.5 nM. Calculating the ratio of the ACE2 KD and the antibody KD shows that both patients have potent neutralizing antibodies with KD ratios close to or even higher than 10. As you can see from the data, this approach provides comprehensive and—importantly—quantitative information of the functional immune response to SARS-CoV-2. Future Platform Potential Now, at the moment both the affinity and concentration binding curve as well as the receptor binding competition assay are run as separate experiments. As a brief outlook though, I just wanted to show you that we are currently working on combining these two assays into one single experiment by measuring neutralization curves at different concentrations of labeled ACE2. This approach will provide affinity and concentration of antibodies and their neutralization potential in one single assay with as little as 150 µL of serum per sample. So watch out for that, we will put the data online as soon as we have finished the assay development work. Neutralization Assay Types Before I summarize this presentation, I just wanted to reiterate that compared to the gold standard cell-based virus neutralization assay, our assays provide you with the flexibility to address the demand for a safe, rapid and reproducible test, that is easy to use and interpret. Our approach provides exceptionally granular information about the functional immunity of a COVID-19 patient or vaccinated individual by determining antibody affinity, concentration and neutralizing potency. Summary In summary, as you have hopefully seen from our data—measuring what really matters is of vital importance to understand the functional immune response to SARS-CoV-2. Based on measurements performed directly in serum, which enabled us to quantitively profile the molecular interactions via which neutralizing antibodies interfere with the binding of SARS-CoV-2 to host cells, we were able to develop rapid and safe affinity-based neutralization assays that enable decision-making based on measuring the functional immunity. This applies to the evaluation of vaccine efficacy against different mutant variants, the identification of lead candidates for therapeutic or vaccine development and the selection of the most suited donors for convalescent plasma therapy. Our technology enables researchers to profile the functional immune response of COVID-19 patients or vaccinated individuals with confidence. So, thank you very much for your attention, I am really looking forward to answering any questions you may have. Thank you, and back to you Haris. Outro Haris: Thank you very much for that insightful presentation Sebastian. Before we move onto the Q&A portion off the webinar, I just want to remind everyone that our next webinar: Population-wide immune responses to SARS-CoV-2: Insights from quantifying the antibody response will be presented by Prof Adriano Aguzzi on the 23rd of March. Prof Aguzzi is a leading professor of Neuropathology at the University of Zurich. (Sign up here) His webinar will outline the challenge in understanding the humoral immune response to COVID-19, predicting the degree of immune protection resulting from previous infection, vaccination, and other treatments. It will look at the limitations of current techniques, and the work that was conducted using novel microfluidic assays to quantitatively profile the affinity and concentration of anti-COVID-19 antibodies in the serum of convalescent COVID-19 patients. I will now start asking Sebastian some of your questions, as a reminder there is still time to submit them through the chat box and we will try and go through as many as we can.