Location: Animal Disease Research Unit
2023 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.
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 fiscal year (FY) 2023 progress for project 2090-32000-043-000D, titled, “Identifying Effective Immune Responses and Vaccine Development for Bovine Anaplasmosis”.
In support of Objective 1, to characterize the immune response that correlates with protection from infection or disease, ARS researchers in Pullman, Washington, completed development of assays to measure Fc mediated, antibody-dependent monocyte and neutrophil phagocytosis. The Fc, or invariant region of antibodies interact with the Fc-receptor present on all innate immune cells. This interaction results in a variety of immune effectors, including phagocytosis and activation of a variety of immune cells.
Using these assays, ARS researchers determined that the Fc region of anti-A. marginale antibodies mediate phagocytosis by monocytes and neutrophils at the time A. marginale infection is controlled. A high level of monocyte phagocyte is expected, though the potential role of neutrophils in the control of infection is unexpected.
Next, they compared the Fc-mediated response directed against two A. marginale proteins, Msp5 and Msp1b. The ARS researchers determined that the Fc mediated antibody response for monocyte and neutrophil phagocytosis is greater for antibodies that recognize Msp1b as compared to Msp5. This is important because the anti-Msp1b antibody response is likely to have a role in protective immunity, while the anti- Msp5 antibody is thought to play a minor role in protective immunity. Enzyme-linked immunoassays (ELISAs) to determine if there is a correlation between antigen-specific antibody response and the Fc mediated effector function are complete and data analysis is in progress.
Ultimately completion of this objective relies on cloning, expressing and purifying many A. marginale proteins which are vaccine candidates. Toward facilitating this difficult and labor-intensive work, ARS researchers installed a fast protein liquid chromatograph (FPLC) unit in the lab. FPLC requires a great deal of technical expertise, which our lab has developed in this past year. Additionally, they cloned 11 genes and have protocols to express and purify seven of those proteins. These will be used in assays to measure the functional antibody response to top priority vaccine candidates.
In support of Objective 2, to develop a vaccine platform for A. marginale antigen expression, ARS researchers determined that A. marginale has no basal level of protein synthesis in sucrose phosphate glutamate medium. Additional glutamate added to the buffer is insufficient to induce protein synthesis. The reserchers are in the process of doing a metabolic pathway analysis of A. marginale to predict required nutrients.
Accomplishments
1. Rickettsia bellii, part of the bacterial community normally present in ticks, inhibits the ability of Anaplasma marginale to infect the tick. Anaplasma marginale is a tick borne, bacterial pathogen that causes bovine anaplasmosis, a high impact, production limiting disease of cattle. Few tools are available for preventing this disease in part due to large knowledge gaps in our understanding of interactions between the tick, its microbiome, and the pathogen. Rickettsia bellii, a component of the tick microbiome, resides in over 25 species of ticks. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University in Pullman, Washington, determined that A. marginale is less able to establish an infection in tick cells in the presence of R. bellii and that an established R. bellii infection inhibits A. marginale growth. This interaction highlights the importance of the microbiome in pathogen establishment and transmission. This foundational knowledge may lead to the development of new methods to control bovine anaplasmosis by preventing tick transmission, which will directly support cattle producers worldwide.
2. Anaplasma marginale does not use variation of its surface proteins to overcome existing immunity in cattle. Anaplasma marginale is tick borne pathogen that causes bovine anaplasmosis, a high impact, production limiting disease of cattle. Once infected with a strain of A. marginale, cattle mount an immune response that controls, but does not eliminate that strain. Despite this immune response, cattle remain susceptible to infection with additional strains of A. marginale. The mechanisms A. marginale strains use to overcome the existing immunity in an animal to establish infection are unknown. ARS researchers in Pullman, Washington, in collaboration with researchers at the University of Ghana in Accra, Ghana, determined that the ability of an A. marginale strain to overcome existing immunity is not due to variation in the major surface protein that allows for immune escape. These findings have direct implications in designing vaccines and control programs to reduce the impact of bovine anaplasmosis, which will improve cattle health production and thus food security worldwide.
Review Publications
Koku, R., Futse, J.E., Morrison, J., Brayton, K.A., Palmer, G.H., Noh, S.M. 2023. The use of the antigenically variable major surface protein 2 in the establishment of superinfection during natural tick transmission of Anaplasma marginale in southern Ghana. Infection and Immunity. 91(4). Article e00501-22. https://doi.org/10.1128/iai.00501-22.
Aspinwall, J.A., Jarvis, S.M., Noh, S.M., Brayton, K.A. 2023. The effect of Rickettsia bellii on Anaplasma marginale infection in Dermacentor andersoni cell culture. Microorganisms. 11(5). Article 1096. https://doi.org/10.3390/microorganisms11051096.
