Project Description

Preamble

Three profoundly destabilizing scientific ideas ricochet through the twenty-first century. When combined, this trifecta – the atom, the byte, and the gene – elucidate the potential of future medical technologies. As is sciences’ impulse to understand nature, it is technology’s nature to manipulate it. Our project aims to apply these inquisitive cornerstones of the scientific method to synthesize an articulate and novel therapeutic technology.

Over 1 billion people are disproportionately affected by diseases caused by pathogens found in impoverished communities. Despite affecting a significant portion of the global population, these tropical diseases fly under the radar, with limited funding for research in addition to poor education and awareness. These 20 diseases are now categorized as "Neglected Tropical Diseases", or NTDs. The world has committed to eradicating NTDs by the year 2030, as part of the United Nations Sustainable Development Goals. We have identified two NTDs, namely Cysticercosis and Echinococcosis, which demonstrate high prevalence, severe symptoms, difficulty in treatment, and lack of available vaccines. The former is spread through infected pig liver tissue that gets consumed via pork, whereas the latter causes infection through parasitic tapeworm eggs that can be found in any type of food, along with contaminated water or soil. Both are capable of developing into widespread infection, leading to extensive organ damage and death. Globally, it is estimated that cysticercosis and echinococcosis cause over 20,000 deaths and 3 million disability-adjusted life-years annually. These diseases not only significantly impact the health of the communities in which they are prevalent; they perpetuate a cycle of poverty and suffering. Children cannot go to school and receive proper education, adults cannot work to support their families, and these communities suffer grave social and economic consequences under the burden of these debilitating illnesses. Our inspiration stems from the urgent need to protect high-risk communities from the severe health consequences of NTDs.

Despite existing vaccines for livestock, a human vaccine for cysticercosis and echinococcosis remains elusive. A vaccine that targets early stages in the pathogens’ life cycles would be important for conferring protection against these diseases in endemic areas. Our goal is to recognize the antigens that present on the larval forms of these parasites and target them with a vaccine before the parasites are given the opportunity to grow and spread throughout the body (causing dangers to the lungs, liver, and brain, alongside other bodily organs). As such, we aim to design an orally-administered vaccine to target the small intestine, where the larval forms of these parasites reside. By utilizing Adeno-Associated Virus Phage (AAVP) technology, we strive to develop a groundbreaking multi-vaccine that targets both diseases simultaneously. AAVPs show potential in gene therapy given their non-pathogenicity, ability to infect non-dividing cells and most importantly their ability to stably integrate into the host cell’s genome at specific sites. This predictable behavior makes them excellent vectors for administering a vaccine. By leveraging the unique properties of AAVPs, including their hybrid nature combining bacteriophage and mammalian virus properties, we aim to overcome limitations of existing delivery systems to develop safe and effective vaccines.

We plan to equip our AAVP vaccine with a plasmid that encodes for antigens present on the oncospheres from both diseases. By utilizing the AAVP vector, we aim to initiate a robust immune response, combining humoral immunity (B-cell-produced antibodies) and cell-mediated immunity (cytotoxic T-cells and macrophages). Our iGEM team has already developed AAVP-related technology in the past; our project last year involved creating an AAVP that acted as an oncolytic to target B cell lymphomas. Now, we seek to lay the foundation for AAVP vaccine technology as a safe, effective, and low-cost method of producing therapeutics for NTD-impacted communities. In addition to our scientific endeavors, our team aims to educate students, organizations, experts, and the general public about NTDs and their impact.

Introduction

Neglected Tropical Diseases (NTDs) are a set of illnesses that disproportionately affect many third world countries around the world. Each disease brings its own challenges, but all include debilitating symptoms and elusive pathogens that make treatment difficult.

These diseases not only significantly impact the health of the communities in which they are prevalent; they perpetuate a cycle of poverty and suffering. Children cannot go to school and receive proper education, adults cannot work to support their families, and these communities suffer grave social and economic consequences under the burden of these illnesses. Our inspiration stems from the urgent need to protect high-risk communities from the severe health consequences of NTDs.

Two of these diseases stand out as being particularly troublesome: Cysticercosis and Echinococcosis, which cause infection in human hosts via the transmission of the parasite’s eggs in feces or unsanitary drinking water. Once inside the host’s small intestine, the eggs hatch into larval forms, called oncospheres. Oncospheres burrow through the epithelial wall of the small intestine and into the bloodstream, where they travel to other areas of the body.

Upon settling into a tissue, the oncospheres form cysts which can degrade and cause symptoms including soreness, irritation, seizures, and even mortality. Despite current treatments for formed cysts, targeting the oncospheres within the small intestine remains a major obstacle to defending against these diseases. As such, McMasterU has decided to tackle this problem, believing that it is entirely unjust for billions of people to suffer from these ailments because they are unable to access the tools and resources necessary to prevent them.

Beyond this, we wish to contribute to the ever expanding field of synthetic biology by developing a novel treatment that can be utilized to not only prevent the diseases mentioned, but contribute to future therapeutics targeting similarly neglected illnesses.

Given our goal to treat Cysticercosis and Echinococcosis, we have worked to develop an adeno-associated virus phage (AAVP) vector that will carry antigens of both diseases on its coat proteins. Upon interacting with the immune system, the antigens will be presented to T cells via antigen-presenting cells (APCs) and begin a cascade that will result in inflammation in the small intestine. Once inflammation occurs, the oncospheres will be unable to bypass the intestinal wall and be forced into the feces by its host.

Ultimately, our novel therapeutic will prevent the parasite from forming systems inside the host’s tissue, which can result in eventual death. We believe our vaccine will make significant contributions to the way therapeutics are produced, being both novel to the scientific field and modular to allow for modifications when necessary.

The Parasite and How it Infects

The two parasitic diseases we are targeting are Cysticercosis and Echinococcosis. Both involve infection of the host with their larval eggs which then hatch into oncospheres (Figure 1) (Verastegui et al. 2007). Hatching of the oncospheres appears to involve interactions with gastric and intestinal fluids of the host, suggesting the eggs have chemical receptors that respond to the environment by hatching the parasite (Law, 1968).

Figure 1: Anatomical drawing of larval oncosphere

Upon hatching, viable oncospheres migrate towards the intestinal wall of the small intestine and begin the process of penetrating through the mucosal layer. Although this process is not well understood, it appears the secretion of proteolytic enzymes is utilized to degrade proteins found within the intestinal mucosal layer, allowing the oncosphere to make contact with endothelial cells (Zimic et al. 2007). Interaction and adhesion of the oncospheres to endothelial cells is facilitated by microvilli found on the surface of the oncospheres, allowing them to maintain contact long enough for them to penetrate (Verastegui et al. 2007). This process likely occurs with the help of hooks found within the inner envelope of the larvae, allowing them to mechanically cleave the cell membrane, destroying the cell, and clear a path towards the bloodstream.

Once inside the bloodstream, oncospheres travel to different areas of the body until they find a viable tissue to inhabit. To find suitable tissue for implantation, oncospheres environmental cues such as chemical signals to determine where they end up migrating before settling (Swiderski et al. 2018). Again, the use of microvilli and hooks found on the oncosphere allow them to adhere to their target tissue, and then begin to develop into cysts within said tissue (Prodjinotho et al. 2020). Once this happens, these cysts may be attacked by the immune system, causing soreness, irritation, inflammation, and even seizures within the host.

Current Treatments and Obstacles

Given the severity of both diseases, it is imperative that patients receive treatment as soon as possible. However, this may be difficult to do depending on the country the affected individual resides in and their resources for treatment. Moreover, current treatments face the obstacle of not being able to stop the oncospheres of cysticercosis and echinococcosis at its source. Stopping disease progression as early as possible is critical to preserving human health, and this is why a new solution for these NTDs are necessary (Garcia et al., 2014).

With regard to cysticercosis, current treatment strategies rely on the use of anthelmintics, or anti-parasitic drugs, in combination with anti-inflammatory drugs, such as corticosteroids (Prodjinotho et al., 2020). Anthelmintics for cysticercosis include albendazole and praziquantel. Corticosteroids can also be used to combat both diseases, some including prednisolone and dexamethasone. Other medications, such as antiepileptic or anticonvulsant drugs, may also be prescribed to manage seizures. However, surgery is required in cases where the patient does not respond to other treatment methods or has excessive swelling in critical areas such as the brain (Cleveland Clinic, 2022). Surgical procedures include classical extraction, endoscopic surgery, ventricle-peritoneal shunts (VPS) placement, and endoscopic third ventriculostomy (ETV) among others (Hamamoto Filho et al., 2022). Additionally, these are relatively invasive procedures that could have potentially been avoided had earlier treatment been available. In a few cases, treatment is not required at all because no symptoms present themselves or there is no detriment to the quality of the life of the individual.

Current treatment plans for echinococcosis involve surgery with or without the use of chemotherapy. Additionally, treatment also includes antiparasitic drugs and the use of various surgical techniques, much like with cysticercosis. It has been noted that benzimidazoles given to patients for a minimum of two years alongside monitoring their progress for 10 or more years is an effective mode of treatment (Centers for Disease Control and Prevention, 2020). As such, current treatments for echinococcosis can be invasive, inefficient, and lengthy.

Although the current therapeutics used to treat Cysticercosis and Echinococcosis have provided relief for patients, they ultimately do not cure them and can have unwanted side effects. The use of anthelmintic medications can cause allergic reactions in patients, and may also result in the parasites developing drug resistance to the medication (Chai, 2013) As well, the surgical route is more invasive and can cause more grievance than the relief it may provide for the patient. Moreover, these treatments face the obstacle of being unable to stop Cysticercosis and Echinococcosis at its source by targeting and destroying their oncospheres. Therefore, a new solution for these NTDs is necessary if we are to stop the spread of both diseases and preserve the health of people living in these areas (Garcia et al., 2014).

Our Project - AAVP Vaccine

Using our knowledge of parasites and how the immune system works, our team plans to develop a vaccine that will aid patients in mounting an immune response to the parasitic oncospheres that generate symptoms for both Cysticercosis and Echinococcosis. Our vaccine will make use of the adeno-associated virus phage (AAVP), a chimeric virus consisting of the fd filamentous phage and the adeno-associated virus (AAV) that co-infects mammalian cells with other viruses (Hajitou et al. 2007). Given the modularity of the phage, its specificity for target cells and its efficiency in delivering therapeutics, we believe using the AAVP is a great opportunity to generate a safe and efficient vaccine while providing a framework for future iGEM teams to explore other therapeutics using the AAVP.

Figure 2: Schematic design of fdGPS2.1(RGD4C)-Amp plasmid (image generated on Benchling).

To help our phage arrive at the gut, we determined that delivering it orally was the best option because oral vaccines have great potential for inducing protective responses at mucosal layers and systemically, are flexible with storage conditions and can remain viable in various environmental conditions, and are both easier ingestible and less invasive compared to most other vaccines (i.e., do not require a medical professional to administer the vaccine via needle) (Davitte & Lavel, 2015). As our vaccine vehicle, we plan to use poly (lactic-co-glycolic acid) (PLGA) nanoparticles to deliver our phage, serving as flexible delivery vehicles that can be modified to withstand degradation until nearing the target, and generate nanoparticles that increase the total surface area of the vaccine compared to microparticles (Banerjee et al. 2016).

How Our Vaccine Works

Since we are utilizing the oral route, our vaccine must travel through several components of the digestive system before reaching its target, the Peyer’s Patches found in the small intestine (Van Kruiningen et al. 2002). The first major obstacle was the mouth and esophagus, which produce enzymes and mechanical force, respectively, that may damage the vaccine before it arrives in the stomach. As well, the low pH of the stomach made bypassing it challenging as most materials would decompose. However, we plan to modify the PLGA nanoparticles we are using to reduce degradation of the phage via crosslinking of its molecules to provide greater stability (Banerjee et al. 2016). After passing through the stomach, the vaccine would enter the small intestine, which is an environment that reaches a pH of approximately 7.5 over the course of its tract (Evans et al. 1988). Our vaccine must travel to the distal ileum (third segment of the small intestines), where approximately 46% of Peyer’s Patches tissue are located, having a greater potential to elicit an immune response (Van Kruiningen et al. 2002). This is a challenging task but is possible with the help of our modified PLGA nanoparticles which can be adjusted to degrade at higher pH values, releasing our phage once it enters the distal ileum (Banerjee et al. 2016).

Once our phage is released, it will interact with dendrite extensions that belong to dendritic cells that reside in the Peyer’s Patches (Lelouard et al. 2012). Our binding peptide, RGD4C, will bind to a dendritic cell subset called CD11c which play a major role in priming Th2 immune response that can expel the parasites (Rijt et al. 2005). After binding, the dendritic cell engulfs the phage and presents our antigens, EG95 & TSOL18, on its MHC class II to T-helper (Th) cells found in the Peyer’s Patches. This allows for T cells that pass by to sample the antigen that is presented on the MHC-II. This process is made easier by our adjuvant, which signals that this antigen is foreign and must be taken care of (Salvador et al. 2012). This interaction signals to the Th-cells that a pathogen is present and must be eliminated, resulting in an immune response being mounted against the parasitic oncospheres (Bonnardel et al. 2015).

Some major cytokines, which are chemical messengers that play a role in activating affector cells, are produced by CD11c. Specifically those that will prime Th cells to elicit Th2 immune pathway, this includes IL-4, IL-5, IL-9, and IL-13. These cytokines also induce the second player, B cell. They promote the proliferation of B cell and shift its antibody subset to Immunoglobin E (IgE) which will then activate more immune cells including eosinophil, mast cells, and basophil that are the primary players during a parasitic infection (Walker & McKenzie. 2017).

Th cells are essential during immune response as they determine the fate of the battle. Unless they sense the danger is over, they will keep activating and stimulating more immune cells (Dettmer. 2021). After the infection is over, they will begin to enter the contraction period, where most of them will kill themselves. This is to preserve energy and avoid unnecessary cell deaths. Only less than 10% of Th cells live as memory cells (Farber et al. 2014). Meaning when the same pathogen reinfects the body, it will be able to act faster without the whole process of DC activation all over again. Cytokines IL-7 and IL-15 will keep these memory cells proliferating slowly yet continuously, even without constant infection, meaning that vaccines designed to target T cells can create independent and long-lasting immunity (Geginat et al. 2001). Besides from memory T cells, memory B cells will also be produced at the end of the vaccine exposure where they will settle down in the lymph nodes and waiting for the actual parasites to infect the body (Dettmer. 2021).

Figure 3: Diagram of immunological pathway involved in combating parasitic diseases.

Future Directions

Although we could only complete so many tasks within one iGEM cycle, we have several plans for future research and hope to implement them if given the opportunity. Firstly, we will continue to optimize our protocols until we successfully assemble our AAVP plasmid. Once we achieve this, we will transform our plasmid into competent bacterial cells and generate a phage product which will undergo morphological and functional analysis to determine if it is viable for vaccine use.

Our dry lab subgroup worked on several projects that were not completed during the cycle but are a priority for future research. One of these involves applying what we know about our vaccine vehicle to the lab by experimenting with the composition of the PLGA nanoparticles to generate a viable vehicle. We plan to use crosslinking methods that will optimize stability as it passes through the digestive system, while also maintaining its immunogenicity to aid in eliciting an immune response in the body (Banerjee et al. 2016). Another project our team will investigate is building a cost-efficient bioreactor to generate phage particles in third-world countries. Doing this will allow these areas to produce our vaccines to fight parasitic infection and reduce the overall mortality rate.

Beyond this, our team will continue to reach out to non-profit organizations that aid third-world countries and gather more information on the factors associated with Cysticercosis and Echinococcosis. Our hope is that we will coordinate collaborations with these groups to spread information about NTDs, and gather funding for these locations to provide them with resources and tools to combat these ailments.