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

Location: Animal Disease Research

2017 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
In the first year of this new project, we are working toward the completion of Subobjective 1A of Objective 1, to identify the surface proteins of Anaplasma marginale that mediate adhesion to the tick midgut. The first step in meeting this objective is to construct a phage display library. Phage display libraries, in general, are composed of a population of viruses engineered to display proteins from the bacterial pathogen of interest. For this project, we will use the phage display library to display all known and predicted surface proteins of A. marginale. This is a new technique for our laboratory. We have acquired the knowledge and capacity for growing and handling phages. Importantly, we have PCR amplified all genes of interest, and cloned them, as a population, into T7 phages. Currently we are in the process of evaluating the library to determine the frequency of each member of the library. Next we will optimize the conditions used for the assays that will be used to identify the A. marginale adhesins.

Accomplishments
1. Discovered that administering lipids to ticks causes up-regulation of the tick immune system and reduces pathogen colonization. Tick borne pathogens cause a variety of severe diseases in both humans and animals, including Rocky Mountain spotted fever, Lyme disease, and human and bovine anaplasmosis. A lack of understanding of the tick immune system and particularly the interaction between the pathogen and tick immune system is a major impediment to the development of methods to prevent tick borne diseases. In collaboration with researchers at the University of Maryland in Baltimore, Maryland, and the University of Sao Paulo, Brazil, ARS researchers at Pullman, Washington, have determined that the tick innate immune system is markedly different than that of well-studied insects, such as Drosophila. Importantly, pretreatment of ticks with lipids derived from pathogens stimulates the tick immune system and reduces the ability of these pathogens, including Anaplasma species to colonize the tick. These findings provide the foundation for developing effective methods to prevent tick borne diseases.

2. Overcoming Anaplasma-specific immune suppression as an alternative method to control bovine anaplasmosis. Once an animal is infected with Anaplasma marginale, it generally remains infected for life and treatment of disease relies on the use of antibiotics, though complete clearance of the pathogen is difficult even when high doses of antibiotics are used for extended time periods. One reason for this long-term persistent infection is the functional exhaustion of the population of immune cells (CD4+ T cells) required to prevent or control the disease. Until now, the mechanisms that mediate this functional exhaustion were unknown. In collaboration with colleagues at Hokkaido University, Soporo, Japan, and Washington State University in Pullman, Washington, ARS researchers at Pullman, Washington, determined that molecules expressed on the surface of the immune cells (PD-1 and LAG-3) contribute to the A. marginale-specific immune suppression. Blockade of these receptors helps restore the function of these immune cells. These findings indicate that therapies, such as antibody treatment, can potentially be used to replace or reduce the use of antibiotics in livestock to control bovine anaplasmosis.

3. Identifying conserved epitopes for use in a broadly protective vaccine against bovine anaplasmosis. Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterial pathogen of cattle that causes economic losses to cattle producers throughout the world. This pathogen is widespread in the U.S., with greater than 80 percent of cattle in some regions of Texas being infected. One of the constraints of vaccine development is the variability of A. marginale strains such that one vaccine is not protective against all strains. In collaboration with colleagues at Washington State University in Pullman, Washington, and the University of Ghana in Legon, Ghana, ARS researchers at Pullman, Washington, have identified a vaccine candidate that is broadly conserved among geographically diverse strains, including those from North America, Mexico, Ghana and Australia. Importantly, this vaccine candidate is recognized by T cells, is surface exposed and antigenic. This finding is important for the development of a vaccine that is broadly protective against diverse A. marginale strains.

Review Publications
Capelli-Peixoto, J., Carvalho, D.D., Johnson, W.C., Scoles, G.A., Fogaça, A.C., Daffre, S., Ueti, M.W. 2017. The transcription factor relish controls anaplasma marginale infection in the bovine tick rhipicephalus microplus. Developmental and Comparative Immunology. doi: 10.1016/j.dci.2017.04.005.
Okagawa, T., Konnai, S., Deringer, J.R., Ueti, M.W., Scoles, G.A., Murata, S., Ohashi, K., Brown, W.C. 2016. Cooperation of Pd-1 and LAG-3 contributes to T-cell exhaustion in anaplasma marginale-infected cattle. Infection and Immunity. doi: 10.1128/IAI.00278-16.
Deringer, J.R., Forero-Becerra, E.G., Ueti, M.W., Turse, J.E., Futse, J.E., Noh, S.M., Palmer, G.H., Brown, W.C. 2016. Identification of a cell epitope that is globally conserved among outer membrane proteins (OMPs) OMP7, OMP8, and OMP9 of anaplasma marginale strains and with OMP7 from the A. marginale subsp. centrale vaccine strain. Clinical and Vaccine Immunology. doi: 10.1128/CVI.00406-16.
Schetters, T., Bishop, R., Crampton, M., Kopacek, P., Lew-Tabor, A., Maritz-Olivier, C., Miller, R., Mosqueda, J., Patarroyo, J., Rodriguez-Valle, M., Scoles, G.A., De La Fuente, J. 2016. Cattle tick vaccine researchers join forces in CATVAC. Parasites & Vectors. 9:105.
Shaw, D.K., Brown, L.J., Reif, K.E., Smith, A.A., Scott, A., McClure, E., Sundberg, E., Snyder, G., Ueti, M.W., Pedra, J.H. 2017. Infection-derived lipids elicit a novel immune deficiency circuitry in arthropods. Nature Communications. doi:10.1038/ncomms14401.