Location: Animal Disease Research
2022 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
In the first year of this project, we initiated work on Objective 1, to characterize the immune response that correlates with protection from infection and/or disease. The first step of this objective involved acquiring new techniques in the lab and developing protocols. We have successfully isolated peripheral blood mononuclear cells from cattle, learned about the interpretation of flow cytometry data. Additionally, we have established methods to isolate neutrophils and enrich for immune cell populations using negative selection. Competitive enzyme-linked immunosorbent assays (ELISAs), to measure the antibody response to A. marginale in immunized and infected animals through time are complete. Thus, we have well-defined set of samples collected through time that will be used in identifying the correlates of protection to A. marginale.
Progress was achieved on Objective 2, which is to develop a vaccine platform for A. marginale antigen expression. Toward completing this objective, and in support of Sub-objective 2A, we have immunized animals with Coxiella burnetii Nine Mile Phase II bacterial lysates that express A. marginale proteins. The animals immunized with these lysates developed a strong immune response to the C. burnetii proteins, but a very weak to non-detectable immune response to the A. marginale proteins. This was unexpected because C. burnetii lysates should bias the immune response toward antibody production. We measured the amount of one of the A. marginale proteins in the lysates and determined it was present in level comparable to A. marginale outer membrane extracts, indicating the sufficient amounts of the A. marginale proteins were present to induce an antibody response. Our next step will be to formulate the bacterial lysates differently. This work was performed in collaboration with researchers at Washington State University in Pullman, Washington, under ARS project number 2090-32000-043-002S.
Progress was achieved on Sub-objective 2B, which is to develop media that supports A. marginale replication in the absence of host cells. We are working on developing the foundational methods for this objective and have developed a method to isolate and visualize large number of A. marginale grown in tick cells. Additionally, we have determined that a methionine analog, L-Homopropargylglycine (HPG), can be taken up by A. marginale and used in protein expression. This analog can be detected with a fluorescent label. This will allow us to measure protein incorporation by A. marginale without the use of radioisotopes during testing of various nutrients that could support the growth of A. marginale.
Accomplishments
1. An attenuated mutant of Anaplasma marginale provides protection against disease. Effective vaccines and other methods are needed to prevent bovine anaplasmosis. ARS researchers in Pullman, Washington, in collaboration with researchers at Kansas State University in Manhattan, Kansas, developed a mutant of A. marginale that does not cause disease. All animals immunized with the mutant and then challenged with A. marginale were protected from disease. This mutant opens the door for development of a safe, live attenuated vaccine to prevent bovine anaplasmosis.
2. Anaplasma marginale uses sugar residues on to invade the tick midgut. Entry of A. marginale into the tick midgut is the first step required for transmission of the pathogen. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University in Pullman, Washington, determined that A. marginale alters the sugars displayed on the midgut surface and uses those sugars to gain entry to midgut cells. These findings are an important step in understanding how the pathogen enters tick cells and may lead to identification of pathogen and tick targets that can be used to prevent pathogen transmission and thus disease.
3. Anaplasma marginale requires iron for growth in tick cells. Anaplasma marginale cannot survive or grow outside of the tick, thus for successful tick transmission the pathogen must acquire all of its nutrients directly from tick cells. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University in Pullman, Washington, determined that A. marginale cannot grow if iron levels are reduced in the tick cells. These findings indicate that disruption of iron acquisition by the tick cell or pathogen is a possible method to prevent bovine anaplasmosis.
Review Publications
Solyman, M.M., Ujczo, J., Brayton, K.A., Shaw, D.K., Schneider, D.A., Noh, S.M. 2022. Iron reduction in Dermacentor andersoni tick cells inhibits Anaplasma marginale replication. International Journal of Molecular Sciences. 23(7). Article 3941. https://doi.org/10.3390/ijms23073941.
Koku, R., Herndon, D.R., Avillan, J., Morrison, J., Futse, J.E., Palmer, G.H., Brayton, K.A., Noh, S.M. 2021. Both coinfection and superinfection drive complex Anaplasma marginale strain structure in a natural transmission setting. Infection and Immunity. 89(11). Article e00166-21. https://doi.org/10.1128/IAI.00166-21.
Sidak-Loftis, L.C., Rosche, K.L., Pence, N., Ujczo, J.K., Hurtado, J., Fisk, E.A., Goodman, A.G., Noh, S.M., Peters, J.W., Shaw, D.K. 2022. The unfolded-protein response triggers the arthropod immune deficiency pathway. mBio. Article e00703-22. https://doi.org/10.1128/mbio.00703-22.
Vimonish, R., Capelli-Peixoto, J., Johnson, W.C., Hussein, H.E., Taus, N.S., Brayton, K.A., Munderloh, U.G., Noh, S.M., Ueti, M.W. 2022. Anaplasma marginale infection of Dermacentor andersoni primary midgut cell culture is dependent on fucosylated glycans. Frontiers in Cellular and Infection Microbiology. 12. Article 877525. https://doi.org/10.3389/fcimb.2022.877525.
Scoles, G.A., Lohmeyer, K.H., Ueti, M.W., Bonilla, D., Lahmers, K.K., Piccione, J., Rogovskyy, A.S. 2021. Stray Mexico origin cattle captured crossing into Southern Texas carry Babesia bovis and other tick-borne pathogens. Ticks and Tick Borne Diseases. 12(5). Article 101708. https://doi.org/10.1016/j.ttbdis.2021.101708.
Hove, P., Madesh, S., Nair, A., Jaworski, D., Liu, H., Ferm, J., Kleinhenz, M.D., Highland, M.A., Curtis, A.K., Coetzee, J.F., Noh, S.M., Wang, Y., Genda, D., Ganta, R.R. 2022. Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis. PLoS Pathogens. 18(5). Article e1010540. https://doi.org/10.1371/journal.ppat.1010540.
