Description

Koi fish (Cyprinus rubrofuscus) are familiar ornamental fish synonymous with many Asian cultures as well as having huge decorative appeal worldwide for their many aesthetically pleasing varieties. Like with many high-density fish farms, the breeding of these picturesque fish means increased exposure to infectious disease. One particularly disruptive disease is Koi herpes virus disease (KHVD) which is caused by the highly pathogenic Koi Herpes Virus (KHV) also called cyprinid herpes virus-3. Mass mortality in Koi usually results from infection. Our project aims to develop a stress-free easy to administer vaccine against this virus.

Our plan is to engineer live bacterial vaccines that will be administered to the fish through the oral route. We will develop and engineer systems that allow bacteria to produce antigens only upon arrival in the iron-deficient environment of the fish gut, while we will also design a system to allow bacteria to present the antigen on the cell surface to illicit the immune response.

Background & Inspiration

Koi and our Community

iGEM has been part of our school now for three years. This year we are very happy to have the opportunity to continue to build its legacy at SUIS. Our teacher leaders have always tried to ensure the work the BioBuilder club and iGEM teams do is relevant to the whole school community, thus when the team began to assemble in February and March we had the objective of choosing an area of research that would again get all members of our school and surrounding community together. We began to look at issues in the Asian and Chinese news which could be tackled. One particularly nice day in March we decided to have our team meeting outdoors on our small campuses Chinese traditional garden. This garden is a central part of our school's community, open to all students and faculty and is host to some innovative lessons throughout the year. During this meeting, we were distracted by a member of the school maintenance department who proceeded to feed the numerous colorful koi fish which reside in the gardens pond. Our discussion quickly turned to the relevance of this particular fish to Asian culture, particularly Chinese and Japanese folklore. We entertained our teacher with the story of the koi fish leaping over the Lung Men Dragon Gate in the Yellow river and how if a koi masters these rapids it is said to turn into the dragon. and how it is a symbol of fortune and perseverance. With that we decided to look into the nutrition of these fish and an early idea of using synthetic biology for producing essential nutrients for the fish feed industry was explored.

A short time after beginning the research into koi fish nutrition and health we set up a meeting with the head of operations of the school Mr. Wang, who would be responsible for maintaining the pond and garden. We discussed with Mr. Wang the running costs of the pond and the general procedures in place for maintaining the fishes' health. He informed us that they are fed every morning with food purchased specifically for boosting the fish immune system by containing amylose and lecithin in their ingredients. When asked about the mortality rate of the fish Mr. Wang said it depends on the year and that in 2017 there was a high mortality rate possible due to the warmer summer and the fact they did not respond in time to cleaning the pond. That year they needed to replace the entire fish stock twice costing around 8000 RMB. Since then they have improved their procedures into monitoring the water quality and using better quality food for boosting health. We also asked whether these fish were vaccinated against any specific disease and found out that these koi are relatively cheap and are thus not vaccinated. Their immune defense is based entirely on the adjuvants they receive from their feed.

Koi fish Histoiry and Industry

Koi (Cyprinus rubrofuscus) is actually a mutated form of carp which develops patches of colors which can create a mosaic appearance of white, red, black and gold, depending on the variety. Koi thrived along with feudal dynasty for thousand years in China and people believe that having the spirit of koi near you will attract fortune and spiritual benefits becoming the personification of tenacity and purposefulness. It has become the subject of many traditional art works, especially in Japan, as well as appearing in popular culture in both Asian and the West such as beer advertisements and tattoo designs below.

The breeding of koi for their beautiful colors and patterns can be traced back as early as the 18th century [1], Japanese rice farmers began to cultivate koi because they had unique colors and patterns. Japanese attach great importance to the breeding of koi. The popularity of the Japanese Koi quickly grew all around the world, and koi still represent a symbol of purpose and success in Asia while also becoming a trademark of Asian culture adapted by the west. This has led to breeding all over the world and the creation of over one hundred types of Koi fish which are grouped into thirteen general classes [2].

Nowadays Three markets for koi breeders exists. There is the mass market which is generally for cheap, colorful and almost small fish. Majority of such fish are bought by people who would usually class themselves as pond owners rather than Koi keepers. The fish colors are diverse they grow big and may become friendly in the pond. We found that our own koi pond falls into this category. The smallest market - is what one could variously call the "top-end", the specialist, serious or professional end of the market. This market is ultimately why the hobby exists. It sets standards and establishes views and opinions. Fashionable breeds pictured below such as the Sanke (left image), Showa (center), or Tancho (right image) are sought after. A quick google search for purchasing these breeds will show some are valued in the thousands of dollars. The "middle market", within which most Koi keepers (pond owners) fall. These are people who start off in the hobby in a small way, but soon get the bug. It is a market which itself has much influence on suppliers and people looking to enter or develop in the hobby.

Koi and cyprinid herpes virus-3

With greater demand comes an expansion of current koi fish breeding industry. Now koi fishes are farmed in high density in freshwater and exposed to environment which brings many potential new diseases. Additionally, other factors like environmental stress and international trade also can lead to the emergence and spread of diseases. Koi Herpesvirus Disease (KHVD) is the name of the disease caused by the cyprod herpes virys-3. Our research found that it is a quite common disease that can disrupt the Koi Fish industry. This pathogen causes lethargy and gasping movements of the fishes in shallow water suffering from suffocation. The most prominent lesions for the fishes are the gill, skin, kidney, spleen, liver and gastrointestinal systems, which is caused by the loss of lamellae [3]. Currently, although KHVD has been reported by many fisheries in many countries, there is no effective treatment toward KHVD that minimize the economic loss.

We thus decided to focus on this topic as our iGEM 2019 project. We felt the connection between Asian and Chinese culture and our school's community offered us a topic that our BioBuilder club could continue to increase awareness of iGEM and synthetic biology education through engagement of as many people in our school as possible. Since these early days of this iGEM season we have developed our project through extensive research on vaccine development and delivery in aquaculture.

Oral vaccination application

As part of our project development we explored vaccination of fish. Unlike human, which is susceptible to bacteria and virus due to the various infection routes, fishes, which reside in an aquatic environment, are more likely to be invaded through mucosal route. Vaccination for most aquaculture species are available and is generally achieved through three routes. Injection through the intraperitoneal or intramuscular route, by immersion or dipping, or by oral administration by mixing with food.

Despite of the high efficacy of injection based vaccination, it is associated with stress to the fish due to excessive handling, the same can be said for dipping. The oral route was attractive to us because it is can have comprehensive use (both small and large sized fish can be vaccinated), can be part of a mixed immunity approach by the additional of immune boosting adjuvants (as seen in the fish food above), and poses little harm to fish with respect to animal welfare. Some challenges did present themselves to us however, such as the need to protect our vaccine from antigen degradation when passing through fish’s intestine and discovering the appropriate dosage which constitute a good immunological protection. We saw iGEM as an opportunity to develop a recombinant bacterial vaccine delivery system.

Bacterial Vaccine system

We discovered that there are several approaches we could of taken to develop our vaccine including using:

- Inactivated vaccines

- Attenuated live vaccines

- Recombinant coat protein expressed in bacteria.

- DNA vaccine

We felt synthetic biology gave us an opportunity to design and develop a recombinant bacteria which expressed an antigen to illicit immunity against KHVD. We also thought about developing a DNA vaccine but were unsure whether the regulatory bodies would allow us to peruse it. Thinking of our lab resources and the iGEM medal criteria we felt recombinant bacteria was a perfect strategy. One of the major advantages of using live attenuated vaccine-carriers is that the expression of the antigens can be under the control of a constitutive promoter or be engineered to respond to environmental cues. They can also be encapsulated and thus overcome the obstacles faced by antigen alone since environment at mucosal surfaces consists of degradative enzymes and extreme pH, which prevents free antigens from reaching target cells. Furthermore, free antigens alone are usually less immunogenic due to poor uptake by mucosal cells. Enteric pathogens are known to survive the mucosal environment by their intrinsic protective mechanisms, and hence are able to protect the heterologous antigen carried with them. Since fish is the experimental unit, this kind of bacterial vector is preferred. We quickly got to the literature to find potential antigens we could incorporate into the design.

Our aim thus came to be:

To engineer E Coli, so that it is capable of expressing glycoprotein of ORF81 protein of KHV and producing live vaccine for Cyprinid Herpesvirus 3 to induce protective immunity in koi fish via oral vaccination.

References:

Azizi, A., Ghunaim, H., Sirskyj, D., Fallahi, F., Le, H., & Kumar, A. (2013). Delivery of immunogens to mucosal immune system using an oral inactivated cholera vaccine: A new approach for development of oral vaccines. Human Vaccines & Immunotherapeutics,9(7), 1445-1448.

Embregts, C., & Forlenza, M. (2016). Oral vaccination of fish: Lessons from humans and veterinary species. Developmental and Comparative Immunology,64, 118-137.

Gerdts, V., Mutwiri, G., Tikoo, S., Babiuk, L., & Revues Inra, I. (2006). Mucosal delivery of vaccines in domestic animals. Veterinary Research,37(3), 487-510.

Kotton, Camille N., & Hohmann, Elizabeth L. (2004). Enteric Pathogens as Vaccine Vectors for Foreign Antigen Delivery. Infection and Immunity,72(10), 5535-55347.

Michel, Benjamin, Fournier, Guillaume, Lieffrig, Francois, Costes, Berenice, & Vanderplasschen, Alain. (2010). Cyprinid herpesvirus 3. Emerging Infectious Diseases,16(12), 1835-1843.

Mutoloki, S., Munang'Andu, H., & Evensen, A. (2015). Oral Vaccination of Fish - Antigen Preparations, Uptake, and Immune Induction. Frontiers in Immunology,6, 519.

Shata, M., Stevceva, L., Agwale, S., Lewis, G., & Hone, D. (2000). Recent advances with recombinant bacterial vaccine vectors. Molecular Medicine Today,6(2), 66-71.

Srinivasan, K., Brusic, V., August, T., & Entzeroth, Michael. (2004). New technologies for vaccine development. Drug Development Research,62(4), 383-392.