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[Fluidity One-W Serum] SARS-CoV-1 및 SARS-CoV-2에 대한 항체의 교차 반응성 (Cross-reactivity of antibodies against SARS-CoV-1 and SARS-CoV-2)
인성크로마텍(주)
Date : 2021.08.19
분류 : Analytical Products > Fluidic Analytics

 

Cross-reactivity of antibodies against SARS-CoV-1 and SARS-CoV-2

 

Published on 01 Jul 2021

 

 

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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. 

 

 

 

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 (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.

 

 

AP-0017_Figure_1A.jpg

 

AP-0017_Figure_1b.jpg

 

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.

 


AP-0017_Figure_2.jpg

 

Figure 2: Cross-reactivity of CR3022 (anti-SARS‑CoV‑1) and AS35 (anti-SARS‑CoV‑2) to the spike S1 subunits of SARS‑CoV‑1 and 

SARS‑CoV‑2.

 

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 crossreactive antibodies in patients

or vaccinated individuals, as well as to rapidly evaluate therapeutic antibodies against the emerging mutant variants of SARS‑CoV‑2. 


 

 

References

 

Huang, Y.; Yang, C.; Xu, X. feng; Xu, W.; Liu, S. wen. Structural and Functional Properties of SARS‑CoV‑2 Spike Protein: Potential 

Antivirus Drug Development for COVID‑19. Acta Pharmacol. Sin, 2020, 41 (9), 1141–1149. 

 

Sabarimurugan, S.; Dharmarajan, A.; Warrier, S.; Subramanian, M.; Swaminathan, R. Comprehensive Review on the Prevailing 

COVID‑19 Therapeutics and the Potential of Repurposing SARS‑CoV‑1 Candidate Drugs to Target SARS‑CoV‑2 as a Fast-Track 

Treatment and Prevention Option. Ann, Transl, Med, 2020, 8 (19), 1247–1247. 

 

Tian, X.; Li, C.; Huang, A.; Xia, S.; Lu, S.; Shi, Z.; Lu, L.; Jiang, S.; Yang, Z.; Wu, Y.; Ying, T. Potent Binding of 2019 Novel Coronavirus

Spike Protein by a SARS Coronavirus-Specific Human Monoclonal Antibody. Emerg. Microbes Infect, 2020, 9 (1), 382–385. 

 

Yuan, M.; Wu, N. C.; Zhu, X.; Lee, C. C. D.; So, R. T. Y.; Lv, H.; Mok, C. K. P.; Wilson, I. A. A Highly Conserved Cryptic Epitope in the

Receptor Binding Domains of SARS‑CoV‑2 and SARS‑CoV. Science, 2020, 368 (6491), 630–633. 

 

Majumder, J.; Minko, T. Recent Developments on Therapeutic and Diagnostic Approaches for COVID‑19. AAPS J, 2021, 23 (14), 1–22. 

 

Ter Meulen, J.; Van Den Brink, E. N.; Poon, L. L. M.; Marissen, ;W. E.; Leung, C. S. W.; Cox, F.; Cheung, C. Y.; Bakker, A. Q.;

Bogaards, J. A.; Van Deventer, E.; Preiser, W.; Doerr, H. W.; Chow, V. T.; De Kruif, J.; Peiris, J. S. M.; Goudsmit, J. Human Monoclonal

Antibody Combination against SARS Coronavirus: Synergy and Coverage of Escape Mutants. PLoS Med, 2006, 3 (7), 1071–1079. 

 

 

 

Appendix

 

Equation used to calculate Rh :

 

AP-0017_Equation.jpg

 

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