Description

Abstract

The highly pathogenic avian influenza virus sweeps through the world. Current biosecurity and prevention strategies (e.g. intravenous vaccination) are insufficient and logistical nightmares, resulting in deaths of millions of poultry worldwide.

With Nanobuddy we propose a novel solution by engineering Limosilactobacillus reuteri, a native species in the lung and intestinal microbiome of chickens, to secrete broadly neutralizing nanobodies against bird flu. These nanobodies bind to a conserved region of the virus’s hemagglutinin receptor, blocking it from entering cells.

We researched several facets of our project: we investigated L. reuteri’s ability to secrete nanobodies and explored via lung-microbiomics L. reuteri’s presence in Dutch chickens. We explored the efficacy of the anti-avian influenza nanobodies and proposed an in-silico nanobody design workflow.

Additionally, we developed a temperature sensitive biosafety module and performed extensive human practice to refine our final product. Nanobuddy, administered as a convenient spray, might revolutionize the fight against avian influenza.

Scope of the Problem

What is Avian Influenza?

Avian influenza, also known as bird flu, is a collective title for various influenza A viruses that infect birds. Belonging to the family Orthomyxoviridae, these single-stranded, negative-sense RNA viruses threaten both human and animal health alike [1]. The viruses are transmitted by the feces of infected wild aquatic birds, such as geese, swans, ducks, and seagulls. Several bird species are becoming increasingly more susceptible to avian influenza, including chickens, waders, turkeys and starlings [2]. Factors which influence the susceptibility of avian influenza spreading to other bird species, such as poultry, are shown in figure 1.

Figure 1: Factors increasing the risk of spread of avian influenza to domestic birds. Adapted from [2]

The influenza A virus contains 8 RNA segments, comprising a genome encoding 11 viral proteins. Out of these, the receptor-binding membrane fusion glycoprotein hemagglutinin (HA) and the enzyme neuraminidase (NA) respectively facilitate the attachment and release in the host cells. The different subtypes of avian influenza are classified according to the antigenicity of these two surface glycoproteins [3, 4]. So far eleven NA (N1-N11) and eighteen HA (H1-H18) subtypes have been found in nature, H17N10 and H18N11 being the latest discoveries. Combination of the different NA and HA subtypes exhibit two types of pathogenicity, resulting in two types of infections – HPAI (highly pathogenic avian influenza) and LPAI (low pathogenic avian influenza). Although most avian influenza viruses are of the LPAI type, the H5 and H7 viruses, once infecting poultry, have evolved to become a HPAI virus causing 100% mortality with hemorrhaging, endothelial cell infection and oedema [1].

The attachment and binding of hemagglutinin is unique to species, resulting in the differences between human specific influenza and avian influenza. For survival of the virus, regular antigenic alterations take place in the hemagglutinin. Minor antigenic changes occur a few years apart to avoid antibody neutralization. This is known as antigenic drift and is responsible for formation of new avian influenza variants and epidemics. Major antigenic changes are caused by gene reassortment and are known as antigenic shifts. This results in the virus being able to affect another species, increasing the risk of new pandemics [4].

Figure 2. Genetic reassortment in animals. Adapted from [1] Created with BioRender.com

Avian flu in Mammals

Once HPAI viruses have emerged in poultry, more specifically in chickens, they can easily spread to avian or non-avian hosts. Humans are not an exception here. HPAI viruses can diversify themselves due to the process of genetic reassortment [5], which is possible because of the segmented structure of the influenza genome. If a chicken is infected with two individual strains of influenza A viruses, newly assembled viral particles will be produced, each originating from another strain. The reassortant will contain properties of both parental strains. This results in rapid evolution of influenza viruses, increasing the probability of zoonotic infections [6].

Cross-species transmissions to humans have been seen from wild birds, swine, and poultry. Earlier swine was considered to be a necessary intermediate for the virus to be transmitted to humans but direct cases of transmission from avian sources were seen in the late 90s [7].

The European Center for Disease Prevention and Control states a few criteria which are essential in diagnosing avian influenza in humans. The person usually has fever and symptoms of acute respiratory infection or dies from an unexplained acute respiratory illness. Furthermore, there should be detection of influenza A nucleic acid or specific antibody response in the clinical specimen of the patient [8].

The first case of HPAI H5N1 transmission to humans came out in 1997. Since then, the H5, H6, H7, H9 and H10 have been shown to cross species barriers. Out of these HPAI H5N1 and LPAI H7N9 are particularly concerning due to the severity of infection caused [1]. Figure 3 shows countries with zoonotic events.

Figure 3. Countries with zoonotic infections. Brackets show the number of confirmed cases. Adapted from [1]

The first case of inter species transmission to humans was reported in Hong Kong in 1997 as mentioned earlier. Since then several other cases have been reported throughout the major continents, especially from contact with poultry. In a particular case in China, 375 human infections had been reported of which 115 fatalities. Even though cases of human infections have been seen emerging from animals, no cases of human-to-human transmission has been reported yet [7].

The virus can enter humans through multiple ways. These include rubbing the eye, inhalation of dust or droplets and eating undercooked meat of infected animals. A common misconception is that any contact with affected poultry can infect humans with the influenza virus. The virus spreads to humans only with intensive direct contact like working in live bird markets and slaughter houses or playing with infected birds under unhygienic conditions [9].

The risk of the virus spreading among humans might be low, but immunologists are raising awareness that we should address this epidemic quickly to prevent the virus from uncontrollably mutating. For example, the other HPAI H7 variant only needs 3 mutations to become specific to humans [20]. This might not be the case for the H5 variant, but there are increasing concerns about the possibility of zoonotic infections that might lead to the next pandemic [21, 22].

Agriculture and Avian Influenza in the Netherlands

With 2,398 outbreaks in poultry and over 46 million birds culled, the current HPAI epidemic season is the largest one observed in Europe in history. The Netherlands ranks second out of all other European countries in the number of outbreaks. Between March 16th and June 10th 2022, there were 629,294 affected poultry in 13 establishments [10]. Figure 4 shows the number of HPAI affected poultry and establishments in Europe between the aforementioned dates.

Figure 4. Number of affected establishments and poultry in Europe between 16 March and 10 June 2022. Adapted from [7]

The poultry in The Netherlands are mostly affected by the H5N1 strain [10]. Figure 5 shows the number of H5N1 infections in the current and last avian influenza seasons. The high number of poultry culled in the EU has been attributed to the fact that as soon as a H5 or H7 virus outbreak is detected on a farm, all its poultry is culled immediately [11]. Although this move has been cited as controversial by many due to the killing of healthy birds, it helps to keep the outbreaks in check.

Figure 5. Number of animals culled at poultry farms in the Netherlands for avian influenza season 2020-2021 and 2021-2022. Data on 2021-2022 available as of September 9 2022. Adapted from [8, 9].

In a country like the Netherlands, where 19% of the land is covered with water and the coastline is over 450 km long [14], bird flu is an unavoidable problem. The poultry farmers and the government alike are currently not adept at handling the problem. For example, there have been talks about the government closing down poultry farms in the Dutch wetlands [15]. As mentioned before, the avian influenza virus spreads to poultry from wild water birds. Considering the massive coastline of the country, banning poultry farms in the wetlands should reduce the number of avian influenza outbreaks in poultry but it would ultimately lead to loss of livelihood and imbalance in the supply of poultry. Although effective, this approach is running away from the problem rather than solving it. Dutch farmers have been fencing off their poultry and looking into new ways to keep wild birds away. Strict biosecurity measures have been taken in the farms to keep everything hygienic to keep the virus from entering, without much avail [15]. The problem got so big that HPAI has become endemic in the EU. Figure 6 shows how animals need to be culled throughout the whole year to prevent outbreaks. We are not talking about avian flu seasons anymore, and are approaching a situation where it would be present permanently. These biosecurity protocols include various hygiene measures, but also the culling and caging of poultry, as well as transport restrictions for the industry. Considering that these biosecurity measures are not working efficiently enough to effectively keep the virus out, governments and industry alike are looking at alternatives such as vaccination to solve this worldwide problem.

Figure 6. Animals culled in the Netherlands as a result of avian influenza outbreaks in 2021/2022. Adapted from [12]

Current Strategy: Vaccination

Poultry vaccination programs have been carried out to address the ever increasing emergence of novel HPAI viruses. This tactic serves as an effective tool against the spread of HPAI viruses, more specifically in connection with sudden outbreaks of influenza [5]. In Vietnam, the vaccination program has drastically reduced the extent of HPAI H5N1 spread in the poultry business by combining it with an improved biosecurity program [16, 17]. Unfortunately, poultry vaccination programs did not succeed yet in eradicating influenza viruses in the poultry industry. Successful implementation of a vaccination program faces several challenges which have detained the process of influenza eradication [5].

In some circumstances, the significant labor expenses related to vaccination can be prohibitive. This is especially true for broiler chickens, whose brief lifespan discourages the use of vaccines [5]. Numerous elements can also affect vaccine effectiveness and lessen vaccine-induced protection, including co-infection with other diseases, nutritional status of the bird, and age [2]. Furthermore, due to the reassortment changes in the virus over time, poultry vaccines require frequent updating. Concerns over the vaccination’s potential to hasten this process is also raised by the absence of sterilizing immunity, which requires neutralizing antibodies at the site of infection that typically completely prevents infection of respiratory viruses [18]. Lastly, the vaccination process has significant effects on poultry export markets since countries that vaccinate their chickens against influenza viruses frequently face a ban from other countries due to concerns that vaccination may mask an active infection [2]. Promoting trade is also the reason that the EU has banned vaccination of livestock all together to this date; remember this, it will be important later.

The Wageningen Bioveterinary Research situated in Wageningen, has been constantly working on developing new solutions to the bird flu problems here in the Netherlands. They had developed inactivated vaccines for H5 and H7 sub types, but they are not useful for emergency vaccinations due to each chicken needing to be vaccinated separately with multiple doses [11]. Now that influenza season lasts year long [15], inactivated vaccines are suboptimal. They are also working on other strategies related to live attenuated vaccines, live vector vaccines and synthetic vaccines each with their own challenges. Though the main challenge, as mentioned by Riks Maas from WBR, is to comply with Europe’s vaccination regulations [11]. It is not possible to vaccinate your poultry using a vaccine for avian influenza due to these regulations. These regulations were initially implemented because viral disease had been mostly eradicated in 1991 [23]. Therefore, we could stop with vaccinating animals: saving money on vaccines and manpower, as well as making trade easier within EU countries. When an outbreak would take place, the strategy would be to cull and/or cage the livestock in the nearby area, and lay down transport restrictions surrounding the affected areas. The reason trade becomes easier can be found back in the ability to make a distinction between infected, and vaccinated meat before crossing the borders. This is also referred to as the DIVA principle. When animals are vaccinated, it is difficult to make this distinction, which hindres trade. Thirty years later, we can safely say that this policy is severely outdated and should change with regards to avian influenza, as this problem cannot physically, and more importantly ethically be prevented through current strategies [31].

Besides several vaccination programmes, no other solution has been implemented yet to protect poultry against influenza A viruses. Over the course of time, researchers have looked into different biological fields in an attempt to tackle the limitations of vaccination programmes. Overall, vaccination is not always the best course of action due to the fact that it would take time for the birds to gain immunity, giving time for the virus to affect them.

Our Solution

A Novel Nanotherapeutic Prevention Strategy Against Highly Pathogenic Avian Influenza

The avian influenza crisis has never been more relevant than right now. Rapidly increasing numbers of animals and people are affected, while there is no solution ready to be implemented. This problem involves a lot of different stakeholders, perspectives, needs, and safety concerns. With Nanobuddy, we therefore aimed to create a safe, responsible, and most importantly: effective solution to the avian influenza crisis in the poultry industry, to save millions of needlessly culled poultry lives.

We aimed to design and engineer an alternative to conventional vaccination techniques, providing the world the possibility to properly address and tackle this crisis. Nanobuddy provides an upscalable nanotherapeutic prevention strategy that can rapidly protect poultry against the highly pathogenic avian influenza virus variant (H5N1): the main virus strain raging through the Netherlands, as well as the rest of the world.

We take a whole different approach to protecting poultry, moving away from the popular viral prevention framework that is used to address most epidemics. Rather than utilizing the immune system of the poultry, our Nanobuddies provide a protective layer of our nanotherapeutic to disease transmission hotspots, which can prevent poultry from infection by the virus. Here, we will discuss the specifics and an elaborate explanation to our solution.

We are using antigen neutralizing single domain antibodies, also known as nanobodies, which are the variable domains of the heavy chain antibody often derived from camelid species (figure 7) [24]. These nanobodies are very small (15 kDa) and are therefore often able to bind to more conserved regions of antigens. Due to their small size, strict monomeric state, robustness, and easy tailoring, they are being used more frequently by research groups to design innovative multi-domain constructs to explore novel applications. A visual representation of nanobody and antibodies is included in figure 7. The nanobody that we use, the alpaca derived R1a-B6 [25], has repeatedly proven in literature to have broadly neutralizing capabilities in vivo against highly pathogenic avian influenza H5N1, H2N2 and H9N2 [25, 26 27]. This nanobody binds to a conserved part of the Hemaglutanin receptor, therefore being less prone to becoming redundant as the result of a mutation in the viral epitope. Moreover, one of these studies has also shown that a single intramuscular injection in mice of an adeno-associated viral (AAV) vector encoding R1a-B6 nanobodies was able to provide complete protection against lethal challenge with both H1N1 and avian influenza H5N1 [26].

Figure 7. Antibody and nanobody structures. Adapted from [28] and created using BioRender.

When intranasally administrating another nanobody, researchers have demonstrated how the nanobodies were effectively able to control homologous influenza A virus replication in vivo [19]. These researchers found that the intranasal administration of nanobodies strongly reduced H5N1 virus replication in the lungs and protected mice from morbidity and mortality after a lethal challenge with the H5N1 influenza virus. Moreover, a bivalent version of the nanobody they used, as visualized in figure 7 on the far right, was at least 60-fold more effective than the monovalent Nanobody in controlling virus replication [19]. These results are also complemented by studies with similar results doing the same research on SARS-CoV-2 [32, 33, 34, 36], and the respiratory syncytial virus [35].

Considering that intranasal administration of nanobodies has strong effects in the prevention of various diseases, and that there are nanobodies which are able to broadly neutralize highly pathogenic avian influenza viruses, we decided to use these tools to protect poultry against the virus. We will use a microbe, Limosilactobacillus reuteri, to directly administer nanobodies in the microbiomes of poultry themselves. The microbe will mainly colonize the respiratory tract, and secondly the gastrointestinal tract. We have decided to target these specific ‘disease transmission hotspots’ as these are the locations where poultry is considered to be most susceptible for viral infection and transmission [37, 38, 39].

Protein excretion of very large proteins is difficult, therefore a very useful and powerful feature of our nanobodies is their small size. As our delivery organism we use L. reuteri, and make use of secretion signals native to this species to extracellularly secrete nanobodies. We take advantage of a L. reuteri native signal peptide [29]. Literature suggests that using the the first 27 codons from the Cnb region (a collagen-binding protein), which functions as a transporter, is able to excrete proteins with very high efficiency (named S9 in the original study) [29]. Protein secretion using the collagen-protein is thought to be carried out using an ABC-transporter system in L. reuteri [30]. We will use this signalling peptide to secrete our nanobodies in L. reuteri as is graphically shown in figure 8. Using this native excretion pathway, our modified L. reuteri will be able to excrete nanobodies in the relevant microbiomes of poultry.

Figure 8. An overview of the L. reuteri signalling pathway used to secrete nanobodies.

We are using mainly using L. reuteri because of two reasons: their natural presence in the microbiome, and their probiotic nature. L. reuteri is a well-studied probiotic (Generally Recognized As Safe: GRAS labeled) bacteria which colonizes a large group of mammals [40]. In addition, a multitude of articles show the presence of L. reuteri in the chicken lung microbiome [41, 42, 43, 45] and the chicken gastrointestinal tract [46, 47, 48]. Some of these articles also show the enduring presence of L. reuteri throughout the lifetime of poultry living in different environments, further elucidating that this bacteria is able to live in the lung microbiome relatively with stability [41, 43]. L. reuteri is also repeatedly present in microbiomes of other major species of poultry besides chickens. Namely, turkey gastrointestinal tracts [50], duck gastrointestinal tracts [51], and goose gastrointestinal tracts [49]. Lastly, L. reuteri shows immunostimulatory functions when used as a lung probiotic in chickens, further demonstrating that L. reuteri is an excellent host organism for our applications [44]; showing both effective and safe colonization when administered.

To administer our nanobody secreting L. reuteri strain to the poultry, we will make use of a spray. For this spray, already existing spray-vaccination infrastructure can be used (e.g. the spray machines used to disperse doses of regular vaccine), giving a lot of farmers and veterinarians the ability to quickly implement this solution if they would like to. Given the above provided literature, the nanobodies secreted by our bacteria will provide a wall of protection. This will be able to block the virus from infecting the poultry. Using our nanotherapeutic prevention strategy, farmers are easily able to quickly protect their flocks against the highly pathogenic virus.

This strategy might have some potential upsides to using a conventional vaccination strategy, as we are not making use of the immune systems of the poultry themselves. This means that we are not dependent on the immune response, and would most likely only need a single dose of spray. This is also useful considering the ability that has to be made between infected and vaccinated poultry. Since we are not vaccinating, and are only administering a nanotherapeutic, this distinction can be made without a problem. In addition, our solution might improve on vaccination considering that the virus will be targeted and neutralized before it actually enters the chicken cells. After regular vaccination, the virus can still enter the host, replicate, and possibly spread even further. A vaccine strategy is in fact mostly focused on treating the therapeutic symptoms of a disease, decreasing mortality and morbidity rates, rather than preventing the infection itself. Using our strategy, the chance of the virus actually making it to the host should be diminished significantly, providing another edge over vaccinations. Lastly, our solution could also work together with already existing vaccination strategies, further improving protection rates against the highly pathogenic virus. There is no reason why our strategy cannot work together with a vaccination strategy. As our stakeholders confirmed, there will not be a single solution to this world-wide problem. More likely, it will be a combination of several solutions taking infections rate down more effectively together. Our solution can be a good addition to this multi-faceted approach to get rid of this epidemic once and for all.

In our project, we are testing and developing this solution through four main pillars:

1. Choie of host-organism:
2. Ensure that our bacteria indeed lives in the Dutch poultry microbiome by carrying out a lung microbiome analysis, and review the safety of using it for our purposes.
3. Nanobody secretion:
4. Prove that our engineered bacteria can create and secrete monovalent, and bivalent anti highly pathogenic avian influenza nanobodies. The latter of which has not yet been synthesized in literature.
5. Nanobody efficacy:
6. Show that our nanobodies can bind to a variety of Hemagglutanin subtypes (e.g. H5).
7. Biocontainment:
8. Develop a killswitch sensitive to temperature, light, and an external non-toxic chemical signal to ensure one of our main key values: safety.

Safe-by-design

In the above text, we have included an elaborate problem description which we created after doing an extensive literature review. To ensure the safety of Nanobuddy for animals, humans and the environment, Nanobuddy was designed with a safe-by-design approach. We took the utmost regard when designing the project and our GMO to prevent any harm from occurring due to our efforts. Not only did we design an elaborate kill switch, but we also took various other measures to ensure the safety of our project and people that we worked with. More information on our safe-by-design approach can be found on our Safety page.

Conclusion

Without a definitive solution on hand, the poultry industry and governments alike are pleading for the introduction of vaccination strategies. Vaccinations however, are also suboptimal as the only suitable vaccinations for this highly pathogenic variant have to be introduced intravenously; through an injection, often more than one. Considering we would have to conventionally vaccinate one hundred million poultry in the Netherlands alone, there has to be a better way to swiftly protect our poultry sufficiently.

With Nanobuddy we propose a novel solution by engineering Limosilactobacillus reuteri, a native species in the lung and intestinal microbiome of chickens, to secrete broadly neutralizing nanobodies against bird flu. These nanobodies bind to a conserved region of the virus’s hemagglutinin receptor, blocking it from entering cells. This novel approach to protecting livestock from viral disease might be the solution that the world needs right now.

We ensured safety, responsibility and most importantly effectiveness throughout all of the steps in designing our project. Nanobuddy is a project which is created to have a positive impact on the world, and everything living in it.

Key Values

1. Safe
2. - Safe for humans, the environment, and poultry.
3. Responsible
4. - Sustainable, adaptable, and able to work together with already existing solutions.
5. Effective:
6. - Designed to work efficiently, using cutting edge science.
7. Upscalability:
8. - Protect all poultry with ease, all around the world.
9. Animal well-being:
10. - Saving countless of needlessly culled poultry, not just chicken.
11. Human well-being:
12. - Preventing the next pandemic without compromising human health with our solution.

Inspiration

Early on in the project we decided, after brainstorming multiple ideas, to subdivide into smaller groups to evaluate the feasibility of each proposition. The three main projects we decided to develop further on ranged from bioremediation to biodegradable polymer synthesis. However, we ultimately decided to focus on the oral passive immunization of poultry. In part due to its feasibility, but mostly because of the urgency of the problem. At that moment we had noticed more and more outbreaks of avian influenza were appearing in the news, but no one was really talking about how to actually solve this problem. When doing some extensive informal google research, we found that people in the government were also slowly addressing this problem by suggesting strategies such as vaccination. However, even if vaccine strategies could be allowed to be implemented, these vaccinations would be regular intravenous injections. We suspected that this would present tremendous logistical problems considering we have about one hundred million poultry in the Netherlands. We had heard of spray vaccinations, but it appeared that these are not able to be implemented for highly pathogenic avian influenza. There must be another way to protect our poultry; which is where our project originated.

When evaluating this project, one of the first things we looked into was whether we could maybe develop a way to passively immunize chicken through the genetic modification of a feed additive. This feed additive would consist of bacteria that produce virus particles which would stimulate the immune system of chickens. After careful consideration, we found that this would most likely not be efficient enough to address this problem. Therefore, we looked for other ways we could protect chickens without vaccinating them. We realized that we could take a whole new approach to protecting animals for disease, thinking out of the box and the popular viral disease prevention framework. One of our team members, Ronald, read up on a paper about nanobodies which could bind to viruses and deactivate them. Considering the designs previously proposed, where a GMO would secrete viral proteins, he figured that we could also do it the other way around: the GMO secreting the therapeutic against the disease!

This was the moment our project truly took the shape that it has right now. We thought of the plan where this bacteria would enter the lungs of poultry, and secrete the avian influenza deactivating particles. When looking through literature, we found that by spraying anti influenza nanobodies on mice, you could sufficiently protect them from viral infection [19]. Considering we would have literal nanobody producing factories living in chickens, we concluded that this approach would definitely be worth looking into further.

All in all, we decided to move forward with this strategy as it showed a lot of potential and was an interesting take on preventing infection: not utilizing the immune system of the organism itself, but rather building an extra layer of protection.