Project Description

Throughout history, countless pathogens have demonstrated an extraordinary ability to mutate and adapt, often surpassing existing therapeutic tools. This creates a competition in which, while humanity develops new solutions, these pathogens in turn develop new adaptations. In this dynamic, prevention and control become a challenge where delays in implementing effective and efficient measures can lead to local, regional, or even pandemic outbreaks.

In Ecuador, as in many other countries, we have seen how rapidly evolving pathogens can impact both healthcare systems and the economy. In this sense, the threat of pathogens ceased to be something distant for us with a particular case: H5N1 avian influenza. At the end of 2022, H5N1 avian influenza outbreaks began to significantly affect our country. This news was surprising, as Ecuador had previously been considered free of highly pathogenic avian influenza (HPAI)[1]. This was not an isolated event, since a similar situation to that of our country was evident globally. In Ecuador, the virus severely affected poultry (especially chickens), decimating its production in several provinces. This was accompanied by several cullings that were taken as biosecurity measures to control the outbreaks[3], thus affecting the local economy, especially in rural communities that depend on poultry farming for their livelihoods. Shortly after, cases were detected in wild birds, including protected species that inhabit fragile ecosystems such as coastal wetlands and the Galapagos Islands. The threat was not limited to birds; on the coasts of South America, mass deaths of sea lions and other marine mammals were reported, revealing the virus's ability to cross new species barriers and expanding the risk of spread[2].

The impact also reached humans. In January 2023, Ecuador confirmed the first human case of avian influenza: a nine-year-old girl from the province of Bolívar, who developed a severe infection [4]. This event, along with other precedents that compromise not only our country's productive model but also our biodiversity and health, marked a turning point in how this problem was addressed.

Faced with this reality, we united various students, scientists, and professionals from different universities, diverse areas, and different locations in Ecuador. Despite coming from such diverse backgrounds, we all shared a common vision. We all agreed that avian influenza outbreaks were not the only problem; they were rather a symptom of something much larger. In this case, this experience only served to show us that the rapid evolution of zoonotic viruses is not just a technical challenge, but a reality that directly impacts our communities, economies, and ecosystems.

Considering all of this, we understood that the fight against highly virulent pathogens requires equally rapid and adaptable solutions. Thus began our proposal for nothing less than an interchangeable dual immunization platform that can respond both preventively and therapeutically. This is why we decided to use H5N1 avian influenza as a starting point, not only because of its relevance or the context that brought us together as a team, but also because it represents the type of challenges we want to prepare health systems, productive ecosystems, and human communities. This platform offers an option capable of responding not only to current H5N1 variants but also to future mutations and other emerging zoonotic viruses due to its transmissible nature.

We believe that having a fast, flexible, and scalable platform will help mitigate outbreaks more quickly, reducing economic losses, strengthening biosecurity, and protecting both biodiversity and the health of communities.

Initially, as mentioned in the previous section, our main objective was to develop the design of this interchangeable and adaptable immunization platform, which would serve as a model for responding quickly and effectively to emerging pathogens, with avian influenza as our particular case study, with a view to scaling it up to other emerging pathogens in the future.

However, as we developed our project, other objectives and perspectives were added that helped us give our work a more meaningful direction. Among these complementary objectives, we proposed:

We converge on an integrated, probiotic-based platform that pairs immediate passive immunity via secreted cyclobodies (intein-cyclized nanobodies) with durable active immunity via surface display of conserved influenza antigens in Lactococcus lactis. This dual architecture simultaneously addresses the early outbreak protection gap and the need for immune memory, while keeping delivery oral, low-cost, thermostable, and field-ready.

I. Passive arm (cyclobodies)

An inducible pLux/LuxR–AHL cassette with Npu DnaE split inteins and Usp45 secretion yields in vivo post-translational cyclization and export of nanobodies that are thermo- and protease-resistant, suitable for water/feed matrices. The cassette is modular (defined swap sites for Nano-A/Nano-B), enabling plug-and-play retargeting to new variants or even other viruses without rebuilding the backbone.

II. Active arm (L. lactis surface display)

Using PnisA (NICE) decouples growth from production. Usp45 + LPXTG anchoring is selected by epitope geometry/reach: SpaX for the compact LAH–4×M2e module and M6 for full-length NA. Multivalent M2e (4×, multispecies) and the orientation options (LAH↔4×M2e) aim to maximize epitope accessibility and immunogenic breadth, while avoiding interference with the passive arm's HA1/HA2 targets.

What makes this platform stand out

To sum up, AvianGuard offers an accessible, adaptable, and operationally viable route to mitigate avian influenza (and other pathogens), combining instant passive protection with sustained active immunity, delivered as a lyophilized, orally reconstitutable product.

The global spread of the Highly Pathogenic Avian Influenza (HPAI) virus (H5N1), which in rare cases has infected humans, has motivated the search for new antiviral strategies that combine specificity, stability, and broad protection. Our passive immunity approach takes inspiration from the Nanobuddy project (iGEM Groningen 2022) and proposes a cyclobody capable of recognizing conserved regions of the influenza hemagglutinin (HA), a key viral protein involved in host-cell entry.

The cyclobody combines two nanobodies with complementary binding profiles:

Both nanobodies are linked by a flexible (G₄S) linker that reduces steric hindrance and enhances conformational freedom. The construct is cyclized using split inteins (Npu DnaE) from Nostoc punctiforme, which rejoin their N- and C-terminal fragments through a peptide bond, forming a continuous cyclic peptide without external residues.

The resulting construct represents a modular, stable, and cross-protective passive immunization system that can be adapted to other viral antigens by swapping nanobody domains, providing a flexible foundation for rapid antiviral countermeasures.

The active immunity strategy of the AvianGuard platform focuses on developing a Lactococcus lactis based surface display system capable of presenting conserved viral antigens to the host immune system. Using influenza as a model, this approach seeks to elicit broad and durable immune responses at mucosal and systemic levels.

The platform is organized into two main antigenic modules:

Module A (LAH–4×M2e):

integrates the long α-helix of the HA stalk (LAH) with four tandem copies of the matrix protein 2 ectodomain (M2e).

• The M2e sequence (~23–24 aa) is highly conserved and surface-exposed, making it a strong candidate for cross-protective immunity.
• Including four variants (two avian, one swine, one human) enhances coverage across species and influenza subtypes.

Module B (Neuraminidase, ΔTM):

expresses a truncated version of neuraminidase (NA) lacking the transmembrane domain but retaining the head and stalk regions. Antibodies against NA inhibit sialidase activity and restrict viral spread, complementing HA- and M2e-based protection.

Each module incorporates the Usp45 secretion signal to direct extracellular export of the antigen and an LPXTG-type anchoring domain that allows covalent attachment to the cell wall. To optimize surface exposure, two different anchors were used: SpaX, derived from Staphylococcus aureus protein A, ideal for compact antigens such as LAH–M2e, and M6/CwaM6, with spiral stem regions that project larger proteins, favoring NA accessibility on the bacterial surface. Both constructs were linked by flexible (G₄S) sequences that minimize steric interference and preserve antigenic conformation.

Advantages and extensibility:

The final design is a nisin-inducible, surface-anchored vaccine platform based on Lactococcus lactis, displaying conserved influenza epitopes (LAH–4×M2e and NA) in a stable and accessible configuration. This system demonstrates the potential of synthetic biology to develop flexible, broad-spectrum, and reconfigurable mucosal vaccines.

In the future, we aspire to scale up the current system and turn it into a comprehensive biotechnology platform for the immunization and surveillance of zoonotic microorganisms in poultry and other animals of agricultural interest. The dual design based on active and passive immunity allows this design to be converted into a modular tool that can be quickly reused and reprogrammed in response to new emerging pathogens.

In the medium term, we hope to establish collaborations with universities, research centers, and agro-industrial companies to evaluate the functionality of the prototypes in controlled and semi-commercial situations, validating the field performance of our proposal. This will enable in vivo testing with model birds to analyze the immune response generated, as well as the parameters of bioavailability, stability, and the kinetics of action of the system on the probiotic.

Likewise, the design and validation of the type of encapsulation will be proposed, using chitosan or other biopolymer coatings, to improve the stability of the prototype upon reaching the birds' gastrointestinal tract.

These results will serve as the basis for biotechnological scaling and the development of a platform adaptable to different zoonotic diseases, contributing to innovation in animal health and food safety from a sustainable approach.