Peromyscus leucopus , Mus musculus , and humans have distinct transcriptomic responses to larval Ixodes scapularis bites.

Ixodes scapularis ticks are an important vector for at least six tick-borne human pathogens, including the predominant North American Lyme disease spirochete Borrelia burgdorferi . The ability for these ticks to survive in nature is credited, in part, to their ability to feed on a variety of hosts without excessive activation of the proinflammatory branch of the vertebrate immune system. While the ability for nymphal ticks to feed on a variety of hosts has been well-documented, the host-parasite interactions between larval I. scapularis and different vertebrate hosts is relatively unexplored. Here we report on the changes in the vertebrate transcriptome present at the larval tick bite site using the natural I. scapularis host Peromyscus leucopus deermouse, a non-natural rodent host Mus musculus (BALB/c), and humans. We note substantially less evidence of activation of canonical proinflammatory pathways in P. leucopus compared to BALB/c mice and pronounced evidence of inflammation in humans. Pathway enrichment analyses revealed a particularly strong signature of interferon gamma, tumor necrosis factor, and interleukin 1 signaling at the BALB/c and human tick bite site. We also note that bite sites on BALB/c mice and humans, but not deermice, show activation of wound-healing pathways. These data provide molecular evidence of the coevolution between larval I. scapularis and P. leucopus as well as expand our overall understanding of I. scapularis feeding. Significance: Ixodes scapularis tick bites expose humans to numerous diseases in North America. While larval tick feeding enables pathogens to enter the tick population and eventually spread to humans, how larval ticks interact with mammals has been understudied compared to other tick stages. Here we examined the transcriptomic response of a natural I. scapularis rodent host ( Peromyscus leucopus ), a non-native I. scapularis rodent host ( Mus musculus ), and an incidental host (humans). We find that there are differences in how all three species respond to larval I. scapularis , with the natural host producing the smallest transcriptomic signature of a canonical proinflammatory immune response and the incidental human host producing the most robust signature of inflammation in response to the larval tick. These data expand our understanding of the pressures on ticks in the wild and inform our ability to model these interactions in laboratory settings.

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks.

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick must remain attached to the skin of the host and complete its multi-day feeding process to acquire its blood meal. Exposing animals to ticks is a common practice for studying host responses to tick bites and tickborne diseases. We developed the procedure, conducted the first human research study, and published the findings on exposing human volunteers to uninfected larval Ixodes scapularis ticks. This article describes the methodology used to construct the containment dressing, how to apply and secure the ticks to the host, how to maintain the dressing, and how to remove the ticks from the host. Exposing volunteers to tick bites is an experimental procedure and must be performed under a clinical research protocol approved by the appropriate regulatory authorities. This method allows for translational research to better understand the human response to tick bites and foster the development of diagnostics, prevention, and therapies for tickborne diseases.

Antiphospholipid autoantibodies in Lyme disease arise after scavenging of host phospholipids by Borrelia burgdorferi.

A close association with its vertebrate and tick hosts allows Borrelia burgdorferi, the bacterium responsible for Lyme disease, to eliminate many metabolic pathways and instead scavenge key nutrients from the host. A lipid-defined culture medium was developed to demonstrate that exogenous lipids are an essential nutrient of B. burgdorferi, which can accumulate intact phospholipids from its environment to support growth. Antibody responses to host phospholipids were studied in mice and humans using an antiphospholipid ELISA. Several of these environmentally acquired phospholipids including phosphatidylserine and phosphatidic acid, as well as borrelial phosphatidylcholine, are the targets of antibodies that arose early in infection in the mouse model. Patients with acute infections demonstrated antibody responses to the same lipids. The elevation of antiphospholipid antibodies predicted early infection with better sensitivity than did the standardized 2-tier tests currently used in diagnosis. Sera obtained from patients with Lyme disease before and after antibiotic therapy showed declining antiphospholipid titers after treatment. Further study will be required to determine whether these antibodies have utility in early diagnosis of Lyme disease, tracking of the response to therapy, and diagnosis of reinfection, areas in which current standardized tests are inadequate.

A Randomized, Double-Blinded, Placebo-Controlled, Phase 1 Study of a Replication-Defective Herpes Simplex Virus (HSV) Type 2 Vaccine, HSV529, in Adults With or Without HSV Infection.

BACKGROUND: Herpes simplex virus 2 (HSV2) causes genital herpes in >400 million persons worldwide. METHODS: We conducted a randomized, double-blinded, placebo-controlled trial of a replication-defective HSV2 vaccine, HSV529. Twenty adults were enrolled in each of 3 serogroups of individuals: those negative for both HSV1 and HSV2 (HSV1-/HSV2-), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2-). Sixty participants received vaccine or placebo at 0, 1, and 6 months. The primary end point was the frequency of solicited local and systemic reactions to vaccination. RESULTS: Eighty-nine percent of vaccinees experienced mild-to-moderate solicited injection site reactions, compared with 47% of placebo recipients (95% confidence interval [CI], 12.9%-67.6%; P = .006). Sixty-four percent of vaccinees experienced systemic reactions, compared with 53% of placebo recipients (95% CI, -17.9% to 40.2%; P = .44). Seventy-eight percent of HSV1-/HSV2- vaccine recipients had a ≥4-fold increase in neutralizing antibody titer after 3 doses of vaccine, whereas none of the participants in the other serogroups had such responses. HSV2-specific CD4+ T-cell responses were detected in 36%, 46%, and 27% of HSV1-/HSV2-, HSV1±/HSV2+, and HSV1+/HSV2- participants, respectively, 1 month after the third dose of vaccine, and CD8+ T-cell responses were detected in 14%, 8%, and 18% of participants, respectively. CONCLUSIONS: HSV529 vaccine was safe and elicited neutralizing antibody and modest CD4+ T-cell responses in HSV-seronegative vaccinees. CLINICAL TRIALS REGISTRATION: NCT01915212.

Hydroa vacciniforme-like lymphoproliferative disorder: an EBV disease with a low risk of systemic illness in whites.

Patients with classic hydroa vacciniforme-like lymphoproliferative disorder (HVLPD) typically have high levels of Epstein-Barr virus (EBV) DNA in T cells and/or natural killer (NK) cells in blood and skin lesions induced by sun exposure that are infiltrated with EBV-infected lymphocytes. HVLPD is very rare in the United States and Europe but more common in Asia and South America. The disease can progress to a systemic form that may result in fatal lymphoma. We report our 11-year experience with 16 HVLPD patients from the United States and England and found that whites were less likely to develop systemic EBV disease (1/10) than nonwhites (5/6). All (10/10) of the white patients were generally in good health at last follow-up, while two-thirds (4/6) of the nonwhite patients required hematopoietic stem cell transplantation. Nonwhite patients had later age of onset of HVLPD than white patients (median age, 8 vs 5 years) and higher levels of EBV DNA (median, 1 515 000 vs 250 000 copies/ml) and more often had low numbers of NK cells (83% vs 50% of patients) and T-cell clones in the blood (83% vs 30% of patients). RNA-sequencing analysis of an HVLPD skin lesion in a white patient compared with his normal skin showed increased expression of interferon-γ and chemokines that attract T cells and NK cells. Thus, white patients with HVLPD were less likely to have systemic disease with EBV and had a much better prognosis than nonwhite patients. This trial was registered at www.clinicaltrials.gov as #NCT00369421 and #NCT00032513.

Xenodiagnosis Using Ixodes scapularis Larval Ticks in Humans.

Xenodiagnosis is the use of a natural vector to detect the presence of an organism, and xenodiagnosis using Ixodes ticks has long been used by entomologists in Lyme disease research to provide evidence of the host's infectious status with Borrelia burgdorferi. We developed the methodology and performed the first human research study using uninfected larval Ixodes scapularis ticks to assess evidence of B. burgdorferi infection. Here, we describe in detail the methodology used for the procedure. Xenodiagnosis using Ixodes ticks in humans remains an experimental method and must be performed under an approved clinical research protocol.

Transcriptome Assessment of Erythema Migrans Skin Lesions in Patients With Early Lyme Disease Reveals Predominant Interferon Signaling.

Background: The most common clinical manifestation of early Lyme disease is the erythema migrans (EM) skin lesion that develops at the tick bite site typically between 7 and 14 days after infection with Borreliella burgdorferi. The host-pathogen interactions that occur in the skin may have a critical role in determining outcome of infection. Methods: Gene arrays were used to characterize the global transcriptional alterations in skin biopsy samples of EM lesions from untreated adult patients with Lyme disease in comparison to controls. Results: The transcriptional pattern in EM biopsies consisted of 254 differentially regulated genes (180 induced and 74 repressed) characterized by the induction of chemokines, cytokines, Toll-like receptors, antimicrobial peptides, monocytoid cell activation markers, and numerous genes annotated as interferon (IFN)-inducible. The IFN-inducible genes included 3 transcripts involved in tryptophan catabolism (IDO1, KMO, KYNU) that play a pivotal role in immune evasion by certain other microbial pathogens by driving the differentiation of regulatory T cells. Conclusions: This is the first study to globally assess the human skin transcriptional response during early Lyme disease. Borreliella burgdorferi elicits a predominant IFN signature in the EM lesion, suggesting a potential mechanism for spirochetal dissemination via IDO1-mediated localized immunosuppression.

Long-term Follow-up of Patients With Lyme Disease: Longitudinal Analysis of Clinical and Quality-of-life Measures.

BACKGROUND: Lyme disease is the most common vector-borne disease in the United States. Some patients report persistent or intermittent subjective symptoms of mild to moderate intensity after antibiotic treatment for Lyme disease. We sought to evaluate trends in clinical and quality-of-life (QOL) measures in a cohort of patients with Lyme disease enrolled in a natural history study at the National Institutes of Health from 2001-2014. METHODS: QOL was measured using the self-administered 36-item Short Form Health Survey (SF-36) during study follow-up. Primary outcomes included mean physical (PCS) and mental (MCS) health QOL composite scores and reporting long-term (≥2 years) symptoms, adjusted for Lyme disease stage and severity at diagnosis. RESULTS: Overall, 101 patients with an average follow-up time of 3.9 years (range, 0.5-11.3 years) were included. At first visit, overall mean QOL scores were below the US population mean for both PCS (45.6 ± 10.4) and MCS (47.3 ± 11.5) but increased to just above the national average after 3 years of follow-up for both PCS (50.7 ± 9.6) and MCS (50.1 ± 10.0). Baseline QOL scores were lowest in those with late disease (P < 0.01) but also increased by the end of follow-up to national averages. In multivariate analysis, the only factors significantly associated with long-term symptoms or lower QOL scores were other comorbidities unrelated to Lyme disease. CONCLUSIONS: Comorbid conditions can play a role in the reporting of long-term symptoms and overall QOL of Lyme disease patients and should be considered in the evaluation of these patients. CLINICAL TRIALS REGISTRATION: NCT00028080.

Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study.

BACKGROUND: Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans. METHODS: Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals. RESULTS: Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient. CONCLUSIONS: Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result. Clinical Trials Registration NCT01143558.

Detection of molluscum contagiosum virus (MCV) DNA in the plasma of an immunocompromised patient and possible reduction of MCV DNA with CMX-001.

Molluscum contagiosum virus (MCV) is a poxvirus that causes localized papules in healthy persons. We evaluated a woman with severe immunodeficiency and disseminated MCV. During treatment with CMX-001, an antiviral with activity against other poxviruses, MCV DNA was detected in 20% of plasma samples. When the patient was not receiving CMX-001, MCV DNA was detected in 50% of samples. We also noted improvement in warts on her fingers during CMX-001 therapy. Although MCV is caused by direct inoculation of virus into skin in healthy persons, in a severely immunocompromised person MCV DNA was present in blood and may spread by viremia.

Characterization and treatment of chronic active Epstein-Barr virus disease: a 28-year experience in the United States.

Chronic active EBV disease (CAEBV) is a lymphoproliferative disorder characterized by markedly elevated levels of antibody to EBV or EBV DNA in the blood and EBV RNA or protein in lymphocytes in tissues. We present our experience with CAEBV during the last 28 years, including the first 8 cases treated with hematopoietic stem cell transplantation in the United States. Most cases of CAEBV have been reported from Japan. Unlike CAEBV in Japan, where EBV is nearly always found in T or natural killer (NK) cells in tissues, EBV was usually detected in B cells in tissues from our patients. Most patients presented with lymphadenopathy and splenomegaly; fever, hepatitis, and pancytopenia were common. Most patients died of infection or progressive lymphoproliferation. Unlike cases reported from Japan, our patients often showed a progressive loss of B cells and hypogammaglobulinemia. Although patients with CAEBV from Japan have normal or increased numbers of NK cells, many of our patients had reduced NK-cell numbers. Although immunosuppressive agents, rituximab, autologous cytotoxic T cells, or cytotoxic chemotherapy often resulted in short-term remissions, they were not curative. Hematopoietic stem cell transplantation was often curative for CAEBV, even in patients with active lymphoproliferative disease that was unresponsive to chemotherapy. These studies are registered at http://www.clinicaltrials.gov as NCT00032513 for CAEBV, NCT00062868 and NCT00058812 for EBV-specific T-cell studies, and NCT00578539 for the hematopoietic stem cell transplantation protocol.

Kinetics of serum cytokines after primary or repeat vaccination with the smallpox vaccine.

BACKGROUND: The smallpox vaccine is associated with more serious adverse events than any other live attenuated vaccine in use today. Although studies have examined serum cytokine levels in primary vaccine recipients at 1 and 3-5 weeks after vaccination with the smallpox vaccine, serial measurements have not been performed, and studies in revaccinated subjects have not been conducted. METHODS: We analyzed cytokine responses in both primary vaccine recipients and revaccinated subjects every other day for 2 weeks after vaccination. RESULTS: Primary vaccine recipients had maximal levels of granulocyte-colony-stimulating factor on days 6-7 after vaccination; peak levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptor 1, interferon (IFN)-gamma, IFN-inducible protein-10 (IP-10), interleukin (IL)-6, and tissue inhibitor of metalloproteinases-1 on days 8-9 after vaccination; peak levels of soluble TNF receptor 2 and monokine induced by IFN-gamma (MIG) on days 10-11 after vaccination; and peak levels of granulocyte-macrophage-colony-stimulating factor on days 12-13 after vaccination. Primary vaccine recipients were significantly more likely to have higher peak levels of IFN-gamma, IP-10, and MIG after vaccination than were revaccinated subjects. Primary vaccine recipients were significantly more likely to have fatigue, lymphadenopathy, and headache, as well as a longer duration of these symptoms and more hours missed from work, compared with revaccinated subjects. CONCLUSIONS: The increased frequency and duration of symptoms observed in primary vaccine recipients, compared with revaccinated subjects, paralleled the increases in serum cytokine levels in these individuals. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00325975.