Investigating the Human Immune Response to Ixodes scapularis Tick Bites

National Institute Of Allergy And Infectious Diseases

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Abstract

Tick-borne diseases (TBDs) are a serious public health problem in the United States, with the number of reported cases more than doubling during 2004 to 2016. The deer tick (Ixodes scapularis) is the vector of at least 7 pathogens that cause human diseases: Lyme disease (Borrelia burgdorferi and Borrelia mayonii), anaplasmosis (Anaplasma phagocytophilum), babesiosis (Babesia microti), Borrelia miyamotoi disease (Borrelia miyamotoi), ehrlichiosis (Ehrlichia muris eauclairensis), and tick-borne encephalitis (deer tick virus/Powassan virus). Lyme disease accounts for more than 80% of the reported cases of tickborne diseases, with estimates of over 476,000 cases of Lyme disease been diagnosed and treated per year in the US. Ticks are obligate blood-feeding arthropods and require a blood meal at every active life stage. To acquire its blood meal, an ixodid tick must remain attached to the skin of a vertebrate host and complete its multi-day feeding process. Tick saliva is composed of a large variety of proteins that are differentially expressed throughout the process of tick feeding. Some of these proteins have anti clotting, anti-platelet, vasodilatory, and immunomodulatory activities that allow successful feeding and pathogen transmission. The skin response to tick salivary proteins has been implicated in tick rejection as certain animals (e.g., rabbits, guinea pigs) that are repeatedly infested develop an immune response against tick proteins. Resistance to tick bites is manifested by reduced numbers and body weights of engorged ticks or tick death in subsequent infestations in these animal models and consequently protection against Borrelia burgdorferi transmission. Most people will not feel a tick bite, as tick bites do not tend to cause itching or immediate skin irritation. But people who have had many tick bites can become sensitized to tick bites over time, and this can protect against Lyme disease. Little is known about the nature of the protective immune response. We have combined our expertise to study the response to tick bites in a well standardized clinical setting to identify critical aspects of the human innate and adaptive immune responses in skin and blood following exposure to uninfected Ixodes scapularis ticks; and monitor the acquisition of a tick-associated skin immunity, including itch. This research could lead to the identification of tick salivary proteins that are targets of host immunity and might serve as targets for an anti-tick vaccine. The Lyme Disease Studies Unit at NIAID has been involved for many years in studies to examine the use of larval I. scapularis ticks in xenodiagnosis of B. burgdorferi infection and have extensive experience with placement of larval I. scapularis ticks in humans. The Vector Molecular Biology Section at the Laboratory of Malaria and Vector Research, and the Tick-Pathogen Transmission Unit, at the Laboratory of Bacteriology at Rocky Mountain Laboratories (RML), have extensive experience in working with the tick Ixodes scapularis, the isolation of Ixodes salivary components, molecular aspects of tick salivary proteins and the understanding of guinea pig immune responses to tick exposure, tick rejection and the role tick salivary proteins on tick rejection. Major accomplishments of the past year include concept of the project and the creation of the collaborative group, with the successful application to the Bench to Bedside grant program for the research. Since the Bench to Bedside approval on November 24, 2020, we have worked on developing all the details of the clinical research protocol, and on obtaining the necessary regulatory approvals. We have written the investigational device exemption (IDE) and have received US Food and Drug Administration (FDA)s approval for the study. The clinical protocol is currently undergoing approval by the National Institutes of Health (NIH) Intramural Institutional Review Board. We have also continued to develop and verify the procedures and techniques that will be necessary in the laboratory to support the project. We have established the protocol to isolate RNA from human skin to perform RNA sequence analysis. We tried different strategies to disrupt the skin and isolate RNA of high quality for sequencing. This protocol will be used for the biopsy samples we will get from human subjects in our study. We developed an ELISA assay to measure the antibody response in humans to ticks salivary proteins. We dissected tick salivary glands and prepared salivary gland homogenates and tested sera of human subjects previously exposed to tick bites or human subjects with no previous history of tick exposure. We cloned and expressed several tick salivary proteins to use them as antigens for Western blot analysis. We developed the Western blot assay using first salivary gland homogenate and then expressed and purified recombinant tick salivary proteins to evaluate its potential as biomarker for tick bite . We have established a pipeline to do de novo assembly of tick transcriptomes and to perform proteomic analysis of tick tissues using resources from the NIH High Performance Computer Systems. This will be used to perform tick-related sample processing and evaluation; bioinformatic and gene expression analysis of Ixodes scapularis larvae fed on humans that will be generated from this study. Additionally, the Tick-Pathogen Transmission Unit has been working on developing an assay to test the presence of peptides containing alpha galactose on the salivary glands of ticks. Secretion of proteins containing alpha galactose into humans during tick feeding have been related red meet allergy in the US. Larval ticks are being prepared by Dr. Sam Telford from a laboratory-maintained tick colony at Tufts Veterinary School, following the procedures described in our FDA-approved IDE. Dr. Telford is also supporting Dr. Tirloni in developing the procedures for transfer and maintenance of the Ixodes tick colony used in the IDE to RML. A tick room was recently remodeled and upgraded at RML to accommodate the tick colony. Along with Dr. Craig Martens at NIAID Research Technologies Branch, we are working to develop and run a panel of molecular assays to detect tick-borne pathogens.