Performing Department
(N/A)
Non Technical Summary
This research project addresses the recurring outbreaks of Highly Pathogenic Avian Influenza (HPAI) in the U.S. poultry industry, which cause significant economic losses and threaten the global food supply. Current methods to control HPAI, such as culling and vaccination, have limitations.To combat this issue, the project aims to develop a novel approach using recombinant Lactobacillus reuteri, a common probiotic bacterium. We will engineer L. reuteri to produce anti-influenza nanobodies and cell-penetrating influenza interfering peptides in the poultry's gastrointestinal tract. These bioengineered bacteria are designed to act as a first line of defense against influenza infection in poultry.This innovative approach aims to reduce the susceptibility of poultry to HPAI and other influenza strains, thereby safeguarding the poultry industry, global food security, and public health. If successful, this project will provide an alternative to traditional vaccines, offering immediate protection, stability, high specificity, and scalability.
Animal Health Component
25%
Research Effort Categories
Basic
65%
Applied
25%
Developmental
10%
Goals / Objectives
The major goal of this project is to develop a novel platform for the prevention and control of Highly Pathogenic Avian Influenza (HPAI).This will be achieved through a dual strategy:Utilizing a recombinant Lactobacillus reuteri (L. reuteri) strain as a delivery system for protective nano-antibodies (nAbs).Utilizing a recombinant L. reuteri strain as a delivery system for cell-penetrating influenza interfering peptides (CPPIIPs) targeting conserved epitopes across multiple H5N1 HPAIV proteins.The project objectives are:Engineer Potent nAbs against H5N1 HPAI.Engineer CPPIIPscapable of disrupting H5N1 HPAI viral replication in vivo.Develop recombinant L. reuteri strains secreting probiotic vectored nanobodies (pvAbs) and CPPIIPs (pvCPPIIPs) that confer protection against H5N1 HPAI challenge in chickens.
Project Methods
The project will utilize a multi-faceted approach to Highly Pathogenic Avian Influenza (HPAI) prevention and control.Engineering ofL. reuteriStrainsThe project will involve engineering the bacteriumLactobacillus reuterito express anti-influenza nanobodies and cell-penetrating influenza interfering peptides. The goal is for these bioengineered bacteria to act as a first line of defense against influenza infection in poultry.The project will utilize a design-build-test-learn (DBTL) cycle, integrating machine learning and design of experiments (DOE) to optimize the nAb secretion capabilities of theL. reuteristrains.Development of Anti-Influenza Nanobodies and PeptidesThe project will involve a multi-faceted approach to identify and optimize nanobodies and peptides with antiviral properties.Nanobodies:Alpaca immunization and phage display library screening will be used to identify broadly neutralizing nanobodies against H5N1 HPAI.The project will create a synthetic nAb library by incorporating degenerate codons into the complementarity-determining regions (CDRs) to enhance the potential to produce high affinity nAbs.Selected nAbs will be engineered for intracellular targeting by incorporating cell-penetrating peptide (CPP) sequences and potentially nuclear targeting sequences.Cell-Penetrating Influenza Interfering Peptides (CPPIIPs):A library of cell-penetrating peptides will be generated to target protein-protein interaction domains of influenza viruses.The hypothesis is that virus-derived peptides can disrupt these protein-protein interaction domains, impairing viral function and reducing replication.Wewill engineer tandem constructs of CPP(NLS)-PB1 domain α peptides to disrupt the PB1-PA interaction, a prime target for inhibiting influenza virus replication.Additional CPP(NLS)-IIPs will be designed to target key interaction domains within M1 and NP proteins.Each CPP(NLS)IIP construct will be designed with or without an N-terminal signal peptide (SP) sequence, allowing for either secretion or intracellular retention.Evaluation of EfficacyThe efficacy of the engineeredL. reuteristrains will be evaluatedex vivoin chicken enteroids andin vivoin chickens challenged with H5N1 HPAI.Ex vivo studies:Colonization and antiviral efficacy studies will be conducted using a streamlined chicken 2D enteroid model of the intestinal epithelium.The 2D enteroids will be exposed to recombinantL. reuteripvAb strains, and viral replication will be assessed.In vivo studies:The experimental design will mirror a practical application in commercial chicken farming, using spray administration at hatch and oral administration via drinking water.Bacterial colonization efficiency and impacts on the gut microbiome will be assessed.Chickens will be challenged with H9N2 LPAIV and H5N1 HPAIV, and antiviral response will be evaluated.Virus shedding, clinical signs, and tissue samples will be analyzed.NGS will be performed to determine antigenic drift and changes in the virus progeny genome.Large-Scale ProductionThe pvAb and pvCPPIIP candidates that show the most promise will be further refined to improve their production process.Fermentation Process Optimization:Fed-batch fermentation experiments will be conducted to regulate bacterial metabolism and maintain optimal metabolic conditions.Design of experiments (DOE) and statistical modeling will be used to identify the best conditions.Critical process parameters (CPC) such as pH, inoculum size, temperature, and agitation rate will be optimized.Lyophilization Optimization:The most effective cryoprotectant formulations and concentrations will be identified to mitigate viability losses post-lyophilization.The optimization process will include freezing, primary drying, and secondary drying stages.Design of experiments (DoE) will be used to optimize sample moisture content and drying kinetics.Scale-up and production readiness:The efficiency of fermentation, downstream processing, and lyophilization will be assessed to identify challenges related to scale-up.The end-to-end production process will be replicated in triplicate batches at 100L scale.Efficacy of large-scale produced pvAb and pvCPPIIP candidates:The antiviral profiles of the large-scale products will be assessed against H5N1 HPAIV in 2D chicken enteroid cells and in vivo in chickens.Data AnalysisData will be analyzed by ANOVA, and treatment means will be separated by Tukey's multiple comparison tests.All data will be evaluated for approximately normal distribution and similar group variance.Transformations, such as log10, will be used to correct for skewness.Longitudinal analyses will provide relationships between groups and outcomes over all time points.