Team:Technion-Israel/Contribution

Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged at the end of 2019. On March 2020, the World Health Organization (WHO) characterized COVID-19 as a pandemic. As of October 2020, there are about 36 million active cases of COVID-19 and around 1 million confirmed deaths from 235 countries. WHO has published general hygiene guidelines and recommended to maintain social distancing, but specific treatments or vaccines have yet to be found.^{[1] [2][3][4]}

Cellular entry of SARS-CoV-2 depends on binding of the viral spike proteins to angiotensin-converting enzyme 2 (ACE2) receptor presents on the membrane of lung cells and other human cells. The spike protein is responsible for host recognition via interaction between its receptor binding domain (RBD) and ACE2. Targeting the virus-host interface, where mutational adaptations of the virus are arguably less likely, could potentially present durable, broad-spectrum treatment.^[1][5]

In our project, we created a self-care product with the aim of lowering the infectivity of SARS-CoV-2 with decoy molecules that were design to effectively bind the virus.

In one aspect of our work, we focused on Sybodies as decoy molecules. Sybodies are small (12-15 kDa) synthetic single domain antibody fragments which contain the unique structural and functional properties of naturally occurring heavy chain only antibodies (Fig. 1). Besides, they are stable, and inexpensive to produce in bacteria and yeast.^[1][6]

Fig. 1: Conventional antibodies, Camelid antibodies and a nanobody

In our work, we focused on Sybody #15 and Sybody #68 developed by Walter JD et al. ^[1] using an unconventional, expeditious in vitro process. Combining both of these Sybodies can result in a higher affinity bond with the viral spike, with substantial inhibition of RBD interaction with ACE2 better than individually.^[1]

Firstly, our iGEM project was first to use Sybodies targeting the spike proteins of SARS-CoV-2 by either linking them to Bacillus subtilis spores (Fig. 2). The production potential of the Sybodies was shown by successfully synthesizing them fused to a His-tag in E. coli KRX. We have registered the new bio-bricks and shared our protocols with the iGEM community with the iGEM community.

Fig. 2: Incorporation of sybodies in the final product

Secondly, we aimed to demonstrate the versatility of the described therapeutic options. We managed to successfully insert Sybody #68 into Bacillus subtills pY79 genome using pBSC1 bacterial expression vector for the first time. We are the first iGEM team to direct Bacillus subtilis spore surface display for such a purpose (skin application against viral transmission).

Fig. 3: pET-9d vector and the Sybodies

Fig. 4: pBS1c vector and the Sybodies

In another aspect of our project, we tried to optimize the ACE2 receptor’s ability to bind more efficiently to the spike protein. Saccharomyces cerevisiae, a vastly used yeast by the industry and researchers, was recruited for scanning libraries of ACE2 variants. For this purpose, a cloning method called AGAP – Any Gene Any Plasmid – that enables the cloning of any gene (or combination of linear DNA fragments) into any conceivable vector in a single step without the need for ligase or molecular cloning kits ^[7]. While this method is commonly used by researchers, the iGEM community rarely makes use of it. Moreover, accessing information and protocols of this method is relatively difficult. Here we present a simple and easy-to-use protocol with our tips and information in order to simplify the AGAP method for future iGEMers.

AGAP Manual

References

1. Walter JD, Hutter CAJ, Zimmermann I, et al. Sybodies targeting the SARS-CoV-2 receptor-binding domain. bioRxiv. 2020. doi:10.1101/2020.04.16.045419
2. Coronavirus disease (COVID-19). World Health Organization website. Https://www.who.int/emergencies/diseases/novel-coronavirus-2019
3. Advice for the public. World Health Organization website. Https://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-public
4. WHO Director-General’s opening remarks at the media briefing on COVID-19 - 11 March 2020. World Health Organization website. Https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020
5. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181(2):271-280.e8. doi:10.1016/j.cell.2020.02.0526.
6. . Understanding Nanobodies. Ablynx, a Sanofi company website. Updated December 19, 2019. Accessed July 12, 2020. Https://www.ablynx.com/technology-innovation/understanding-nanobodies/
7. Joska TM, Mashruwala A, Boyd JM, Belden WJ. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile. J Microbiol Methods. 2014;100:46-51. doi:10.1016/j.mimet.2013.11.013