Location: Animal Disease Research
Project Number: 2090-32000-039-35-A
Project Type: Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Aug 31, 2021
East Coast Fever (ECF) is a tick-borne disease of cattle caused by the apicomplexan parasite, Theileria parva. Transmission of T. parva by Rhipicephalus appendiculatus ticks leads to pulmonary edema and respiratory failure in susceptible cattle. Currently, prevention predominantly relies on the infection and treatment method (ITM) vaccine, in which cattle are simultaneously infected with live T. parva sporozoites and treated with long-acting oxytetracycline. While the ITM vaccine elicits immune protection, production of live sporozoite stabilates is expensive, difficult to standardize, and limits widespread adoption by smallholder farmers. Thus, there is a critical need to develop a next generation vaccine against T. parva. To develop improved vaccines, the Animal Disease Research Unit (ADRU, ARS-USDA) and members of the international East Coast Fever consortium are interested in characterizing the humoral immune response to T. parva vaccination by sequencing natively paired immunoglobulin genes expressed in single B cells from vaccinated cattle. Cattle from ECF endemic areas contain antibodies that neutralize sporozoite infectivity in vitro. The primary target of these antibodies, and neutralizing murine monoclonal antibodies, is the highly conserved, major sporozoite surface protein, p67. When used in vaccination trials, recombinant p67 induced immunity in approximately 50% of cattle following T. parva sporozoite needle challenge. In field trials, this was reduced to approximately 25%. No correlations were revealed between sporozoite neutralizing titers and immunity to ECF, and differences in IgG1 and IgG2 isotype responses were not observed between immune and susceptible cattle. Additionally, approximately 30% of bovine immunoglobulins contain exceptionally long CDR3H regions with up to 62 amino acids in length. The role of these antibodies in sporozoite neutralization is unknown. Our goal is to address these knowledge gaps for vaccine development to T. parva and other diseases impacting cattle.
At USDA-ARS ADRU: One group of Holstein cattle will be immunized and boosted multiple times with p67 vaccine formulations. A second group of cattle will be immunized with vaccine vector or adjuvant only, as negative controls. Blood will be collected at multiple time points following each immunization, and the antibody response to the p67 antigen assessed via ELISA, western blot, and/or ELISPOT assays. For these assays, recombinant p67 antigen will be generated and utilized. At each time point, white blood cells will by cryopreserved. Once cattle have developed a strong immune response to p67, cryopreserved peripheral blood mononuclear cells (PBMC) and p67 antigen will be sent to Pirbright. Concurrently, ARS will continue development of monoclonal antibodies to bovine CD19, CD38, and CD138, which are markers of plasma cells (antibody secreting cells). These antibodies and/or hybridomas will also be shipped to Pirbright. The laboratory personnel at Pirbright will sort bovine plasmablasts from the cryopreserved PBMC using ER Tracker dye staining in conjunction with surface Ig labelling, p67 tetramer labeling and/or use of ARS monoclonal antibodies to CD19, CD38, and CD138. Using well-established methods in their lab, they will then sequence immunoglobulin pairs from isolated plasmablasts and generate recombinant antibodies. This will likely result in 20-100 antibodies. Those antibodies will be shipped back to ADRU for further testing. At that point, ARS will screen the antibodies for reactivity with p67 using ELISA and sporozoite neutralization assays. Any antibodies that react with p67 will be expanded and further tested.