Research Project: Identification of Tick Colonization Mechanisms and Vaccine Development for Anaplasmosis

Location: Animal Disease Research

2021 Annual Report

Objectives
Anaplasma marginale is a tick-transmitted, obligate intracellular bacterial pathogen of ruminants. Survival and ongoing transmission of this pathogen requires entry and replication in specific cells types in the ruminant host and tick vector, both of which serve as potentially effective sites of intervention. First, in the ruminant host, induction of an immune response that blocks A. marginale adhesion and entry into erythrocytes, the primary host cell, would result in protection from challenge. Second, delivery of an immune response to the tick midgut targeting A. marginale adhesins and the corresponding midgut receptors could prevent colonization of the tick midgut, thus preventing or limiting ongoing transmission. However, little is known about the molecules and mechanisms required for entry either into bovine erythrocytes or the tick midgut, which is the first barrier to tick colonization. We propose to use a phage display library as an unbiased screen to identify A. marginale surface proteins that mediate adhesion to either bovine erythrocytes or Dermacentor andersoni tick midguts. The ability of the identified adhesin candidates to bind their respective host cells will then be confirmed. The tick midgut is biologically unusual and thus likely has unique surface receptors that could additionally be targeted by the bovine immune response. Consequently, candidate receptors for the A. marginale midgut adhesins will be identified using pull-down assays. Specific binding between each A. marginale adhesin and its corresponding midgut receptor candidate will be confirmed. Because antibody is the most likely effector molecule, the ability of bovine antibody to block binding of the adhesin to its target cell will be tested in vitro. If successful, immunization and challenge experiments will be done to determine the in vivo efficacy of these two approaches. Objective 1: Identify the determinants of tick colonization of A. marginale in the host with the long-term goal of blocking transmission. • Subobjective 1A: Identify the A. marginale proteins that mediate adhesion to the D. andersoni midgut. • Subobjective 1B: Identify D. andersoni midgut receptors that serve as the binding partners for the A. marginale adhesins. • Subobjective 1C: Determine if bovine antibody directed against A. marginale adhesins and D. andersoni midgut binding partners will reduce A. marginale midgut colonization. • Subobjective 1D: Determine the efficacy of immunization against A. marginale adhesins and tick midgut receptors in blocking D. andersoni colonization. • Subobjective 1E: Determine the amount of heterogeneity among the A. marginale adhesins and midgut receptors proteins in A. marginale and North American populations of Dermacentor spp, respectively. Objective 2: Develop a safe and efficacious vaccine for Anaplasma marginale using novel platforms and techniques. • Subobjective 2A: Identify A. marginale adhesins for bovine erythrocytes. • Subobjective 2B: Determine if immunization against adhesins will confer protective immunity to challenge with A. marginale.

Approach
In Objective 1 we will identify the determinants of tick colonization of A. marginale in the host with the long-term goal of blocking transmission. Due to the unusual nature of the A. marginale outer membrane, algorithms to identify outer membrane proteins are often inaccurate and identification of functionally relevant vaccine targets is difficult due to the obligate intracellular nature of A. marginale. Thus, we will use a phage display library to perform an unbiased screen of the A. marginale proteome to identify midgut adhesins. The functional significance of the adhesin candidates will then be determined using a combination of adhesion assays, immunofluorescence and competitive inhibition of A. marginale invasion of tick cells. It is possible the initial phage display library will be of low diversity. If this is the case, an alternative phage display systems will be used. Next we will identify D. andersoni midgut receptors that serve as the binding partners for the A. marginale adhesins because both bacterial ligands and their receptors on the midgut epithelial cells could serve as targets of the bovine immune system to disrupt A. marginale colonization of the tick midgut. To identify the midgut receptors, we will use pull-down assays. Once the putative midgut receptors are identified, siRNA will be used to knock-down gene expression of the midgut receptor candidates and A. marginale infection rate and levels will be measured in ticks. Finally, we will determine if bovine antibody directed against the A. marginale adhesins and D. andersoni midgut binding partners will reduce A. marginale midgut colonization in vivo and in vitro. It is possible that individual anti-ligand or anti-receptor antibodies will fail to significantly block A. marginale colonization of DAE cells. If this is the case, mapping of the specific binding domains will be done and the immune response will be directed specifically against the binding domains of each ligand. The focus of Objective 2 is to develop a safe and efficacious vaccine to prevent anaplasmosis. Toward this end, we will identify A. marginale outer membrane proteins that serve as adhesins for bovine erythrocytes using the phage display library developed in Objective 1. Next we will conduct an immunization and challenge trial to determine if the identified adhesins provide protection from challenge.

Progress Report
This is the final report for the project 2090-32000-038-000D which will terminate in September 2021. The new project, titled “Identifying Effective Immune Responses and Vaccine Development for Bovine Anaplasmosis” is currently undergoing NP03 OSQR review. A summary of results for all the expiring project sub-objectives are described below. In support of Objective 1, we developed a phage display library expressing nearly all outer membrane proteins of A. marginale and used it in a non-biased screen to identify candidate A. marginale proteins that mediate adhesion to either tick cells or erythrocytes. Transcriptional profiling was then used to narrow the candidate list. Thus, we have identified a group of five outer membrane proteins, with previously unknown function that mediating adhesion and entry of A. marginale to either red blood cells (RBCs), tick cells or both. Additionally, these adhesins are conserved among A. marginale strains and will serve as primary vaccine targets going forward. Next, we identified groups of tick genes and their protein products that A. marginale potentially exploits in order to invade, replicate and be transmitted by ticks. These gene products may serve as targets of anti-tick and/or anti-pathogen interventions. First, we used existing databases to assemble D. andersoni genes likely required for A. marginale to bind and invade tick cells. Invasion of A. marginale into tick cells is likely mediated, in part, by glycosylated proteins. We identified and sequenced all genes encoding glycosylation enzymes and confirmed the expression of these enzymes in ticks and cultured cells. This will lay the foundation of the next experiments determining the requirement of these enzymes and the identification of midgut receptors required for A. marginale invasion. Iron is an essential nutrient with limited availability to living organisms. We determined that iron is required for A. marginale replication in the tick. Thus, disruption of genes and proteins involved in tick iron acquisition and metabolism are potential targets to disrupt both the tick and the pathogen. We identified and sequenced 13 genes likely involved in iron uptake and metabolism by D. andersoni ticks. We then induced iron starvation in the cells and measured the response of these genes in an effort to identify receptors involved in iron uptake. However, unexpectedly, no putative iron uptake genes were up-regulated in response to iron reduction. Thus, whole transcriptome analysis will next be done to identify potential iron transporters. Finally, we developed a number of techniques that will be critical for improving our understanding of the interactions between the tick and the pathogen and thus develop effective interventions. These techniques include the develop of an artificial membrane feeding system, which reduce animal use and allow for specific testing of anti-tick or anti-pathogen interventions in a highly controlled manner. We developed a technique to store viable A. marginale without the host cells. This critical technique is potentially widely applicable for obligate, intracellular bacterial pathogens and allows for highly repeatable, quantitative infection and infection blocking assays in tick cell culture. This work will help lay the foundation for the next project plan. Finally, we developed a primary cell line derived from D. andersoni midgut cells and determined this cell line is morphologically similar to digestive cells and, importantly is permissive to A. marginale infection. This will help ensure experiments involving tick, pathogen interactions are biologically relevant by minimizing experimental artifacts that can occur by using immortal cell lines. In support of objective 2, during the life of this project we have determined that Omp7, Omp8, and Omp9 are high priority vaccine candidates because they share a highly conserved T-cell epitope that is present in A. marginale strains in N. American and South Africa. Additionally, Am779 and OmpA, an A. marginale adhesin, are highly conserved among A. marginale strains in South Africa and in the case of OmpA, South Africa and Ghana. Together these data suggest that it may be possible to develop a single, cross-protective vaccine rather than a multitude of strain-specific vaccines. One major limitation in development of an effective vaccine to prevent A. marginale is the poor immunogenicity of proteins expressed in E. coli, which is the standard method for producing bacterial recombinant proteins. To address this limitation, in collaboration with researchers at Washington State University, we determined that the attenuated Coxiella burnetii Nine Mile Phase II (C. burnetii NM II) can express A. marginale proteins. Additionally, multiple immunizations with the bacterial lysates that include the A. marginale vaccine candidates does not produce excessive inflammation and skin damage at the immunization site, which can occur with multiple exposures to wild type C. burnetii. The additional benefit of using C. burnetii NM II is that is produces the type of immune response thought to be required for protection against A. marginale. Thus, the residual C. burnetii proteins will serve as an adjuvant that will correctly bias the immune response. The next steps will be to determine if the immunized animals have an antibody response directed against the A. marginale proteins, then express a broad array of vaccine candidates and test their protective capacity.

Accomplishments
1. Tick salivary gland extracellular vesicles dictate tick feeding success and influence disease severity of tick-borne pathogens. Ticks and borne diseases cause heavy losses to the U.S. cattle industry. Methods to control ticks and prevent transmission of tick-borne diseases are limited to management practices and use of acaricides, which are expensive and partially effective. In a large, multi-institutional collaborative effort, ARS researchers in Pullman, Washington, helped determine that ticks, including D. andersoni, secrete extracellular vesicles during feeding. These vesicles modulate the dermal immune system, facilitate tick feeding and influence the outcome of disease, in a pathogen-dependent manner. This paradigm shifting work will lead to targets for an anti-tick vaccine and help guide development of a vaccine to prevent bovine anaplasmosis.

2. Identification of targets within the tick for control of ticks and tick-borne diseases. Ticks and borne diseases cause heavy losses to the U.S. cattle industry. Methods to control ticks and prevent transmission of tick-borne diseases are limited to management practices and use of acaricides, which are expensive and partially effective. The many knowledge gaps in our understanding of the basic tick biology and physiology limits our ability to develop new strategies for controlling ticks and tick-borne diseases. ARS researchers in Pullman, Washington, in collaboration with colleagues at Washington State University in Pullman, Washington, and the University of Idaho in Moscow, Idaho, identified and sequenced a comprehensive set of tick genes potentially involved in iron metabolism, developed a method to reduce iron levels in tick cells and determined the response of these genes to iron reduction. This information will be essential for determining the mechanism and molecules used by ticks to acquire iron, an essential, limiting nutrient. These mechanisms and molecules could serve as rationale and broadly applicable targets for controlling ticks and tick-borne diseases.

Review Publications
Solyman, M., Brayton, K.A., Shaw, D.K., Omsland, A., McGeehan, S., Scoles, G.A., Noh, S.M. 2020. Predicted iron metabolism genes in hard ticks and their response to iron reduction in Dermacentor andersoni cells. Ticks and Tick Borne Diseases. 12(1). Article 101584. https://doi.org/10.1016/j.ttbdis.2020.101584.
Negretti, N.M., Ye, Y., Malavasi, L.M., Pokharel, S.M., Huynh, S., Noh, S.M., Klima, C.L., Gourley, C.R., Ragle, C.A., Bose, S., Looft, T.P., Parker, C., Clair, G., Adkins, J.N., Konkel, M.E. 2020. A porcine ligated loop model reveals new insight into the host immune response against Campylobacter jejuni. Gut Microbes. 12(1). Article 1814121. https://doi.org/10.1080/19490976.2020.1814121.
Chávez, O.S., Wang, X., Archer, N., Hammond, H.L., McClure, E.E., Shaw, D.K., Buskirk, A.D., Ford, S.L., Morozova, K., Clement, C.C., Lawres, L., O'Neal, A.J., Mamoun, C., Mason, K.L., Hobbs, B.E., Scoles, G.A., Barry, E.M., Sonenshine, D.E., Pal, U., Valenzuela, J.G., Sztein, M.B., Pasetti, M.F., Levin, M.L., Kotsyfakis, M., Jay, S.M., Miller, L., Santambrogio, L., Pedra, J.H. 2021. Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection. Nature Communications. 12. Article 3696. https://doi.org/10.1038/s41467-021-23900-8.
Barbosa, I.C., André, M.R., Amaral, R.B.D., Valente, J.D.M., Vasconcelos, P.C., Oliveira, C.J.B., Jusi, M.M.G., Machado, R.Z., Vieira, T.S.W.J., Ueti, M.W., Vieira, R.F.C. 2020. Anaplasma marginale in goats from a multispecies grazing system in northeastern Brazil. Ticks and Tick Borne Diseases. 12(1). Article 101592. https://doi.org/10.1016/j.ttbdis.2020.101592.