Summary
Non Technical Summary
The parapoxvirus Orf virus (ORFV) has long been recognized for its unique immunomodulatory effects on various animal species, and ORFV preparations are used in Veterinary Medicine as a preventive and therapeutic immunomodulatory agent. However, the functions (genes, proteins, and mechanisms of action) evolved by ORFV to modulate host immune responses are poorly understood. We have identified novel ORFV genes with putative virulence and immunomodulatory functions that lack similarity to other viral or cellular proteins. We hypothesize these ORFV genes encode for novel immunomodulatory proteins (ImPs).The objectives of this study are to: 1) identify novel ImPs of ORFV and,determine the molecular mechanism(s) by which ORFV ImPs interfere with immune signaling pathways and2)determine function(s) of ORFV ImPs in the natural host, including assessment of ORFV ImP gene-deleted viruses for vaccine vector potential.We anticipate that successful completion of these studies will have broad impact on immunovirology and on current understanding of the immunomodulatory properties of ORFV. The work will identify and characterize proteins with novel and broad based immunomodulatory activities suitable for use in a variety of animal species potentially offering advantages over existing immunotherapies and positively impacting formulation of improved vaccines, vaccine vectors and adjuvants. In addition, an improved understanding of novel viral immunomodulatory functions and host responses in the skin may suggest new and improved strategies for controlling viral and other diseases targeting skin and mucosal surfaces.
Objectives & Deliverables
Goals / Objectives
The objectives of this study are: 1)Identification and functional characterization of novel ORFV immunomodulatory proteins(ImPs). and2)Determine function(s) of ORFV ImPs in the natural host, including assessment of ORFV ImP gene-deleted viruses for vaccine vector potential.
Challenges
Project Methods
1)Identification and functional characterization of these novel ORFV ImPs.Novel ORFV ImPs will be characterized by using recombinant ORFV ImP gene-deleted viruses (OV-IA82ΔImP) and transiently expressed ImPs in RNA Seq, real time- PCR and pathway analysis of cellular gene expression, rapid screening assays to assess immune signaling pathways, and immunoprecipitation/mass spectrometry to identify potential cellular protein targets of ImPs. Predicted ImP-affected cellular pathways will be examined further to define specific ImP target(s) and function: western immunoblots using pan-and phosphor-specific antibodies will be used to define expression and activation state of key pathway components; reciprocal co-immunoprecipitations and confocal microscopy will be used to identify ImP-cellular pathway interacting proteins; and functional assays (CRISPR knock outs/siRNA knock downs, specific pathway inhibitors or pathway product(s) assays) targeting key steps in the pathway will be used to define ImP target(s) and function within the affected pathway.2)Determine function(s) of ORFV ImPs in the natural host, including assessment of ORFV ImP gene-deleted viruses for vaccine vector potential.To assess the immunomodulatory functions of ORFV ImPs in sheep, parameters of infection/disease/host response will be compared between mock infected and animals inoculated with OV-IA82ΔImP or its revertant OV-IA82-RVImPFlagusing a natural host (sheep) model of infection. Viral loads and extent and timing of histological changes and composition of the inflammatory infiltrates in lesions will be compared using RT-PCR, histology and immunohistochemistry (IHC). And, RNA Seq analysis and ELISA assays of skin biopsy samples will be used to compare global gene expression patterns and measure levels of target cytokines, other immune mediators or other products of potentially affected pathways. To evaluate effects of a given ImP on vaccine vector performance, animals will be vaccinated with ORFV viruses containing single ImP gene deletions (OV-IA82ΔImP) and expressing a target antigen, Green Florescent Protein (GFP). Humoral and cellular immune responses against GFP will be assessed using serological assays and IFN-γ ELISpot assays to evaluate quality, magnitude and duration of host response to the target antigen.?
Principle Investigator(s)
Planned Completion date: 30/06/2024
Effort: $500,000.00
Project Status
COMPLETE
Principal Investigator(s)
National Institute of Food and Agriculture
Researcher Organisations
Recipient Organization UNIVERSITY OF ILLINOIS 2001 S. Lincoln Ave. URBANA,IL 61801
United Kingdom