Performing Department
(N/A)
Non Technical Summary
Poultry products are indispensable components of U.S. agriculture. The recent highly pathogenic avian influenza (HPAI) H5 (2022-2025 panzootic) caused tremendous economic losses in the U.S. poultry industry. While biosecurity measures have been crucial in reducing HPAI H5 outbreaks, HPAI vaccines could be strategically implemented to mitigate economic losses. Current HPAI H5 vaccines for poultry rely on generating antibodies against the influenza virus hemagglutinin (HA) and provide protection against antigenically matching strains. However, these vaccines do not adequately protect against variant HPAI H5 viruses with antigenically divergent HAs and typically do not enable practical differentiation of infected animals from vaccinated animals (DIVA). Non-surface proteins of HPAI viruses, which are not targeted by current vaccines, can induce strong T-cell responses that offer broad protection against variant viruses.This project aims to identify non-surface proteins of HPAI H5 viruses that induce broadly protective T-cell responses in chickens and evaluate their potential to enhance current vaccines. Through this work, we aim to uncover novel vaccine targets for HPAI H5 in poultry and assess their potential for improving current avian influenza virus vaccines. These findings are expected to significantly advance strategies for developing more effective HPAI vaccines.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Goals / Objectives
The goal of this project is to identify non-surface proteins of HPAI H5 viruses that induce broadly protective T-cell responses in chickens and determine their potential for enhancing current vaccines. To achieve this, we will leverage a viral vector platform to accomplish three objectives: 1) Define cytotoxic T cell responses against non-surface H5N1 HPAIV proteins, 2) Determine the synergistic efficacy of antibody and T cell responses, and 3) Define components for companion DIVA assays. We expect to identify novel targets for HPAI H5 vaccines in poultry and assess their potential for advancing current avian influenza virus vaccines. We expect these outcomes to significantly advance strategies for developing HPAI vaccines for poultry.
Project Methods
Generation of recombinant NDV vectors:NDV vectors encoding non-surface proteins from a recent H5N1 HPAIV (Clade 2.3.4.4b) will be rescued in cells and propagated in embryonated chicken eggs. The vectors will be verified using immunofluorescence assay (IFA) and Western blot in vitro, as well as through replication assessment in SPF chickens.Defining cytotoxic T cell responses:MHC-homozygous chickens will be immunized intranasally with recombinant NDV vectors. Four weeks post-immunization, T cells will be isolated from various tissues to evaluate cytotoxic CD8 T cell responses. Perforin and IFN-γ levels will be measured as surrogate markers of cytotoxicity, and direct target cell death assays will be performed to confirm functional activity.Synergistic efficacy of antibody and T cell responses:SPF chickens will be immunized with individual or combined NDV vectors and subsequently subjected to a lethal challenge with an antigenically mismatched HPAIV. The ability of T cell responses to enhance protection against variant viruses will be evaluated based on reduction in mortality, clinical scores, and viral shedding. Immunohistochemistry (IHC) will be used to assess T cell presence at local sites pre- and post-challenge.Companion DIVA assays:Production and affinity purification of N1, PB2, PB1, PA, NP, and M1 proteins will be performed. Immunization-specific and infection-specific antibodies will be distinguished using in-house or commercial ELISA assays. Serum samples from the above challenge study will be used to evaluate the proof-of-concept DIVA assay.