Bovine Immunoglobulin Inhibits the Binding

of ACE2 to COVID-19 Spike Protein

Originally presented at the World Microbe Forum

Alexis E. Rose, Adam L. Okerlund, Christopher D. Warner, Christopher J. Detzel

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect enterocytes causing increased gut permeability for both viral and bacterial antigens. Antigen translocation leads to local inflammation and increased systemic inflammation.¹⋅²

Viral entry is driven by the binding of the S1 domain of the SARS-CoV-2 spike protein receptor binding domain (RBD) binding to human angiotensin enzyme 2 (ACE2) expressed on host cells. The binding of ACE2 to RBD is necessary for the fusion of the viral envelope with the host cell membrane to deliver viral RNA. Disruption of RBD-ACE2 binding blocks SARS-CoV-2 entry in cells.³

Serum-derived bovine immunoglobulin (SBI) is an oral supplement composed primarily of IgG, IgA and IgM with broad specificity for microbial and viral antigens.

Due to the high homology, 70-80%⁴, between bovine coronavirus and SARS-CoV-2 spike protein epitopes and the prevalence of bovine coronavirus in the USA it is likely SBI contains antibodies that cross react with SARS-CoV-2 spike protein.⁵

References:

  1. Lamers MM, Beumer J, van der Vaart J, Knoops K, Puschhof J, Breugem TI, et al. SARS-CoV-2 productively infects human gut enterocytes. Science. 2020;369(6499):50-4. doi: 10.1126/science.abc1669. PubMed PMID: 32358202; PubMed Central PMCID: PMCPMC7199907.
  2. Ghosh SS, Wang J, Yannie PJ, Ghosh S. Intestinal Barrier Dysfunction, LPS Translocation, and Disease Development. J Endocr Soc. 2020;4(2):bvz039. doi: 10.1210/jendso/bvz039. PubMed PMID: 32099951; PubMed Central PMCID: PMCPMC7033038.
  3. Du L, He Y, Zhou Y, Liu S, Zheng BJ, Jiang S. The spike protein of SARS-CoV–a target for vaccine and therapeutic development. Nat Rev Microbiol. 2009;7(3):226-36. doi: 10.1038/nrmicro2090. PubMed PMID: 19198616; PubMed Central PMCID: PMCPMC2750777.
  4. Tilocca B, Soggiu A, Musella V, Britti D, Sanguinetti M, Urbani A, et al. Molecular basis of COVID-19 relationships in different species: a one health perspective. Microbes Infect. 2020;22(4-5):218-20. doi: 10.1016/j.micinf.2020.03.002. PubMed PMID: 32194253; PubMed Central PMCID: PMCPMC7102648.
  5. Gomez DE, Arroyo LG, Poljak Z, Viel L, Weese JS. Detection of Bovine Coronavirus in Healthy and Diarrheic Dairy Calves. J Vet Intern Med. 2017;31(6):1884-91. doi: 10.1111/jvim.14811. PubMed PMID: 28913936; PubMed Central PMCID: PMCPMC5697193.
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Objective

Determine if serum-derived bovine immunoglobulin (SBI) can bind to the SARS-CoV-2 RBD protein and disrupt RBD-ACE2 binding.

Methods

RBD-SBI Binding ELISA
ELISA was developed to determine SBI binding to RBD. A
96-well plate was coated with RBD, washed, blocked, and
incubated with SBI. Detection was performed using
HRP-conjugated antibodies against bovine IgG, IgA, or IgM. The reaction was visualized by the addition of TMB and stopped with sulfuric acid (H2SO4). Absorbance was read at 450 nm using a microplate reader. Control experiments were done to ensure specific binding of Ig to RBD protein (Figure 1 B,C).

RBD-ACE2 ELISA
To determine if SBI disrupts ACE2-RBD binding, a
second ELISA was developed. Again, a plate was coated with RBD, washed, blocked, and incubated with rhACE2. Detection of the rhACE2 protein was performed using an anti-HisTag-HRP.

Based upon the rhACE2 standard curve (Figure 2 A), the 40 ng/well ACE2 condition was chosen for evaluating SBI treatments on inhibition of ACE2-RBD binding. Prior to incubation with rhACE2, SBI, TBS or fish gelatin (FG) treatments were added to the wells at concentrations of 1-5 mg/well. Fish gelatin (FG) was used as negative control protein. Percent inhibition of RBD-ACE2 binding was quantified as follows:

All components of the ELISA were individually coated in wells and probed with anti-HisTag-HRP (Figure 2 B) to ensure the specificity of the HisProbe-HRP.

Figure 1 – Bovine IgG, IgM, and IgA each specifically binding to RBD protein (Panel A). The binding affinities were qualitatively demonstrated using a modified ELISA technique. ELISA control conditions (B-D) confirmed specific binding of SBI Ig (Complete ELISA). SBI Ig had a higher affinity for RBD protein (Complete ELISA) when compared to the blocking protein (-antigen, +block). Effective blocking is demonstrated by comparing wells treated with blocking protein (-antigen, +block) to wells not treated with blocking protein (-antigen, -block). Specific binding of the detection antibodies is demonstrated by comparison of Complete ELISA curve to No SBI line. Error bars represent ∓ one standard deviation from triplicate data.  Absorbance units (A.U.) measured at 450 nm.

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Results

  • Absorbance increased proportionally to SBI concentration for IgG, IgM and IgA isotypes, demonstrating binding of immunoglobulins to immobilized RBD protein. (Figure 1 A)
  • Control experiments verified effective blocking and specific binding and detection of SBI IgG, IgM and IgA to RBD protein. (Figure 1 B-C)
  • SBI treatments reduce binding of ACE2 to RBD proportionally to SBI concentration. Percent inhibition increased from 5.6% to 31.4% for the treatments in Figure 3.
  • FG negative control treatments did not inhibit ACE2 binding to RBD.
  • Paired t-test determined SBI and FG treatment groups to be statistically different (p<0.01).

Figure 2 – An RBD-ACE2 binding ELISA was developed to test for the ability of SBI to act as an antagonist. An ACE2 curve was established to verify ELISA performance and to determine assay sensitivity (Panel A). To determine specificity of the detection antibody for rhACE2, the HisProbe-HRP antibody was tested against each ELISA component (Panel B). Error bars represent standard deviation and **** indicates p<.0001.

Figure 3 –  SBI inhibits binding of ACE2 and SARS-CoV-2 spike protein. The fish gelatin (FG) control showed no inhibition of ACE2-RBD binding. Error bars represent standard deviation from mean absorbance measurements used to calculate percent inhibition.

Conclusion

  • Immunoglobulins in SBI bind to SARS-CoV-2 spike protein.
  • SBI inhibits the binding of ACE2 to SARS-CoV-2 spike protein.
  • Binding and neutralization of RBD by SBI is likely driven by cross reactivity of immunoglobulins against bovine coronavirus and SARS-CoV-2 spike proteins.
  • Further in vitro work is necessary to determine if SBI’s disruption of RBD-ACE2 binding can reduce SARS-CoV-2 entry into epithelial cells.

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