Team:Technion-Israel/Human Practices

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Introduction

In December 2019, a local outbreak of pneumonia was detected in Wuhan, China. The pathogen of this pneumonia has been named “Severe Acute Respiratory Syndrome Coronavirus 2” (SARS-CoV-2). In comparison to SARS-CoV, which caused an outbreak in 2003, the novel SARS-CoV-2 has a stronger transmission capacity[1]. On 11th March 2020, the World Health Organization (WHO) announced COVID-19 (the disease caused by SARS-CoV-2) outbreak as a pandemic. As no vaccine nor treatment was approved, we wanted to be a part of the global fight against SARS-CoV-2. To achieve this goal, rather than working exclusively on the scientific front, efforts have also been invested to raise the awareness of the public to the escalating pandemic since we consider all of us as a community that seeks to diminish the impact of COVID-19. Due to the regional health restrictions, we tried to accomplish the awareness campaign in creative ways, such as publishing a Kahoot !game for elementary school kids, creating fun and productive videos, and giving tips about “COVID-19 time”.

Kahoot! Game in Elementary Schools

Overview

Regarding the spreading of the pandemic worldwide, we found it is important to raise the juvenile’s awareness of the situation in elementary schools. To achieve this goal in a fun and attractive way, we created a Kahoot! game. Kahoot! is an online game-based questionnaire platform that made it possible for us to deliver the idea entertainingly.

The schoolteachers who helped us declared that the game was very exciting, educational, and delivered the message to the children in an entertaining atmosphere.

We reached out to three elementary schools:

1
Sadot elementary school in Karkur
2
Dror elementary school in Haifa
3
Nordau elementary school in Netanya

Video clips

At the beginning of our project, COVID-19 was new and mysterious to the public. We understood that raising public awareness of the virus should be a major part of our project. There is no doubt that everybody is "in the same boat". That is how the idea for the video "Corona around the world" was born. In this video, the word “Coronavirus” was pronounced by people from different countries in their native tongue to show the unity of the world when facing this virus. The ultimate goal of this video was to create a feeling of mutual responsibility among the viewers. Essentially, uniting all people under the same purpose, and hopefully, raise one's smile during these hard times.


The second video we made was the "Wash your hands challenge". We noticed that most people do not know how to wash their hands properly. Therefore, this video was designed to show and teach people how to wash their hands correctly in a way that would appeal to young viewers. To do so, gloves and white paints were used to show the important parts of hands that are supposed to be covered when washing hands. By combining several ways of washing, every area on hands can be cleaned decently. This video is crucial for our project because our product's goal is to reduce the infection by the virus, and hand washing is undoubtedly an important measure. Fortunately, the video reached many people (~39.2k views) and hopefully changed their conception about hand washing.


The third video we made was the "Quarantine" video. As part of our country's effort to reduce the infection rate, two lockdown periods have lasted for about a total of two months. These lockdowns posted a huge obstacle in our project because we could not continue our lab work and progress with our research. Nevertheless, an opportunity to introduce our team was found by producing a funny video that shows what our team members were doing during the lockdowns’ time. The difficulties that came from the lockdowns turned out to inspire us to show our team members for the first time, advertise our project, and inspire other people to do different things at home.


In that spirit, we also made a series of images containing suggestions for several occupational options for quarantine time, meant to help people break the daily routine and enrich their time at home. New images were published twice a week, 12 images in total. We hope those efforts eased the quarantine for some of the people and helped them stay safe at home in these difficult times. Shown below are a few examples of our images.







The Project Influence and Connection to the World

On 20th September 2020, over 30.6 million COVID-19 cases and 950,000 deaths have been reported to the World Health Organization (WHO). From 14th till 20th September, there were almost 2 million new cases of COVID-19, which represents a 6% increase compared to the previous week, and it was also the highest number of reported cases in a single week since the beginning of the pandemic[2]. This pandemic has influenced the everyday life of almost everyone in the world for over 9 months. There are a lot of attempts all over the world to develop a remedy for COVID-19, such as a medicine or a vaccine, but none of them have been approved to date, though the some vaccines are undergoing human trails.

When we chose our project, it just got clear that this pandemic will not be resolved quickly and will affect billions of lives around the globe. As a result, we decided to join the worldwide-shared effort against it, to act. We have unanimously decided that our project must help the world overcome this challenge.

Our product is not a conservative treatment, as it is not a treatment at all. Due to the limited knowledge we have, there is no doubt that we cannot end this pandemic on our own. However, we do want to help the public in whatever ways we can. Our project is aimed to create a prophylactic tool that could help to lower the rate of infection and thereby flattening the curve. The design of our product is adapted to the public preferences as much as possible, which can be shown in our survey description in this page. All adjustments were made in the hope to increase the usability of our product and the number of potential users, thus increasing our product’s global influence as well. For example, we chose our way of delivery according to the respondents’ answers and gave up on the idea to add an indication to the virus binding due to the low support rate.

Alongside joining the worldwide-shared effort against this pandemic, ACT. could also be used, as a platform, against possible future pandemics. The specificity to SARS-CoV-2 in our product was derived from the choice of "bait" molecules (the Sybodies and the ACE2 proteins) that are specific to the coronavirus (project description). The core idea of the project, of stopping the virus on the external surface of the body before it gets inside and infects the user, can be adjusted and used for stopping other viruses. In addition to the product itself, we wanted to help in every other way we could. To do so, we created a series of videos with various purposes. More details can be found in the Video clips part of this page.


Product Responsibility

As shown on our Design page, our project contains 5 different elements, each one of them was carefully selected for its purpose while paying attention to the safety of its use on the human body. The Hydrogel, that serves us as the basis of the cream, was selected due to its unique thermodynamic properties, as explained in its Results page. Lufton M. et al[3] presents the various tests that were made to show the safety of using this Hydrogel on the skin in general and for medical applications in particular.

The options for carriers are Bacillus subtilis spores and Microgel beads.

Bacterial spores, in general, are being used as probiotic supplements in animal feeds, human dietary supplements as well as in registered medicines[4]. Specifically, Bacillus subtilis is a Generally Recognized as Safe (GRAS) approved bacterium that is used worldwide in the food industry. Moreover, the robust spores of Bacillus spp. are compliant to simple and cost-effective production processes, in which the end products are stable for long periods[5].

The Microgel is created according to the research “Microgels for efficient protein purification“[6]. The Microgel beads contain coordinatively bound Nickel molecules intended to attach the His-tag tail fused to our proteins. Unbound Nickel can have harmful effects on the human body[7]. However, as mentioned in our safety form and in the consultation with Dr. Fischer company, according to the FDA regulations products defined as “Skin protectant drug product” can contain bound nickel molecules[8].

The two options for baits are the Sybodies and the ACE2 proteins.

Sybodies are synthetic single-domain antibodies that have qualities make them ideal for a practical binder[9]. ACE2 protein is a human protein, which serves as the binding site of the coronavirus in the human body[10]. Proteins might be problematic to use in medicine because of the strong immune response these foreign proteins induce[11]. However, in our project, both proteins are only meant for skin application, so immune response will not be a problem. Moreover, in our product they are bound to the Microgel with a coordinative bond[12] or connected to the spores’ surfaces, so the chances of them coming loose are minor.

In conclusion, all the parts that are used to create our product are safe for external use.

Integrated Human Practices

Introduction

Despite the strict limitations due to the escalating pandemic of COVID-19 in Israel, we tried our best to look beyond the lab because of the huge effect on our project. To achieve this, we consulted with some pharmaceutical companies, experts, and conducted two public surveys to shape our product.

Consulting with Pharmaceutical Companies

To learn more about pharmaceutical products and their characteristics, we approached several Israeli pharmaceutical companies. Due to the COVID-19 limitations, the meetings were conducted virtually via ZOOM.


Dr. Fischer Meeting


Dr. Fischer is a multi-national pharmaceutical company specialized in research & development (R&D) and manufacture of skin and health care products that are marketed worldwide. We have met Sharon Gertler (R&D Team leader at Dr. Fischer) and Nurit Harel (Co-Chairman at Fischer pharmaceuticals) and consulted with them about dimming the color of the product, smell, skin nourishment materials addition and regulations.

For the sake of solving the color issue, Dr. Fischer team suggested adding color-bleaching molecules. On the other hand, such an addition might reduce the product’s efficiency. Also, they advised us to consider the dilution of the beads in the thermo-responsive gel, which might result in a non-visible color. In total, Dr. Fischer team thought that the color could be a feature instead of a bug since it can be an indicator of the “efficiency of appliance”. Therefore, adding more color should be considered especially among children who are likely to leave some areas uncovered. Besides, the possible color of the product could potentially produce a skin tone shade when combined with pinkish skin tone, thus acting as a concealer. In consequence, we decided to keep the product’s color if there is any.

As for masking the product’s smell, adding a bit of fragrant perfume would be enough. The team suggested avoiding scented products since it is not prevalent. If adding a fragrance is a must, giving the product a light fragrance that will last for a short period can solve the problem. Therefore, our team concluded that not to add any perfume to the product since it is not necessary.

Regarding skin nourishment, Dr. Fischer team suggested using Glycerin and Hyaluronic acid as moisturizes. However, they advised us not to add any because the product’s features are enough since the thermo-responsive gel provides a barrier for the skin because of its structure that acts as a semi-permeable layer. Thus, preventing the penetration of harmful substances into the skin and the evaporation of water from the skin. Subsequently, a decision of keeping the formulation without any nourishment materials was taken.

In respect of regulation, Dr. Fischer team advised us to reconsider defining our product as a cosmetic one since it includes bounded Nickel. Cosmetic and Over-The-Counter (OTC) products undergo different regulations. According to European cosmetics regulation, Nickel is almost completely prohibited[13]. As for the FDA regulations, Nickel is approved in implants[8]. Appropriately, a new definition of the product as a skin protectant drug product was taken.

To conclude, the meeting with Dr. Fischer was productive since we learned how to overcome obstacles that face the pharmaceutical industry such as dimming a color, adding perfume, adding nourishment materials, and the importance of clearly declaring which administrative regulations are we looking to meet (FDA/EU as skin-care/OTC-Drug). Eventually, we concluded that our product is good enough as it is and there is no need for unnecessary additions.

Surveys

Our project aims to flatten the curve of COVID-19 infection by creating a gel-typed OTC product meant for external application. As a para-pharmaceutical product, the product needs to comply with the relevant FDA regulations. Nevertheless, for it to become popular and worthwhile, it also needs to meet the consumers’ desires and expectations. With these goals in mind, we published two public surveys. The goal of the first survey was to shape our product according to the needs and preferences of consumers. The second one was aimed to hear consumer’s opinions about specific details of our designed final product.


Results of the first survey

According to the results of the first survey, we deduced that our potential consumers were willing to use a product similar to the one we intended to create (68%) (Fig. 1). Most of the respondents admitted that they are good at acquiring new habits (70%) (Fig. 2), such as applying new external-use products. However, they proclaimed to be better at it when an important issue is in hand (83%) (Fig. 3).

Most of the potential users declared that they will probably use such a product more than 2-3 times a day (54%) (Fig. 4). They desired the product to be delivered as a cream (38%) over a spray (23%), nasal spray (16%), gloves (12%) or masks (10%), according to a multiple-choice question (Fig. 5). Moreover, a washable product was found to be more popular (73%) (Fig. 6).

Examples of answer-analysis for questions from the first survey:

Table 1: Countries answered the survey

Row Labels

Count of country

Canada

4

China

5

Columbia

2

Czech Republic

1

France

3

Finland

7

Georgia

3

Germany

15

India

5

USA

5

UK

2

Australia

7

Israel

1100

Grand total

1159

  1. If there was a product actively preventing getting infected with COVID-19, would you use it?

    Fig. 1: Results of Q1 of survey 1


  2. Are you good with acquiring new habits?

    Fig. 2: Results of Q2 of survey 1


  3. Count of When you decide some action is important to you, how persistent are you with doing it?

    Fig. 3: Results of Q3 of survey 1


  4. How often would you remember to use it?

    Fig. 4: Results of Q4 of survey 1


  5. What is the most comfortable way of using such a product in your opinion?

    Fig. 5: Results of Q5 of survey 1


  6. If the product would be removed from your hands when washing them, would you remember using it again every time after washing them?

    Fig. 6: Results of Q6 of survey 1



Results of the second survey

As a cream is the preferable way to deliver such a product, we chose to continue researching about thermo-responsive gels as they can perform a quick reversible transformation between liquid and gel under temperature stimulation. According to the second survey, the preferable hydrogel’s solidifying time is 0-20 sec (71%) (Fig. 7). Hence, we aim to optimize the properties of our hydrogel to reach a solidifying time of 20 sec. Most of the potential buyers (72%) are willing to buy the product even if it costs more than alcohol-based sanitizers (Fig. 8) to maintain a healthy microbiome as alcohol-based sanitizers harms the microbiome of the skin if applied frequently. Also, 69% do not mind if the product contains Bacillus subtilis spores (Fig. 9). 51% of responders do not mind using a product with a slightly yellowish/teal color resulting from the Microgel particles (Fig. 10). Furthermore, adding fragrance is non-preferable if it reduces product efficiency (72%) (Fig. 11).

  1. The product solidifies in a few short moments on the skin, forming an undetectable thin film. I prefer the hardening time to be _____ seconds.

    Fig. 7: Results of Q1 of survey 2


  2. I am willing to pay more for a product that can do the job of alcohol-based sanitizers but without damaging the beneficial bacteria of the skin.

    Fig. 8: Results of Q2 of survey 2


  3. I don’t mind using a product that contains harmless Bacillus subtilis spores (dormant bacteria, harmless to humans).

    Fig. 9: Results of Q3 of survey 2


  4. I’m willing to use a product that is transparent with a slightly yellowish/teal color.

    Fig. 10: Results of Q4 of survey 2


  5. I prefer the product to have aromatic materials, even if such an addition might reduce its efficiency.

    Fig. 11: Results of Q5 of survey 2


Consulting with Experts

Consulting with Dr. Yotam Bar-On

To understand more about the ACE2-Spike protein binding mechanism, we reached out to Dr. Yotam Bar-On, a virologist from the Ruth and Bruce Rappaport Faculty of Medicine at the Technion. He suggested the idea of using the complement system in our project. Also, he recommended us to consult with his colleague from the field, Dr. Ron Diskin and Prof. Ofer Mandelboim, who can help us with further questions.

Consulting with Dr. Ron Diskin

We reached out to Dr. Ron Diskin as was recommended by Dr. Yotam Bar-On, to learn more about the suitability of ACE2 as an effective “decoy protein” for the SARS-CoV-2 Spike protein. However, he advised us not to use the complimentary system since this system could be affected by various factors, which makes the product not plausible. He suggested another biological binding option, mentioning a novel synthetic antibody, which can bind the Spike proteins and disassemble them.

Consulting with Prof. Ofer Mendelboim

We also reached out to Prof. Ofer Mendelboim as was recommended by Dr. Yotam Bar-On. He explained to us why the complementary system would not work for our purpose. Also, he pointed out that using specific antibodies is irrelevant because of the cream consistency. Moreover, he suggested using a Lentivirus expressing Spike proteins for testing the binding efficiency.

Consulting with Associate Prof. Boaz Mizrahi

To find an ideal carrier of the receptors, we consulted with Prof. Boaz Mizrahi from the Faculty of Biotechnology and Food Engineering at the Technion, who is an expert in the biomaterials field. He suggested using Ni-poly NTA microgels as a vehicle for ACE2 receptors since the microgels contain nickel molecules, and therefore, have high affinity to His-tagged proteins. Moreover, he advised us to make the product more consumer-friendly by integrating the Microgel with a thermo-responsive polymer. As a result, we chose microgel made of Ni-poly NTA as a carrier for our “decoy proteins” (i.e. ACE2). Furthermore, Prof. Boaz and his students guided us in preparing both the Microgel Beads and the thermo-responsive polymer. In conclusion, both ideas of the Microgel and thermo-responsive hydrogel were included in our project as Prof. Boaz Mizrahi suggested.

Consulting with Dr. Smadar Shulami

To learn more about protein extraction from yeast, we consulted with Dr. Smadar Shulami, the lab manager of Protein and Enzyme Engineering lab at the Faculty of Biotechnology and Food Engineering at the Technion. She gave us guidance regarding protein expression and extraction in yeast and advised us to find the right growing method for reaching a high protein concentration after transformation from E.coli to yeast. In conclusion, the extraction of proteins from yeast should be done in the same way as in bacteria.

Consulting Dr. Justin Walter

After reading Dr. Justin Walter paper[14], we consulted with Dr. Justin Walter at the Institute of Medical Microbiology at the University of Zürich to understand more about the Sybodies expression. He said that any strain of bacteria that is compatible with pBAD promoter (arabinose-inducible) can be used to express the Sybodies. Also, he pointed out that using the MC1061 cell line is important for our upstream cloning process since it relies on control of cell death B protein (ccdB) counterselection. He also suggested Sb#15 and Sb#68 (two Sybodies from his preprint) as the best choices for our product since they can bind to the viral Spike RBD simultaneously.

Consulting Prof. Ester Segal

Concerning the adsorption and/or the diffusion in the desired model, we consulted with Prof. Ester Segal from The Laboratory of Functional Nanomaterials, Biosensors, and Sensors at the Technion. Due to the consultation, we realized that the best course of action would be to find a pre-existing diffusion model to rely on. She sent us two exemplary articles, one of which was written by Sofia Arshavsky Graham (a Ph.D. student in her lab)[15]. Later on, Prof. Segal also kindly guided us regarding the collection of the relevant data for the chosen model.

Consulting Sofia Arshavsky Graham

After consulting with Prof. Ester Segal, we reached out to Sofia Arshavsky Graham, a Ph.D. student from Segal’s lab, regarding the realization of the boundary conditions (of the diffusion equations in her article) in MATLAB. Sofia was extremely kind and helped us with the final steps of the MATLAB code used for the model.

References
  1. Zheng Y-Y, Ma Y-T, Zhang J-Y, Xie X. COVID-19 and the cardiovascular system. Nat Rev Cardiol. 2020;17(5):259-260. doi:10.1038/s41569-020-0360-5
  2. WHO. Coronavirus Disease (COVID-19), Weekly report 20 September 2020. WHO Bull. 2020;Publish Ah
  3. Lufton M, Bustan O, Eylon B, et al. Living Bacteria in Thermoresponsive Gel for Treating Fungal Infections. Adv Funct Mater. 2018;28(40):1801581. doi:10.1002/adfm.201801581
  4. Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28(2):214-220. doi:10.1016/j.fm.2010.03.007
  5. Cheon J, Kim SB, Park SW, Han JK, Kim P. Characterization of L-Arabinose Isomerase in Bacillus subtilis , a GRAS Host, for the Production of Edible Tagatose. Food Biotechnol. 2009;23(1):8-16. doi:10.1080/08905430802671873
  6. Mizrahi B, Irusta S, McKenna M, et al. Microgels for Efficient Protein Purification. Adv Mater. 2011;23(36):H258-H262. doi:10.1002/adma.201101258
  7. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Arsenic, metals, fibres, and dusts. IARC Monogr Eval Carcinog Risks Hum. 2012;100(Pt C):11-465.
  8. Food and Drug Administration. Biological Responses to Metal Implants. 2019 Sep. https://www.fda.gov/media/131150/download
  9. Zimmermann I, Egloff P, Hutter CAJ, et al. Generation of synthetic nanobodies against delicate proteins. Nat Protoc. 2020;15(5):1707-1741. doi:10.1038/s41596-020-0304-x
  10. Hamming I, Timens W, Bulthuis M, Lely A, Navis G, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004;203(2):631-637. doi:10.1002/path.1570
  11. Devaux CA, Rolain J-M, Raoult D. ACE2 receptor polymorphism: Susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome. J Microbiol Immunol Infect. 2020;53(3):425-435. doi:10.1016/j.jmii.2020.04.015
  12. Schellekens H, Jiskoot W. Immunogenicity of therapeutic proteins. In: Pharmaceutical Biotechnology: Fundamentals and Applications. 4th ed. New York, NY: Springer New York; 2013:133-141. doi:10.1007/978-1-4614-6486-0
  13. Conti M, Falini G, Samorì B. How Strong Is the Coordination Bond between a Histidine Tag and Ni - Nitrilotriacetate? An Experiment of Mechanochemistry on Single Molecules. Angew Chemie Int Ed. 2000;39(1):215-218. doi:10.1002/(SICI)1521-3773(20000103)39:1<215::AID-ANIE215>3.0.CO;2-R
  14. 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
  15. Arshavsky Graham S, Boyko E, Salama R, Segal E. Mass Transfer Limitations of Porous Silicon-Based Biosensors for Protein Detection. ACS Sensors. 2020. doi:10.1021/acssensors.0c00670




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Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel

    • igem2020.technion@gmail.com