Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-027-039-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2026
End Date: May 31, 2027
Objective:
Surveillance and countermeasure development for high-consequence zoonotic pathogens that threaten agricultural systems are limited by immune profiling tools that lack resolution, scalability, and cross-species compatibility. To address this gap, this project adapts two complementary platforms for high-consequence zoonotic pathogens: B-TOF highly multiplex serology to define scalable, cross-species antibody signatures of exposure and vaccination, and CODEX highly multiplexed spatial tissue imaging to map immune cell organization and pathogen-associated microenvironments in tissues. By applying these tools to banked human blood specimens and viral challenge animal model specimens, we will generate interpretable reference data, immune signatures, and validate a scalable platform for future cohort expansion that advances USDA One Health surveillance, preparedness, and countermeasure evaluation.
This work will be accomplished through the following aims:
1. Develop and validate multiplex BTOF serology panels for zoonotic pathogens of high consequence using recombinant viral antigens, enabling simultaneous IgG, IgM, and IgA detection across humans and animal models/host species.
2. Establish multi-species and multi-matrix compatibility of BTOF for serum, plasma, and minimally invasive mucosal samples.
3. Demonstrate feasibility for B-TOF implementation to support future field surveillance for humans, livestock, and wildlife populations.
4. Establish CODEX/PANINI workflows for inactivated FFPE tissues to enable high-plex spatial immune mapping with zoonotic pathogen localization.
Approach:
B-TOF Multiplex SerologyB-TOF (Bead-based Time-of-Flight) is a next-generation multiplex serology platform capable of measuring tens of thousands of antibody–antigen interactions in a single assay run. It uses uniquely mass-tagged barcoded beads to enable ultra-high multiplexing, low sample volume requirements, and rapid antigen adaptation.Within this project, B-TOF will characterize antibody breadth, isotype patterns, and cross-reactivity using banked specimens, supporting scalable surveillance across diverse hosts and pathogen variants. Early activities will establish SOPs and validate broad antigen panels for zoonotic pathogens of concern (e.g., EBOV, CCHF, NiV, HeV), and confirm performance under approved inactivation conditions (Aim 1). Following panel validation, performance across matrices and species will be explored, including serum, plasma, and mucosal/minimally invasive samples, with paired animal and human data collected for the same pathogens (Aim 2). Reference datasets with signal ranges across species, sample types, and exposure/vaccine groups will serve as pilot data for larger-scale human and animal serosurveillance implementation (Aim 3).CODEX Spatial Tissue ProfilingCODEX (CO-Detection by indEXing) is an iterative, high-plex imaging platform that measures 30+ protein markers on a single fixed tissue section while preserving tissue architecture at single-cell resolution. Using a core immune and structural antibody panel previously optimized for non-human primate (NHP) models, plus pathogen-specific antibodies, we will map immune composition, activation states, and cellular neighborhoods in inactivated samples from laboratory challenge and field studies. CODEX will be paired with same-section viral nucleic acid detection (CODEX/PANINI) to localize latent and replicating viral signals relative to immune microenvironments and tissue pathology. Standardized QC, segmentation, and neighborhood analyses will yield quantitative spatial features linked to protection, pathology, and viral clearance (Aim 4).Integration of Humoral and Tissue Immune DataUsing antibody characteristics from Aim 3 and spatial immune features from Aim 4, we will link tissue immune architecture to antibody profiles and viral challenge outcomes in matched specimens. This integrative framework will support identification of tissue correlates of protection, signatures of harmful inflammation, and features that may translate across species.