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Team:PYMS GZ China/Engineering


Engineering Success
Research

Amidst the COVID-19 pandemic, our team along with the rest of the world realized the urgency of a vaccine against the SARS-Cov-2 virus to restore life back to normalcy. Firstly, we studied the anatomy of the SARS-Cov-2 virus and more specifically the receptor binding domain of the Spike protein located on the outside of the virus. We investigated the entree point of the virus, the ACE2 receptor. Then we researched IgG antibodies and their neutralizing effect on viruses to stop bacterial and viral infections.

Imagine

Hypothesizing our vaccine, we envisioned isolating a small portion of the RBD of SARS-Cov that could be injected into the body to stimulate an immune response to develop immunization antibodies. Through our research, we discovered that the recombinant protein vaccine would be the most effective way to create immunization in the body. Compared to the live virus vaccination, by inserting a small, non-lethal portion of the virus, the body can still produce antibodies in a safer fashion. With this in mind, we wanted to model our vaccine after this method. The recombinant protein vaccine is the best type of vaccine for this virus since the S protein if the virus can infect the body as the live virus does.

Design

Now knowing what we wanted our project to be, we continued our research into designing a SARS-CoV-2 vaccine. Soon, we discovered that making a vaccine did not seem feasible. We realized that we did not have the time to be able to develop a feasible vaccine. To test the effectiveness of our vaccine, we were planning to administer it to rodents and then monitor the antibody development. Given the time constraint, we did not feel morally comfortable injecting a rodent with a vaccine we didn't have 100% confidence in. Furthermore, our team did not have the experience needed to complete experiments that required the Bac-toBac method to generate the needed baculovirus system. Not discouraged, we still wanted our project to revolve around SARS-CoV-2, so we went back to the drawing board.

Research

Since we were no longer creating a vaccine, we continued our research. While conducting our research for the vaccine project, we realized the desperate need for scientists to test the infectivity of different SARS-Cov-2 strains and the validity of vaccines. Recalling this, we shifted our focus to creating a method that would be able to find the binding affinity of variant strains. This led us to research ways to create a general platform that can test the infectivity of all global strains of SARS-Cov-2 to quantify the contagiousness and dangerousness of different strains. We knew we wanted a platform to have multiuse- not only testing the infectivity of the virus variants, but also assessing the effectiveness of vaccines. We kept these two goals in mind as we furthered our research. We learned about assays and, when paired with a marker, it could be used to test infectivity and the success of vaccines in blocking interactions. There are multiple different markers we can use, but with more exploration, we learned about bioluminescence and luciferase that produced more accurate data than alternatives. Currently, to conduct research measuring the infectivity of the virus, experiments must be done in a P3 biosafety laboratory as they use the live virus. We investigated processes that would allow us to observe the virulent behavior of the virus in a P2 or lower biosafety laboratory. The only way to do this is to not utilize the live virus in our experiments. Looking into substitute options, we discovered the safety of pseudoviruses.

Imagine

With our knowledge of the pseudovirus, entry point of SARS-CoV-2, and the assay utilizing the luciferase, we believed we could employ all three to create a platform to test binding affinity and effectiveness of vaccines. The pseudovirus allowed us a way to still be able to experiment with the virus without worry of causing an outbreak as it cannot replicate like normal viruses. By understanding the significance of the ACE2 receptor in SARS-CoV-2 infection, we know what we have to measure to discover the binding affinity. The assay and luciferase will allow us a way to quantitatively compute the binding.

Design

We now know the basic methodology and components we need to execute our project, but there were still gaps in our design. When coming up with our design, we decided on creating a two part system with bacterial and mammalian cells. Reflecting upon our previous knowledge, we selected the use of bacterial (E.coli) cells to replicate the plasmid containing the S-protein. We chose this as bacteria can replicate much faster than mammalian cells. Furthermore, it can be used to exterminate cells that were not properly transfected. Through the use of ampicillin resistance, cells that do not contain the target plasmid will die. The bacterial vessel will optimize project efficiency with its qualities of rapid replication and guaranteed successful transfection. Additionally, using mammalian cells poses problems as it could contain harmful endotoxins that interfere with the experiment later on. We wanted to take advantage of producing plasmids in a bacterial system since scientists have found a way to purify them so they will not contain harmful substances that transfect into human cells that will create side effects or problems. Mammalian cells are later used for pseudovirus packages as it contains machinery to produce the pseudovirus containing the luciferase enzyme and the S protein variant. For our mammalian cells, we selected to use HEK293 for their easy growth and to maintain the high reproducibility of cell lines. Furthermore, they are very accessible for transfection. After the pseudovirus is created, extracted and introduced to another line of HEK293 cells that contain ACE2 receptors, it models how the virus infects humans. Then, the mammalian cells are lysed and contents are observed to measure the binding rates.

Build
Production of SARS-CoV-2 S pseudoviruses
  1. 1.Construct SARS-CoV-2 S and SARS-CoV-2 S-variants plasmids

    Based on the SARS-CoV-2 S plasmid, S variants constructs were generated by PCR-based mutagenesis using corresponding mutant primers. Then mutant S variants plasmids were confirmed by Sanger sequencing.

  2. 2.Package and harvest the SARS-CoV-2 S pseudoviruses

    To generate the SARS-CoV-2 pseudotyped HIV-1 luciferase virus, 5x106 HEK293T cells in 100mm dish were co-transfected with 6 ug pNL4-3.Luc.R-E- and 6 ug recombinant SARS-CoV-2 (S or S-variants) plasmids using the lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. (A: 1ml OPTI-DMEM + 40 ul lipofectamine 3000; B: 1ml OPTI-DMEM + 40 ul P3000 + Plasmids; Mix A and B, incubate 15 min at R.T., then add it into culture dish).

    The transfected cells were replaced by fresh medium (10 ml) about 8 h later. The supernatant containing SARS-CoV-2 pseudoviruses were harvested at 48 h and 72 h after transfection and filtered through a 0.45 um filter. Concentrate viruses with centrifugal ultrafiltration devices, we used 100 ul to dissolve viruses for one package.

  3. 3.Titration of the SARS-CoV-2 S pseudoviruses

    2x104 HEK293T-hACE2 cells were seeded into 96-well plates and infected with 50 ul medium containing different dilution viruses. After incubation for 12 h, the pseudovirus-containing supernatant was removed and replaced with 100 ul fresh medium. After 48 h post-infection, we measured the pseudoviral transduction using the Luciferase Assay Kit (each add 100 ul) by Relative luminescence units (RLU) of Luciferase activity.

  4. 4.Neutralization assays

    2x104 HEK293T-hACE2cells were seeded into 96-well plates. For the neutralization assay, 50 ul medium containing pseudoviruses (~2x104 RLU) were incubated with serial dilutions of sera samples or RBD antibody for 1 h at 37℃, then added to the 96-well 293T-hACE2 cells. After 12 h of infection, a fresh culture medium was added to each well. Luciferase activity was measured 48 h after infection.

Test

After creating the strains of the variant S proteins, packaging the pseudovirus, and infecting the cell, we are able to test the binding affinity of the virus. To measure the bindings, we lysed the infected cells and put the contents in a medium containing the luciferase substrate. This would produce a reaction resulting in biolumense that can be measured through a luminator. By comparing the different results, we will be able to see which strains produced more binding. Additionally, to test the viability of vaccines, after the cells have been infected, we can introduce a serum containing antibodies. Using the same assay and luminator, we will be able to see how the antibodies prevented the binding. For our test, no luminsense indicates that the cells were not infected by the pseudovirus.

References
  1. “Immune Responses and Immunity to SARS-CoV-2.” European Centre for Disease Prevention and Control, 16 July 2020, www.ecdc.europa.eu/en/covid-19/latest-evidence/immune-responses.
  2. Lei, Changhai, et al. “Neutralization of SARS-CoV-2 Spike Pseudotyped Virus by Recombinant ACE2-Ig.” Nature News, Nature Publishing Group, 24 Apr. 2020, www.nature.com/articles/s41467-020-16048-4.
  3. Martin, Karen. “A Crash Course on Luciferase Assays.” GoldBio, www.goldbio.com/articles/article/a-crash-course-on-luciferase-assays.
  4. National Institute of Allergy and Infectious Diseases. “Vaccine Types.” National Institute of Allergy and Infectious Diseases, U.S. Department of Health and Human Services, www.niaid.nih.gov/research/vaccine-types.
  5. Nascimento, I P, and L C C Leite. “Recombinant Vaccines and the Development of New Vaccine Strategies.” Brazilian Journal of Medical and Biological Research = Revista Brasileira De Pesquisas Medicas e Biologicas, Sociedade Brasileira De Medicina Tropical, Dec. 2012, www.ncbi.nlm.nih.gov/pmc/articles/PMC3854212/.
  6. Robson, B. “COVID-19 Coronavirus Spike Protein Analysis for Synthetic Vaccines, a Peptidomimetic Antagonist, and Therapeutic Drugs, and Analysis of a Proposed Achilles' Heel Conserved Region to Minimize Probability of Escape Mutations and Drug Resistance.” Computers in Biology and Medicine, Elsevier Ltd., June 2020, www.ncbi.nlm.nih.gov/pmc/articles/PMC7151553/.
  7. Saplakoglu, Yasemin. “Coronavirus 'Spike' Protein Just Mapped, Leading Way to Vaccine.” LiveScience, Purch, 19 Feb. 2020, www.livescience.com/coronavirus-spike-protein-structure.html.
  8. Sriram Postdoctoral Fellow, Krishna, et al. “What Is the ACE2 Receptor, How Is It Connected to Coronavirus and Why Might It Be Key to Treating COVID-19? The Experts Explain.” The Conversation, 6 Sept. 2020, theconversation.com/what-is-the-ace2-receptor-how-is-it-connected-to-coronavirus-and-why-might-it-be-key-to-treating-covid-19-the-experts-explain-136928.
  9. The Francis Crick Institute. “Structural Analysis of COVID-19 Spike Protein Provides Insight into Its Evolution.” ScienceDaily, ScienceDaily, 9 July 2020, www.sciencedaily.com/releases/2020/07/200709105122.htm.