Location: Animal Disease Research Unit
2024 Annual Report
Objectives
Objective 1: Characterize the immune response that correlates with protection from infection and/or disease.
Sub-objective 1A: Identify the functional antibody profile that predicts protection against bovine anaplasmosis.
Sub-objective 1B: Identify the vaccine candidates against which the protective, functional antibody response is directed.
Objective 2: Develop a vaccine platform for A. marginale antigen expression.
Sub-objective 2A: Determine if A. marginale vaccine candidates expressed by C. burnetii Nine Mile phase II induce protective immunity.
Sub-objective 2B: Develop media that supports A. marginale replication in the absence of host cells.
Objective 3: Devise functional genomics strategies, including gene editing, for developing traits in ruminant livestock to address to enhance disease resistance, enhanced reproductive efficiency, and improve resiliency to ticks, tickborne diseases and other diseases made more prevalent through changing environmental conditions.
Approach
Goal 1A1: Characterize the functional Fc-mediated antibody response produced by immunization of cattle using A. marginale OMPs. Specifically, we will measure antibody dependent cellular phagocytosis by bovine monocytes and neutrophils, antibody dependent complement activation, antibody dependent activation of NK cells and WC1+ 'd T cells, and antibody dependent platelet activation.
Goal 1A2: Identify the functional antibody profile that best predicts protection from disease. Following challenge with A. marginale, nearly all animals immunized with OMPs are protected from severe disease, however, the degree of protection among individual animals tends to be variable. We will leverage this variation to identify the functional antibody profiles that best predict protective immunity.
Goal 1B: Use the functional antibody profile predictive of protective immunity to select vaccine candidates for immunization and challenge trials. We will have identified the Fc mediated effector functions that correlate with protective immunity to bovine anaplasmosis. We will then use these correlates of immunity to identify individual proteins against which the antibodies that mediate these protective immune functions are directed. This will allow us to prioritize the existing vaccine candidates for testing in immunization and challenge trials. The vaccine candidates will be expressed as recombinant protein and used as antigen in the functional antibody assays. We will identify the candidates that elicit an antigen-specific antibody profile that mirrors the profile predictive of protection.
Hypothesis 2A1: Immunization of cattle with A. marginale Omps expressed in C. burnetii phase II induce antibodies that recognize the corresponding native A. marginale proteins. As a proof of principle, we will immunize animals with proteins expressed in C. burnetii phase II and determine if the resulting antibodies bind native A. marginale proteins.
Hypothesis 2A2: The vaccine candidates identified in Sub-objective 1B, when expressed in C. burnetii phase II, induce protection against A. marginale challenge. We will then express the vaccine candidates prioritized in Sub-objective 1B in C. burnetii phase II and determine if they induce protective immunity.
Goal 2B: Develop an axenic growth medium for A. marginale. An efficient method to culture A. marginale in the absence of animals or host cells will allow for the use of OMPs in a vaccine and circumvent the need to identify a subset of proteins and the appropriate formulation to produce a recombinant vaccine. Using a step-wise approach we will identify the nutrients and other components required for A. marginale metabolism as measured first by protein synthesis and then by replication. Once axenic replication is achieved, we will verify expression of a full array of outer membrane proteins.
Progress Report
This report documents FY 2024 progress for project 2090-32000-043-000D, “Identifying Effective Immune Responses and Vaccine Development for Bovine Anaplasmosis”, which began in October 2021.
In support of Objective 1, to characterize the immune response that correlates with protection from infection or disease, ARS researchers in Pullman, Washington, have measured antibody-dependent monocyte and neutrophil phagocytosis that is mediated by the invariant (Fc) domain of antibodies. To accomplish this, they expressed and purified five A. marginale outer membrane proteins that are vaccine candidates. They measured the ability of serum antibodies from cattle with immunity to A. marginale to mediate phagocytosis by neutrophils and monocytes. Overall, phagocytic scores for monocyte mediated phagocytosis were greater than neutrophil mediated phagocytosis. Antibodies against OmpA, Msp1b, and the conserved regions of Omp7/8/9 have the highest levels of monocyte phagocytosis. Antibodies directed against Msp1a, Msp1b and to a lesser degree, conserved regions of Omp7/8/9 mediated the highest levels of neutrophil phagocytosis. One goal of this work is to prioritize vaccine candidates for testing through identifying correlates of immunity. Currently, monocyte phagocytosis directed against conserved regions of Omp7/8/9, Msp1b, and OmpA have a moderate correlation with reduced disease severity. Additionally, neutrophil phagocytosis against Msp1a has a moderate correlation with reduced disease severity. ARS researchers conclude that vaccination should induce isotypes of antibodies that enhance monocyte phagocytosis and are directed against OmpA, Msp1b, and the conserved regions of Omp7/8/9. The next steps involve measuring antibody mediated complement fixation, antigen specific antibody levels, and neutralization.
The major focus of Objective 2, development of a vaccine platform for A. marginale antigen expression, is to develop nutrient medium that supports metabolic activity and ultimately replication of A. marginale. In the long term, this will serve as an alternative method for antigen production for vaccines. A. marginale lacks complete pathways for amino acid biosynthesis, and key enzymes in the Entner-Doudoroff and glycolysis pathway. While the tricarboxylic acid (TCA) cycle, fatty acid synthesis, and purine and pyrimidine biosynthesis pathways are complete. The optimal ion concentrations, carbon sources, and requirements for other nutrients are unknown. ARS researchers have optimized a method for isolation of A. marginale from tick cells and validated a method to cryopreserve the bacteria eliminating the constraint of continuous culture of large numbers of A. marginale. They have identified a buffer that supports A. marginale host cell-free metabolic activity and established positive and negative controls for medium development. Importantly, ARS researchers have determined that pyruvate and succinate are carbon sources for A. marginale. The next steps are to test combinations of carbon sources and other nutrients such as iron to enhance metabolic activity of A. marginale.
Accomplishments
1. Identified Anaplasma marginale surface proteins used for entry into tick cells and bovine red blood cells. A. marginale causes bovine anaplasmosis, a high impact, production limiting disease of cattle found worldwide. Few tools, including recombinant vaccines, are available for preventing bovine anaplasmosis in part due to major knowledge gaps in our ability to select relevant vaccine components. A. marginale is a tick-transmitted, obligate, intracellular pathogen that infects red blood cells in cattle. Thus, a vaccine-induced immune response that prevents the pathogen from entering bovine red blood cells and tick cells could both prevent disease and reduce tick transmission. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University, identified the A. marginale surface proteins that bind tick cells and bovine red blood as part of the process of host cell entry. Identification of these high priority vaccine candidates greatly advances efforts toward development of a vaccine to reduce the impact of bovine anaplasmosis, which will improve cattle health and production and food security worldwide.
Review Publications
Ntsama, F., Noh, S.M., Tizzani, P., Ayangma Ntsama, C.F., Nteme Ella, G.S., Awada, L., Djatche Tidjou, G. 2024. Identification of risk factors on rabies vaccine efficacy from censored data: Pre-travel tests for dogs and cats from Yaounde (2005-2015). Research in Veterinary Science. 174. Article 105278. https://doi.org/10.1016/j.rvsc.2024.105278.
Noh, S.M., Ujczo, J.K., Alperin, D.C., Jarvis, S.M., Solyman, M.S., Koku, R., Akinsulie, O.C., Hoffman, E.E. 2024. Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display. Infection and Immunity. 92. Article e00540-23. https://doi.org/10.1128/iai.00540-23.
Noh, S.M., Ujczo, J.K., Alperin, D.C. 2024. Identification of Anaplasma marginale adhesins for bovine erythrocytes using phage display. Frontiers in Tropical Diseases. 5. Article 1422860. https://doi.org/10.3389/fitd.2024.1422860.