Application Note Assessing Cross-reactivity of antibodies against SARS-CoV-1 and SARS-CoV-2 Published on July 1st, 2021 Authors: Alison Ilsley, Sebastian Fiedler, Viola Denninger, Georg Meisl, Monika A. Piziorska, Anisa Y. Malik, Heike Fiegler, and Tuomas P. J. Knowles Abstract The 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 functional immune response to SARS‑CoV‑2 as well as underlying background immunity and cross-reactivity to less harmful corona viruses. To quantify the immune response of potential cross-reactive antibodies we have used microfluidic antibody-affinity profiling (MAAP) to compare binding affinities of recombinant anti-SARS‑CoV‑1 and anti-SARS‑CoV‑2 antibodies to SARS‑CoV‑1 and SARS‑CoV‑2 spike S1 proteins. By rapidly quantifying the degree of antibody cross-reactivity to different virus variants even in a complex background such as serum, we show that our approach could be exploited to repurpose existing therapeutic antibodies for the treatment of COVID‑19 and to monitor the efficacy of vaccines against changing epitopes in new mutant variants of SARS‑CoV‑2. DOWNLOAD THE APPLICATION NOTE Introduction COVID‑19 is caused by the beta coronavirus SARS‑CoV‑2, one of seven coronavirus species known to infect humans. While the majority of coronaviruses only cause relatively mild diseases, SARS‑CoV‑1, MERS-CoV and SARS‑CoV‑2 all led to major global outbreaks. Based on phylogenetic analyses, SARS‑CoV‑2 was shown to share remarkably high sequence similarity with SARS‑CoV‑1, suggesting that antibodies and compounds previously screened for use against SARS‑CoV‑1 could be lead candidates in the development of therapeutics against COVID‑19.1 A rapid immune response assessment and quantification of potentially cross-reactive antibodies against SARS‑CoV‑2 will, therefore, be a crucial predominant in selecting the best therapeutic candidates. SARS‑CoV‑2 is a single-stranded RNA-enveloped virus, and its genome encodes for several structural and functional proteins including the heavily glycosylated spike protein that covers the external surface of the virus. Similar to other coronaviruses, the spike protein consists of two distinct functional subunits, S1 and S2, that mediate receptor recognition, cell attachment and fusion during viral infection.1 Spike proteins vary significantly between different coronavirus species giving rise to a wide range of hosts and host-cell receptors they bind to.1 In the case of SARS‑CoV‑1 and SARS‑CoV‑2, however, homology modeling of the respective spike proteins revealed a sequence similarity of 75 – 80%, accounting for the fact that SARS‑CoV‑1 and SARS‑CoV‑2 share the same entry mechanisms into the host cells. Both bind to the angiotensin-converting enzyme 2 (ACE2), a receptor that is highly expressed on the surface of human respiratory epithelial cells.3 Based on the high degree of similarity between SARS‑CoV‑1 and SARS‑CoV‑2, it was therefore speculated that cross-reactive epitopes could exist,4 which could be exploited to rapidly repurpose existing therapeutic approaches, or develop new vaccines. For instance, during the initial SARS outbreak in 2002 several monoclonal antibodies including CR3022 were developed against the SARS‑CoV‑1 spike protein with the goal to inhibit entry into the human host cell.5 CR3022 is a neutralizing monoclonal antibody that binds to the receptor binding domain (RBD, residues 318-510) of the SARS‑CoV‑1 spike protein.6 Although the antibody failed to neutralize SARS‑CoV‑2, it was shown to cross-react with a conserved epitope on SARS‑CoV‑2 RBD.3 Moreover, used in combination with another antibody, CR3014, neutralization was achieved due to synergistic binding of different epitopes on the RBD.3 Such a combined antibody therapy was therefore suggested as an option in the treatment of COVID‑19 patients.1,3 To better understand the underlying mechanisms that govern the functional immune response to SARS‑CoV‑2 we have used microfluidic antibody-affinity profiling (MAAP) to assess and quantify the cross-reactivity between CR3022 and the spike S1 proteins of SARS‑CoV‑1 and SARS‑CoV‑2, as well as the cross-reactivity of the two respective spike S1 proteins and a COVID-patient derived neutralizing anti-SARS‑CoV‑2 monoclonal antibody. Our results indicate a unidirectional cross-reactivity with only CR3022 readily recognizing the spike S1 subunits of both viruses, albeit with a ~100 times lower affinity to SARS‑CoV‑2. Importantly, we could not detect any binding of the anti-SARS‑CoV‑2 antibody to the S1 subunit of SARS‑CoV‑1, emphasizing the selectivity for the newly evolved virus. Methods SARS‑CoV‑1 spike S1 (S1N-S52H5, ACROBiosystems) and SARS‑CoV‑2 spike S1 (S1N-C52H4, ACROBiosystems) were reconstituted in 167 μL sterile water to a concentration of 600 μg/mL. Both proteins were labeled with Alexa FluorTM 647 NHS ester (A37573 Thermo Fisher Scientific) by adjusting the pH to 8.3 with 1M NaHCO3 and incubating with the dye at a 3:1 dye-to-protein ratio. Following overnight incubation at 4 °C, the labeled spike S1 proteins were purified via size-exclusion chromatography on an ÄKTA pure system (Cytivia) using a Superdex 200 increase 3.2/300 GL column with PBS (pH 7.4) as elution buffer. For affinity measurements, Alexa FluorTM 647 labeled SARS‑CoV‑1 spike S1 and various anti-SARS‑CoV‑1 spike S1 antibody [CR3022] (ab273073, Abcam) dilutions were mixed at a 1:1 ratio in the presence of 90% serum (H5667, Merck) to yield final concentrations of 10 nM and 0.75 – 250 nM of S1 and antibody, respectively. All samples were incubated for 1 hour at 4 °C prior to measurement and kept at 4 °C throughout the experiment. Because of the high affinity of the antibody, the titration curve was repeated with an antibody concentration ranging from 3 pM to 100 nM and a final SARS‑CoV‑1 spike S1 concentration of 5 nM. For the analysis both titration curves were fitted globally. To determine if the CR3022 antibody binds SARS‑CoV‑2 spike S1, the affinity measurement was repeated with an antibody concentration ranging from 15 pM to 500 nM and a final concentration of SARS‑CoV‑2 spike S1 of 10 nM. The affinity of anti-SARS‑CoV‑2 spike neutralizing IgG1 antibody [AS35] (SAD-S35, AcroBiosystems), to SARS‑CoV‑1 Spike S1 and SARS‑CoV‑2 Spike S1 (S1N-C52H4, AcroBiosystems) was determined in a similar way. The antibody concentration ranged from 15 pM to 500 nM with a final spike S1 concentration of 10 nM. Samples were measured in triplicate at room temperature on the Fluidity One-W Serum using the 1.5 – 8 nm size-range setting. Background fluorescence was corrected for by performing independent measurements of human serum and applying a background subtraction to individual data points obtained in serum. The binding affinity, KD, was generated by non-linear least squares fitting to Equation 1 (See Appendix). Results Cross-reactivity of CR3022 (anti-SARS‑CoV‑1) and AS35 (anti-SARS‑CoV‑2) neutralizing antibody to SARS‑CoV‑1 and SARS‑CoV‑2 spike S1 proteins were determined by titrating the respective antibody against a constant concentration of 10 nM Alexa FluorTM 647 labeled SARS‑CoV‑1 or SARS‑CoV‑2 spike S1 protein. Figure 1: Equilibrium binding curves of (A) CR3022 and (B) AS35 against the spike S1 subunit of SARS‑CoV‑2 (red) and SARS‑CoV‑1 (blue). Measurements were performed in triplicate. The KD was determined by non-linear least squares fitting using Equation 1; for the analysis of two CR3022 binding curves to SARS‑CoV‑1 spike S1, a global fit was applied with the following shared parameters: Rh, free, Rh, complex, stoichiometry and KD. While CR3022 did bind to the spike S1 subunits of both viruses, AS35 was found to exclusively bind to the SARS‑CoV‑2 spike S1 subunit with a KD of 5 nM. Interestingly, quantification of the immune response of CR3022 showed a ~100 fold greater affinity to the SARS‑CoV‑1 antigen (KD < 0.4 nM) compared to the SARS‑CoV‑2 spike S1 protein (KD = 58 nM). These results clearly support previous observations of cross-reactivity between CR3022 and SARS‑CoV‑2 antigens, but also highlight the selectivity of antibodies raised in COVID-patients towards the newly emerged SARS‑CoV‑2 (Figure 2). Conclusion By using microfluidic antibody-affinity profiling on the Fluidity One-W Serum to determine the affinities of CR3022 and AS35 against the spike S1 subunits of SARS‑CoV‑1 and SARS‑CoV‑2, we can evaluate cross-reactivity of an antibody to various antigens even in a complex background like serum. This approach could be used to quantify the immune response of cross-reactive antibodies in patients or vaccinated individuals, as well as to rapidly evaluate therapeutic antibodies against the emerging mutant variants of SARS‑CoV‑2. 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DOI: 10.1371/journal.pmed.0030237 Appendix Equation used to calculate Rh: Where: Rh is the hydrodynamic radius at equilibrium Rh, free is the hydrodynamic radius of the unbound protein Rh, complex is the hydrodynamic radius of the protein-ligand complex [L]tot is the total concentration of labeled species [U]tot is the total concentration of unlabeled species n is the complex stoichiometry (unlabeled molecules per labeled molecule) KD is the dissociation constant Download application note Fill out the form below to download the application note "Assessing Cross-Reactivity of Antibodies Against SARS-CoV-1 and SARS-CoV-2 by Microfluidic Antibody-Affinity Profiling". You can print it, share it with friends and family or just use it as light reading! We hope you enjoy this application note as much as we did creating it!