Implementation

Proposed Implementation

Nanobuddy

With the use of a spray containing a prophylactic engineered probiotic we propose a new therapy against the infection of Dutch poultry with the Highly Pathogenic Avian Influenza (HPAI) virus. Our project Nanobuddy is aimed at the protection of life stock in the Netherlands and reducing the risk of the avian influenza virus mutating and spreading further with the chances of it becoming a new epidemic. Our approach works through the introduction of engineered bacteria back into the lungs and possibly the gastrointestinal track of poultry livestock. Once arrived our tiny ‘buddy’s’ start the secretion of small proteins called ‘nanobodies’ which are small parts of a larger ‘antibody’. Nanobodies similarly as antibodies are able to bind to foreign proteins and neutralise them, our approach would provide the mucus layers of the chickens lungs and intestinal track with an amount of proteins ready to bind to the virus proteins used to enter host cells. By binding to these proteins we expect the virus particle to be hindered or completely blocked in their ability to enter the host cells, thereby preventing infection all together.

Figure 1: Overall scheme of administration

Influenza infection

Influenza infection mainly propagates in the respiratory and/or digestive tracks[2]. Therefore our approach of using an engineered probiotic is expected to target both areas of reproduction of this specific virus. Literature research has shown that both the respiratory and intestinal track are home to our chosen probiotic Limosilactobacillus reuteri[3]. However, to support our choice of chassis we have performed a microbiome analysis on samples obtained from Dutch poultry lungs. This supports the literature findings and can eliminate doubts about differences between studies performed abroad. Ultimately, our plan focuses on the locally implementing our solution, therefore it is very relevant to make sure that our microbe is indeed present in Dutch poultry.

Lung Microbiota Modification

For the creation of our solution we will be working with three different bacteria strains. Initially all of our cloning will be performed using the Escherichia coli strain DH5α, since this lab strain is highly suitable for the incorporation of our genetic constructs. Then we will be using the E. coli strain BL21 which has lower proteolytic activity when compared to our cloning strain[5], this in turn allows us to purify more protein for the testing of our nanobodies and their respective effectiveness towards our prospective goal. Then finally we will be using the bacteria L. reuteri DSM20016 as our carrier organism to provide the protection that we envision for the birds.

In our project we looked into the literature for the most effective promoter sequences and secretion signals that would function within our organism. This to ensure high production of the protective nanobodies and proper secretion into the mucus layer where virus particles need to be blocked from entering.

We foresee however that by adding genetic information into an already native bacterial strain of both the respiratory and intestinal tract, there will be competition between the introduced bacteria and the native ones. An effect which in all likelihood will be detrimental to the ultimate survival of our bacteria in previous mentioned environments, and an effect which will be enhanced due to the strain that the production of our nanobodies will take. However we envision this as an added benefit instead of a down side to our approach, since this will function in addition to our kill-switch as a mode of controlling the retention time of our therapy. For exact retention time of our bacteria and thereby our therapy more research is still needed and also on actual living individuals, which is currently due to time and ethical reasons beyond the scope of our project.

Large-scale Implementation

Once our engineered probiotic is ready for the protection of poultry we envision an easy mode of delivery to the desired poultry through the usage of a spray vaccination machine which produces small droplets. The farmer then only needs to walk through the farm spraying the aerosolised droplets containing the bacteria, when these droplets find their way into the respiratory or intestinal track of the poultry the bacteria are able to survive and can begin by producing the protective nanobodies.

In our project we looked into the possibility of having a temperature and light regulated kill-switch which becomes active when a suppressor chemical like lactose is used up or diffuses enough for the system to become active. Once the kill-switch system becomes active the bacteria will eliminate itself either if it is exposed to light or to a lower temperature than 41 degrees Celsius. We believe that this provides enough protection for the environment against the spread of our probiotic approach. Even when the farmer who uses the product inhales the aerosols due to faulty personal protective equipment or the lack thereof.

We foresee however due to the high mutation rate of influenza viruses that our therapy could become outdated quite quickly when applied on a large scale where a lot of selection pressure arises for the virus to mutate. For this specifically we worked on a modelling project which focuses on the generations of new nanobody sequences which are expected to bind to new or different epitopes of the viral proteins in case of evasion of our strategy through mutation.

The modelling project mainly focuses on three subdivisions; the generation of a database of sequences expected to be useful and realistic in its implementation, the folding of previous mentioned sequences into a structure with high degrees of certainty with the use of a program called NanoNet[4], and the docking of the generated sequences to the desired epitomes with the use of a program called Rosetta. The generation of a nanobody complementary determining regions (CDR) library would require 10^30.88 sequences, which requires an astronomical amount of processing power. By introducing selected mutations in specific amino acid positions within each CDR, based on literature, we reduce the library length to a manageable amount. Also, by further clustering of our data, we can perform a high throughput docking of the folded structures and a sequence-based comparison for binding affinity prediction. With our approach, we’ve provided fundamental steps for a workable system and an initial guide towards more comprehensive models.

Future Prospects

For the future we would like to propose certain experiments and testing to ensure a safe product which can actually be applied to the current avian influenza crisis, without having to worry whether the cure is more dangerous than the affliction.

The additional experiments that we would propose include the integration of all sub parts of the project into the genome of our bacteria, to ensure no regulatory or essential part can be discarded when the bacteria go through mitosis [1]. We would also like to perform clinical trials with our product in a ‘real-life' setting on a small scale with some animals under controlled conditions to see whether Nanobuddy can provide the protection that the poultry so hardly require.

Additionally since a lot of poultry are for the commercial meat industry, we propose experiments that look into the possibility of our bacteria and nanobodies cross-contaminating the meat. We in principle do not expect cross-contamination to occur since the environments where our product should function are not for commercial usage or consumption. Yet additional research into this topic might be valuable since the general public can still have a negative view on the usage of GMO’s, as became clear from the survey that we performed for the Integrated Human Practises part of our project.