Safety & Security

Introduction

Safety and security are two aspects of great concern our team has considered while completing the project. Understanding the potential ramifications of our work and developing ways to avoid negative outcomes for our target population, and the world in general, has been our priority since the beginning of this iGEM cycle. To ensure both needs are met, we have followed an approach that maintains the safety of individuals who will interact with our therapeutic, and secures them against the potentially harmful substances used throughout our project.

Safety Measures

Safety Measures regarding Project Design

Regarding the security of our project and its final outcome, the vaccine we plan to create has several mechanisms that ensure it will not cause harm to human hosts. As the main vehicle of our vaccine, bacteriophages are a safe option because they do not infect human cells unless they are modified to do so (Staquicini et al. 2020). This makes AAVP an appealing option for generating an immune response, capable of modification so it will interact with the cells we want it to. For our project specifically, this modification includes an RGD-4C ligand that allows the AAVP to interact with dendritic cells (DCs) located in the gut. Beyond its modular design, AAVPs have low immunogenicity within human hosts and do not elicit strong immune responses unless they are engineered to do so. This makes them highly safe for a variety of applications including vaccination, drug delivery, and tissue regeneration.

Safety Measures during Lab Experiments

Over the course of our project, there have been several considerations regarding the safety of the experiments and the overall security of its product. First and foremost, the McMasterU team has been committed to safe lab practices over the course of the iGEM cycle, taking care to educate ourselves on lab safety guidelines. While conducting experiments, we have made use of cleaning tools such as cleaning soaps for washing our hands, ethanol and isopropyl alcohol to sterilize our work space prior to experiments, UV light to disinfect surfaces that are commonly used such as biosafety cabinets (BSCs), and bleach to decontaminate solutions and tools prior to disposal. To ensure safe use of bacterial cells and reduction of incidents involving bacterial infection, we have conducted all experiments using E.coli cells in a lab that is acknowledged as containment level 2 (CL2). The CL2 lab we use is highly sterile and specialized for working with samples that can cause mild illness in human hosts. This lab is equipped with the sterilization tools mentioned previously, in addition to BSCs essential for containing bacteria in order to prevent infection.

Safety Measures taken during Modeling

Besides the design of our fdGPS2.1 (RGD-4C) plasmid, we have made sure to be cautious when modeling both the distribution of vaccines in affected areas, and the immune response of the body to our vaccine.

When mapping out locations that are in most need of vaccination against NTDs, we considered several factors that may influence our calculations and predictions. Some of these factors include the age and sex distribution of the population in each area, the cleaning practices of people, and the potential interactions people have with organisms that carry the NTDs. These factors cannot be understated and must be accounted for, otherwise our model would provide inaccurate information but could also result in the potential harm and/or death of individuals who don’t receive aid if our model is used in real-world applications. Likewise, modeling the immune response to our phage carries with it risks that could lead to real-world consequences. For instance, our model may yield data that suggest our vaccine may generate a mild immune response within the body. However, if the model is not carefully designed and developed, it could underscore the real risk our therapeutic may present to patients who need aid. This can be seen with calculations regarding cytokines, which have the potential of proving fatal if their predicted concentrations are lower than their actual concentrations during an immune response to our vaccine (Figure 2). Therefore, we have spent considerable time designing both models, making sure to not overlook any issues that may arise during modeling.

Figure 1: A prediction of the expected antibody response upon injection of the TSOL18 and EG95 proteins. The black line depicts the level of antigen (the injected proteins) while the other lines indicate different types of antibodies. This graphic was obtained from a C-ImmSim simulation (Rapin et al. 2010).

Security Measures

Security Measures of Plasmid Design in Real World Application

While constructing the plasmid that would generate our phage, we also took care to maintain security of our project and the environment around us. Specifically, we worked with antibiotics such as ampicillin and tetracycline, which could generate antibiotic-resistant bacteria if not used carefully. Given the potential risks, antibiotics were only used during experiments that took place within the CL2 lab to minimize the potential increase in prevalence of antibiotic-resistant bacteria in the event of a spill. When disposing of solids or solutions containing antibiotics, we informed the lab technicians and allowed them to properly dispose of our waste. This ensured that lab waste was handled professionally and not disposed of down a drain, in the garbage, or elsewhere that may lead to an increased likelihood of antibiotic-resistant bacteria.

Figure 2: Design schematic of fdGPS2.1 (RGD-4C) with ampicillin resistance genes (designed using Benchling).