Performing Department
Veterinary Research & Extension
Non Technical Summary
Toxoplasma gondii is a highly prevalent zoonotic parasite that causes significant economic losses in livestock through abortions. In sheep, T. gondii is the second most commonly diagnosed cause of infectious abortion world-wide. No drugs nor safe and effective vaccines against T. gondii currently exist for livestock. In previous studies, we have found that a T. gondii dense granule protein (GRA10) is a soluble secreted protein that is essential for growth of intracellular parasites in sheep cells. By analysis of data in Toxodb (Toxoplasma Genomics Resource Database), we have found that GRA10 is expressed during all stages of the parasite, and that it is highly conserved among all T. gondii strains, with orthologies in other important protozoa including Neospora, Babesia, Theileria, Eimeria and Cryptosporidium, suggesting an evolutionarily conserved role of GRA10. We have identified several predictable CD4+ T-cell, CD8+T-cell, and B-cell epitopes in GRA10 protein sequence. Therefore, this project seeks to perform experimental evaluation of the antigenic potential of those GRA10 epitopes in sheep. The knowledge to be gained from this study will be important for designing new effective multivalent vaccines against T. gondii infections that would ultimately increase productivity of the sheep industry.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
To determine the immunogenicity of predicted CD4+, CD8+ and B-cell GRA10 epitopes.Overview: By bioinformatic analysis, we have found that GRA10 possesses several CD8+, CD4+T-cell and B-cell epitopes with the potential of inducing cell-mediated and humoral immune responses. Here, we will evaluate these epitopes to identify those that invoke immune responses in sheep cells.
Project Methods
Overview: By bioinformatic analysis, we have found that GRA10 possesses several CD8+, CD4+T-cell and B-cell epitopes with the potential of inducing cell-mediated and humoral immune responses. Here, we will evaluate those epitopes to identify those that invoke immune responses in sheep cells.A.) In vitro INF-γ production assay to evaluate immunogenicity of predicted CD4+and CD8+ GRA10 epitopes: An effective immune response against T. gondii infection is associated with a robust Th1-biased CD4+ and CD8+ cell-mediated immune response characterized by high levels of IL-12 and IFN-g. Therefore, we will screen the CD4+and CD8+ T-cell GRA10 epitopes for their ability to induce IFN-g production in sheep peripheral blood mononuclear cells (PBMCs) using in vitro assays. The GRA10 peptides will be commercially obtained, and will be reconstituted in dimethyl sulphoxide (DMSO) as stock solutions. PBMCs will be isolated from fresh blood obtained from sheep that will test sero-negative for T. gondii. The PMBCs will be treated with the peptides in culture and incubated for 24 hours. Then RNA will be extracted from the cells for cDNA synthesis followed by real time PCR analysis of IFN-g and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. GAPDH transcripts will be used for normalization. From each category of peptides (CD4+ T-cell or CD8+ T-cell peptides), the peptides that will induce IFN-g transcript expression significantly will be selected for subsequent in vivo testing.B.) In vivo evaluation and deconvolution of predicted GRA10 B-cell epitopes: Four groups of pooled peptides (fiveof each of CD4+, CD8+ and B-cell peptides per pool) will be prepared by mixing equal amounts of each peptide. For each animal, the pooled peptides will be diluted in sterile PBS and emulsified in an equal volume of Freund's Incomplete Adjuvant (FIA). Healthy, adult female sheep that are sero-negative for T. gondii will each be inoculated subcutaneously with the FIA-emulsified pooled peptides. There will be threesheep per group. Control inoculums will consist of sterile PBS (containing a volume of DMSO equivalent to that used in test inoculums) emulsified in FIA that will be administered to threecontrol sheep. Before the initial inoculation of sheep, blood samples will be collected for extracting pre-immune sera. Two booster inoculations will be administered at two weekintervals with the peptide solution emulsified in FIA. Two weeks after the last booster, blood will be collected from sheep for sera extraction. Antibody titers in the pooled sera for each category of sheep will be determined by Enzyme Linked Immuno-Sorbent Assay (ELISA) that will be customized to use the respective pooled peptide solutions (used in the inoculum) as the ELISA antigen. The pooled sera for each group of sheep will be used as the test sera while Horseraddish Peroxidase (HRP)-conjugated anti-sheep antibody will be used as the secondary antibody in the assay. TMB (3,3',5,5'-tetramethylbenzidine) will be used as the chromogenic substrate for HRP. Reaction absorbances will be read at 450 nm to measure the reactivity of the sera to the peptides pool. The peptides pool that will have the highest reactivity to sera will be deconvoluted by using individual peptides from the pool as antigen in the ELISA assay in order to identify the individual peptides that give the strongest reaction to sera.