Background

Overview

• Influenza A virus is a type of RNA virus with high infectivity and mutability, and it is one of the main pathogens causing influenza epidemics in humans. Seasonal influenza viruses infect one-third of the global population each year, causing 3 to 5 million severe cases and 290,000 to 650,000 deaths. The majority of human disease is caused by the influenza virus types A and B. Since influenza B viruses are thought to rely on humans as their primary host, only influenza A viruses are known to cause pandemic outbreaks due to viruses also circulating in animal reservoirs ^[2].The fatality rate of avian influenza can exceed 60%, posing a significant threat to public health[1].People of all age groups can be infected with influenza viruses, but school-aged children usually have the highest infection rate.

Virus Classification and Variation

• Influenza A virus belongs to the Orthomyxoviridae family. Based on the antigenic differences of two key surface proteins - hemagglutinin (HA) and neuraminidase (NA), it can be classified into multiple subtypes^[3]. Influenza viruses mutate frequently, constantly generating new strains, making influenza outbreaks difficult to predict and control. Reassortment among the RNA genome segments from different influenza A viruses during coinfection (antigenic shift) can generate novel influenza viruses with increased pathogenicity and transmissibility, as demonstrated by the 1918, 1957, 1968, and 2009 influenza pandemics^[4].Currently, 18 types of HA (H1-H18) and 11 types of NA (N1-N11) have been discovered, and different combinations form various subtypes, such as H1N1, H3N2, H5N1, H7N9, etc.

Virus Structure

• Core Structure: The virus particle is spherical or filamentous, with a single-stranded negative-sense RNA core, encapsulated in nucleoprotein (NP) and RNA polymerase complex, which determines the genetic characteristics of the virus^[5].
• Surface Proteins:
  • Hemagglutinin (HA): Responsible for recognizing and binding to receptors on the surface of host cells (such as respiratory epithelial cells), it is a key protein for viral invasion of cells and is also the main target for vaccine development.
  • Neuraminidase (NA): Helps newly synthesized virus particles to be released from host cells, promoting viral spread, and is also the target of antiviral drugs such as oseltamivir^[6].
• Envelope:The outer layer of the virus has a lipid envelope derived from the host cell, which enhances the virus's adaptability to the environment.

Transmission Routes

• Human-to-human transmission: Mainly through droplets produced by coughing or sneezing of infected individuals, and can also be contracted by touching the mouth or nose after touching virus-contaminated objects.
• Cross-species transmission Influenza A virus has a wide host range and can infect humans, birds, pigs, horses, seals, and other animals. It is the only influenza virus that can naturally spread between humans and animals. For example, avian influenza virus H5N1 can be transmitted to humans through contact with diseased poultry or their secretions, and pigs may act as "mixers" for different subtypes of viruses to recombine and generate new variants^[7].

Clinical Manifestations

Current Treatment Status

• Vaccination Conventional influenza vaccines are designed to stimulate neutralizing antibodies against immunodominant but highly variable hemagglutinin antigens^[4].
  • Advantages: The most effective measure for influenza prevention, and it has become one of the most frequently used vaccines globally in recent years. Vaccination can significantly reduce the risk of infection and alleviate symptom severity.
  • Disadvantages: There are several shortcomings of the current seasonal influenza vaccines. They must be tailor-made yearly to match the expected circulating strains during the influenza season, since influenza viruses undergo antigenic evolution or antigen drift and shift. The selection of influenza virus strains for seasonal influenza vaccines is made for the northern hemisphere in February and the southern hemisphere in September by the World Health Organization (WHO) to allow sufficient time for vaccine production .Nevertheless, the mismatch between the vaccine and circulating strains results in variable protection[9]. Inherent limitations include suboptimal protection against rapidly changing seasonal influenza viruses and a lack of protection against antigenically novel pandemic influenza^[4].
• Drug Therapy
  • Antiinfluenza Drugs: Such as neuraminidase inhibitors and M2 ion channel inhibitors, serve as first-line medications for preventing and early controlling influenza. These drugs can alleviate symptoms and shorten the disease course to a certain extent ^[10].
  • Disadvantages: Due to selective pressure induced by drugs, viral drug resistance continues to increase, limiting the efficacy of these medications ^[11]. Antiviral drugs such as oseltamivir need to be taken within the first 48 hours of infection (when symptoms first appear) to achieve the best antiviral effect, so timely and effective protection is not easy to get for majority of people.
• Combined Therapy with Chinese and Western Medicines Evidence suggests that adding Chinese patent medicine (CPM) to oseltamivir shortens fever duration, accelerates symptom relief, and reduces hospital stay in adults with influenza. Yet most trials are small, single-centre, and use inconsistent diagnostic criteria; larger, high-quality, multicentre studies are needed to confirm efficacy and safety^[17].

INSPIRATION

Problems to be Solved

Synthetic Biology Applications

SOLUTION

In response to the urgent challenges mentioned above, our team proudly present a synthetic-biology solution:

Flubloker: Synthetic sentry at the nasal gate—variant-proof armor against HxNy.

Scientific Basis

The scientific basis of this project is to utilize secreted single-chain fragment variable (scFv) antibodies to neutralize viruses, thereby blocking the infection process of influenza. The single-chain fragment variable(scFv) is a widely studied type of genetically engineered antibody ^[12]. Unlike traditional antibodies, scFv is a small molecule composed of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) connected by a peptide linker. It retains the complete antigen-binding site and represents the smallest functional unit with antibody activity ^[13]. scFv antibodies are characterized by their small molecular weight, strong penetration ability, short half-life, and low immunogenicity. These features make scFv an ideal choice for antiviral substances secreted by engineered bacteria.

Influenza A virus contains two surface antigenic glycoproteins: hemagglutinin (HA) and neuraminidase (NA), which further subdivide the strains of Influenza A virus ^[14]. Hemagglutinin facilitates viral entry into target cells by recognizing and binding to sialic acid-containing proteins on the host cell surface. It is crucial for the viral infection process and is a major target for anti-influenza virus strategies. Neuraminidase, on the other hand, is essential for viral replication as it enables the release of the virus from host cells ^[14]. In recent years, researchers have increasingly recognized the importance of anti-NA antibodies in reducing the effects of viral infection. Studies have shown that anti-NA antibodies are independently correlated with protection from infection in both field and human challenge studies ^[16].

Based on these findings, we have selected two scFv sequences through literature review: the scFv gene sequences of MEDI8852 and 1G01. MEDI8852 is a human IgG1κ monoclonal antibody derived from human memory B cells that binds to the conserved stalk region of the influenza hemagglutinin (HA) protein ^[15]. 1G01, isolated from an H3N2-infected individual, inhibits neuraminidase activity by directly binding to its active site. Compared with other clonally related monoclonal antibodies, 1G01 exhibits the broadest binding activity, covering all Group 1 NAs (N1, N4, N5, and N8), Group 2 NAs (N2, N3, N6, N7, and N9), and NAs from both lineages of influenza B virus ^[16]. By secreting both of these antibodies simultaneously, engineered bacteria can more comprehensively block the infection and replication processes of the influenza virus, thereby achieving a more effective antiviral effect.

Characteristics

• Our project adopts a nasal spray method, with the nasal cavity acting as the upper respiratory tract, deploying the engineered bacteria in this location allows for rapid detection of the virus and the initiation of a cascade response at the first line of defense against influenza virus infection. Simultaneously releasing single-chain variable fragments (scFv) targeting the two key antigenic surface glycoproteins of the influenza virus (HA and NA), achieving efficient viral clearance. This closed-loop defense mechanism blocks the invasion of the influenza virus at the source, providing more comprehensive protection for susceptible and high-risk populations.
  • For example, for individuals exposed to the virus, the nasal spray method can quickly activate the defense mechanism after viral exposure, achieving efficient viral clearance in the early stages within just a few minutes to several hours. This effectively prevents the virus from spreading within the body, reduces the risk of infection, and offers immediate protection to those exposed.
  • For immunocompromised individuals who may not respond well to vaccines or fail to mount a sufficient immune response, our project provides a direct and effective solution. By releasing scFv through nasal spray, it bypasses the limitations of the human immune system, offering immediate and effective protection for immunocompromised individuals and filling the gap left by traditional vaccines in this special population.
• Overall, our approach rapidly breaks the chain of viral transmission during influenza outbreaks, reducing viral spread in communities and healthcare settings. It provides a convenient and efficient preventive measure for individuals while forming a stronger population-level immune barrier. The nasal spray method is easy to use, requires no specialized personnel, and is suitable for large-scale application, especially in resource-limited areas. Our project offers robust protection for susceptible and high-risk populations and holds significant public health value by pioneering a new synthetic biology approach for influenza prevention and control.

Technical Route

• Genetic engineering strategy
  • The core modification of this project is based on a two-component signaling system (TCS), which constructs a “sensing-response” closed-loop circuit in Lactobacillus rhamnosus GG chassis cells. The viral sensing module is formed by fusing single-chain antibodies (scFv) targeting hemagglutinin (HA) and neuraminidase (NA) with histidine kinase (HK), such as PhoR, to create a transmembrane chimera consisting of scFv-hinge region- (GGGGS)₃-HK. When scFv binds to influenza virus surface proteins, it pulls the extracellular domain of HK, inducing its dimerization and autophosphorylation, thereby transferring the phosphate group to the response regulator protein RR (such as OmpR) and activating the downstream promoter. The antibody response module utilizes RR-sensitive promoters (e.g., OmpC) to drive the secretion of two scFv: medi8852 blocks viral entry, while 1G01 inhibits viral release, creating a synergistic neutralizing effect. To ensure biosafety, a dual suicide system is introduced: the temperature-induced module uses the ROSE RNA thermometer (activated at 30–34°C) or the TlpA temperature-sensitive repressor protein (inactivated at 37°C) to control the expression of lytic enzyme genes; The blue light-induced module is based on the pdawn system , which uses blue light irradiation to release the repressor and express the toxin MazF, enabling on-demand clearance.
• Biosecurity Control
  • Our team prioritizes biosafety. To address risks like environmental exposure and unintended colonization, and to ensure individual treatment autonomy, we designed a dual safety system: “temperature-induced suicide” and “light-induced suicide.” The temperature window (30-37℃) restricts bacterial colonization to the nasal environment. A blue light–induced self-destruction module allows users to terminate treatment at any time and ensures rapid bacterial death if leakage occurs.This biosafety control system ultimately enables precise colonization of the engineered bacteria at the target site, autonomous clearance in the event of environmental leakage or clinical risk, and controllability of the treatment process by individuals.

Experimental Methods

• Engineering bacteria construction using electroporation transformation: Lactobacillus rhamnosus was pretreated with 2% glycine, followed by cell wall disruption using lysozyme at 30 mg/mL to prepare competent cells. Plasmid transformation was performed under conditions of 2 kV/cm, 200 Ω, and 25 μF, and successful transformation was verified via triple colony PCR (VAL-F1/R1 plasmid backbone, VAL-F2/R2 scFv insertion, and 16S rRNA internal control).
• Functional validation of the sensing module included: (1) Phos-tag™ SDS-PAGE detection of HK autophosphorylation, with phosphorylated HK exhibiting delayed migration and high-molecular-weight bands after viral stimulation; (2) Confocal microscopy combined with WGA-Alexa Fluor 633 membrane labeling to dynamically monitor the secretion localization of the scFv-GFP fusion protein under viral stimulation; (3) MOI gradient testing (0.001–10) to quantify response sensitivity.
• Antibody functionality was assessed via plaque reduction assays (using H1N1 inactivated virus), with neutralization rates calculated as: Neutralization rate = [1 −(experimental group plaque count / viral control group plaque count)] × 100% Neutralization rate = [1 − (viral control group plaque count / experimental group plaque count)] × 100%.
• Biosafety validation included temperature response (GFP reporter gene expression and survival rate under 30°C vs. 37°C culture conditions) and blue light induction efficiency (toxin expression and bacterial lysis after 100 nW/cm² irradiation).

INNOVATIONS

The core innovation of this project lies in addressing the existing pain points in influenza prevention and control by achieving a breakthrough across the entire chain, from the point of defense to the mechanism of action:

Defensive Position Innovation

Traditional vaccines rely on the human immune system to generate a specific immune response, which has a delayed onset of action. Antiviral drugs like oseltamivir must be administered within 48 hours of symptom onset and can lead to drug resistance, failing to meet the need for rapid intervention during the initial stages of viral invasion. Therefore, we innovatively shifted the defensive frontline to the “first line of defense” against viral invasion— — the nasal cavity, a critical entry point of the upper respiratory tract. By implanting engineered Lactobacillus rhamnosus bacteria in the nasal cavity, the system can rapidly detect and initiate a response upon viral contact with the host. Compared to traditional methods, this approach achieves early, efficient clearance within minutes to hours, thereby blocking viral spread at its source.

Broad-spectrum Coverage

We selected two single-chain antibodies (scFv), MEDI8852 and 1G01, to target the conserved regions of the HA and NA proteins on the surface of the influenza virus. MEDI8852 binds to the conserved stem region of HA, which remains relatively stable during viral mutations, while 1G01 directly binds to the active site of NA, covering Groups 1 (N1, N4, N5, N8), and Group 2 (N2, N3, N6, N7, N9). This design targeting conserved regions overcomes the limitation of traditional vaccines and drugs, which are constrained by the rapid mutation of viruses, achieving broad-spectrum coverage against multiple influenza A subtypes.

Synthetic Biology Meets Controlled Safety

A “sense-response” closed-loop circuit based on a two-component signaling system (TCS) enables engineered bacteria to automatically initiate antibody secretion upon detecting the virus. Combined with a dual suicide system (temperature-induced and blue light-induced) to ensure biosafety, This design, which combines the precise regulation of synthetic biology with conserved viral targets and is deployed at the source of infection, not only fills the gaps in rapid response and broad-spectrum coverage left by traditional methods but also pioneers a new influenza prevention and control model of source blocking plus broad-spectrum defense, providing a novel approach to addressing viral mutations and sudden outbreaks.

FUTURE WORK

Potential Impact

Our project's FluBlockers intelligent defense system, centered on engineered probiotics, not only pioneers a new biosynthetic paradigm for the prevention and control of influenza A, but also offers an innovative solution that is both broad-spectrum and safe for global influenza pandemics through a three-tier defense design of "virus sensing - signal transduction - response killing". Technically, its cross-species signal transduction mechanism - the scFv-PnpS two-component cross-species signal transduction system and dual suicide safety module - will drive breakthroughs in synthetic biology in cross-border defense fields. Socially, freeze-dried preparations and low-cost production processes are expected to fill the prevention and control gap in developing countries, while the establishment of a nasal biological barrier can significantly reduce the virus transmission rate in public health emergency scenarios, laying a key technical and theoretical foundation for the construction of a global influenza prevention and control network.

Treatment Timing

The FluBlockers intelligent defense system constructed by our project, with its highly sensitive real-time perception ability against Influenza A virus, can trigger a rapid response at the initial stage of the virus invading the nasal mucosa (i.e., the 0-4 hour window period after infection). By using neutralizing antibodies secreted by engineered probiotics to block the adhesion and fusion process between the virus and host cells, the treatment timing is advanced from 48 hours after the appearance of symptoms in traditional drug dependence to the golden intervention stage in the early stage of infection, providing a breakthrough time window for the ultra-early prevention and control of influenza. It is expected to significantly increase the success rate of treatment for high-risk groups and reduce the incidence of severe cases.

Product Form

The primary product form of this project will be a lyophilized nasal spray, suitable for both daily prevention and post-exposure emergency use. Its key advantages include ease of storage, transportation, and self-administration, especially in resource-limited settings. We also plan to develop a modular treatment kit, including refrigerated lyophilized probiotics, disposable spray nozzles, and blue-light activation devices, tailored for deployment in homes, communities, and clinical settings.

For long-term application, we envision novel formulations such as oral probiotic tablets or biodegradable intranasal membrane patches, enhancing user compliance while improving the controllability of colonization duration and preservation of probiotic bioactivity.

Therapeutic Strategy

In future development, we aim to refine the probiotic-mediated therapeutic model by evaluating its broad-spectrum adaptability against various influenza A subtypes (e.g., H1N1, H3N2), and further explore its applicability to other respiratory viruses such as RSV and SARS-CoV-2. If technology permits，we will also try to investigate multi-antibody co-expression, nanoparticle encapsulation delivery, and combination with existing vaccines to achieve full-cycle intervention from prevention to treatment.

Furthermore, we envision upgrading the current nasal administration approach into a “personalized, multi-mode therapy” that integrates wearable blue-light devices, real-time viral load monitoring, and adjustable antibody release, enabling dynamic response and dosage control for optimized efficacy and user convenience.

Reference

[1]Iuliano AD, Roguski KM, Chang HH, Muscatello DJ, Palekar R, Tempia S, Cohen C, Gran JM, Schanzer D, Cowling BJ, Wu P, Kyncl J, Ang LW, Park M, Redlberger-Fritz M, Yu H, Espenhain L, Krishnan A, Emukule G, van Asten L, Pereira da Silva S, Aungkulanon S, Buchholz U, Widdowson MA, Bresee JS; Global Seasonal Influenza-associated Mortality Collaborator Network. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet. 2018 Mar 31;391(10127):1285-1300.

[2]Nachbagauer R, Palese P. Is a Universal Influenza Virus Vaccine Possible? 2020;71(Volume 71, 2020):315-327.

[3]Dou D, Revol R, Östbye H, Wang H, Daniels R. Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement. Front Immunol. 2018 Jul 20;9:1581.

[4]Julie O, Meredith A, Kristine M, et al. Tracking progress in universal influenza vaccine development. Current Opinion in Virology. 2020;40:28-36

[5]Eisfeld AJ, Neumann G, Kawaoka Y. At the centre: influenza A virus ribonucleoproteins. Nat Rev Microbiol. 2015 Jan;13(1):28-41.

[6]McAuley JL, Gilbertson BP, Trifkovic S, Brown LE, McKimm-Breschkin JL. Influenza Virus Neuraminidase Structure and Functions. Front Microbiol. 2019 Jan 29;10:39.

[7]Abdelwhab EM, Mettenleiter TC. Zoonotic Animal Influenza Virus and Potential Mixing Vessel Hosts. Viruses. 2023 Apr 16;15(4):980.

[8]Kalil AC, Thomas PG. Influenza virus-related critical illness: pathophysiology and epidemiology. Crit Care. 2019 Jul 19;23(1):258

[9]Wang W-C, Sayedahmed EE, Sambhara S, Mittal SK. Progress towards the Development of a Universal Influenza Vaccine. Viruses. 2022; 14(8):1684.

[10] De Clercq E. Status presens of antiviral drugs and strategies: Part II: RNA VIRUSES (EXCEPT RETROVIRUSES). Adv Antivir Drug Des. 2007;5:59-112.

[11]de Jong MD, Tran TT, Truong HK, Vo MH, Smith GJ, Nguyen VC, et al. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med. 2005;353(25):2667-72.

[12]Zhang F, Chen Y, Ke Y, Zhang L, Zhang B, Yang L, Zhu J. Single Chain Fragment Variable (scFv) Antibodies Targeting the Spike Protein of Porcine Epidemic Diarrhea Virus Provide Protection against Viral Infection in Piglets. Viruses. 2019 Jan 14;11(1):58.

[13]Yokota T, Milenic DE, Whitlow M, Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 1992 Jun 15;52(12):3402-8.

[14]Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen LM, Santelli E, Stec B, Cadwell G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox NJ, Bankston LA, Donis RO, Liddington RC, Marasco WA. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009 Mar;16(3):265-73.

[15]Ali SO, Takas T, Nyborg A, Shoemaker K, Kallewaard NL, Chiong R, Dubovsky F, Mallory RM. Evaluation of MEDI8852, an Anti-Influenza A Monoclonal Antibody, in Treating Acute Uncomplicated Influenza. Antimicrob Agents Chemother. 2018 Oct 24;62(11):e00694-18.

[16]Daniel Stadlbauer et al. ,Broadly protective human antibodies that target the active site of influenza virus neuraminidase.Science366,499-504(2019).

[17]Li X, Liu B, Duan N, et al. Clinical Efficacy and Safety of Chinese Patent Medicine Combined with Oseltamivir for the Treatment of Adult Influenza: A Systematic Review and Meta-Analysis. The American journal of Chinese medicine. 2022;50:1-19.