Performing Department
(N/A)
Non Technical Summary
Avian influenza poses a serious threat to global poultry health and food security. Current vaccines against the prevalent H9N2 subtype are inadequate, necessitating innovative approaches. Our research aims to develop effective mass vaccination strategies using modified live attenuated vaccines (MLVs).This project will analyze the genetic makeup of the H9N2 virus to understand its evolution and identify key targets for vaccine development. We will create and test improved MLVs, ensuring their safety and effectiveness in preventing disease transmission. Additionally, we will examine how these MLVs stimulate the immune system compared to traditional vaccines, focusing on mucosal, humoral, and cellular responses.Our findings will significantly enhance our understanding of the H9N2 virus and provide valuable tools for rapidly assessing new strains. This knowledge will directly contribute to the development of more effective vaccines against avian influenza, not only using MLVs but also other vaccine technologies.This innovative approach is a departure from traditional methods and could revolutionize poultry vaccination. The potential benefits extend beyond avian influenza, paving the way for similar studies on other respiratory viruses affecting poultry and livestock. By addressing the limitations of current vaccines and developing safer, more effective alternatives,we can protect animal health, safeguard the food supply, and mitigate the economic impact of avian influenza outbreaks.
Animal Health Component
100%
Research Effort Categories
Basic
65%
Applied
25%
Developmental
10%
Goals / Objectives
Avian influenza, a highly contagious disease with significant agricultural implications, poses a severe threat to the poultry industry and national food supply. Effective prevention and control strategies rely on rigorous biosecurity measures and vaccination. Our research focuses on the H9N2 subtype, endemic in vast geographic regions and a known contributor to the emergence of other influenza strains, notably the panzootic H5N1s that reached the Americas in 2022-2023.Despite widespread vaccination efforts, current H9N2 vaccines have proven largely ineffective in eradicating the virus,potentially exacerbating antigenic drift. Building upon prior research funded by the USDA National Institute of Food and Agriculture, our project aims to develop practical mass vaccination strategies using modified live attenuated influenza vaccines (MLVs).Employing phylogenetics, reverse genetics, and antigenic cartography, we seek to comprehensively understand the antigenic properties of H9N2s and optimize mono- and multivariant MLVs for mass administration via spray and drinking water. Rigorous testing will establish the safety, transmission dynamics, and reassortment potential of MLVs in susceptible birds. Additionally, we will assess the protective efficacy of MLVs against viral challenge and investigate the differential immune responses they elicit compared to inactivated vaccines, particularly at the cellular level.This research will significantly advance our understanding of H9N2 antigenic properties, directly contributing to the development of novel MLVs with enhanced immunogenicity, encompassing mucosal, humoral, and cellular immune responses.
Project Methods
Phylogenetic Analysis: We will use publicly available sequences from GISAID to construct a phylogenetic tree of the HA1 region of H9 HA. This will guide the selection of consensus and prototypic H9 HA sequences.Virus Generation: We will synthesize and clone selected HA1 sequences into a reverse genetics vector to generate chimeric H9 HA gene segments in a WF10 backbone.Serum Production: The recombinant viruses will be used to inoculate chickens and quail to produce polyclonal anti-H9 sera.Hemagglutination Inhibition (HI) Assays: HI assays will be performed using the anti-H9 sera against a panel of H9N2 strains to assess antigenic relationships.Antigenic Cartography: HI data will be analyzed using the ACMACS antigenic cartography tool to visualize antigenic differences and identify antigenic clusters.Site-Directed Mutagenesis: Based on phylogenetic, antigenic, and structural data, we will use site-directed mutagenesis to generate additional recombinant viruses with specific amino acid mutations to further refine the antigenic map.Iterative Analysis: We will repeat steps 3-6 as needed to identify additional antigenic clusters and relevant amino acid positions.MLV Generation: We will generate MLV vaccine candidates with modifications on the surface gene segments (HA and NA) to decrease vRNA segment fitness and enhance the immune response. Two platforms will be used: RAM (PB1-M2 and M early stops) and RAM42 (PB1-M42 and M early stops).In Vitro Characterization: We will assess the stability, growth kinetics, and expression levels of MLVs in embryonated eggs and cell culture. We will also evaluate the fitness cost of HA and NA modifications through co-infection studies.In Vivo Assessment (Study 2a and 2b):Transmission and Reassortment Potential: Groups of chickens and quail will be inoculated with monovalent or multivalent MLVs, a mixture of MLVs and wild-type virus, or wild-type virus alone. Naïve contact birds will be introduced to assess transmission. Samples will be collected for virus titration, histopathology, and immunohistochemistry.Immune Response and Protection (Study 3): Chickens will be vaccinated via spray, drinking water, or subcutaneous injection with monovalent or multivalent MLVs or inactivated vaccines. They will be challenged with homologous or heterologous viruses, and samples will be collected for virus titration, histopathology, and immunohistochemistry. Immune responses will be measured by ELISA and HI assays.Sample Collection: Blood, spleen, bursa, cecal tonsils, thymus, and lungs will be collected from chickens vaccinated with multivariant MLV-IL, MLV (without ckIL18), and WIV-adj formulations at 5 and 14 days post-prime and post-boost.Cell Isolation: Peripheral blood mononuclear cells (PBMCs) and cells from collected tissues will be isolated using established protocols.Lymphocyte Proliferation Assays: Cell suspensions will be stimulated with WIV, concanavalin A, LPS, or left unstimulated. Proliferation will be measured using the CellTiter 96 proliferation assay kit.ELISPOT Assays: ELISPOT assays will be performed to quantify total and influenza-specific IgA and IgY antibody-secreting cells (ASC), as well as IFN-g expressing cells.Flow Cytometry: Cell suspensions will be stained with antibodies against various cell surface markers (CD45, Bu-1, KUL01, MHC-II, CD11c, Tgs, CD3, CD4, CD8a, CD8b, CD25) to identify and characterize B cells, T cells, macrophages, dendritic cells, and natural killer cells.