Borrelia miyamotoi Activates Human Dendritic Cells and Elicits T Cell Responses.

The spirochete Borrelia miyamotoi has recently been shown to cause relapsing fever. Like the Lyme disease agent, Borrelia burgdorferi, B. miyamotoi is transmitted through the bite of infected ticks; however, little is known about the response of the immune system upon infection. Dendritic cells (DCs) play a central role in the early immune response against B. burgdorferi We investigated the response of DCs to two different strains of B. miyamotoi using in vitro and ex vivo models and compared this to the response elicited by B. burgdorferi. Our findings show that B. miyamotoi is phagocytosed by monocyte-derived DCs, causing upregulation of activation markers and production of proinflammatory cytokines in a similar manner to B. burgdorferi. Recognition of B. miyamotoi was demonstrated to be partially mediated by TLR2. DCs migrated out of human skin explants upon inoculation of the skin with B. miyamotoi. Finally, we showed that B. miyamotoi-stimulated DCs induced proliferation of naive CD4+ and CD8+ T cells to a larger extent than B. burgdorferi. In conclusion, we show in this study that DCs respond to and mount an immune response against B. miyamotoi that is similar to the response to B. burgdorferi and is able to induce T cell proliferation.

Prevalence and determinants of persistent symptoms after treatment for Lyme borreliosis: study protocol for an observational, prospective cohort study (LymeProspect).

BACKGROUND: After antibiotic treatment of Lyme borreliosis, a subset of patients report persistent symptoms, also referred to as post-treatment Lyme disease syndrome. The reported prevalence of persistent symptoms varies considerably, and its pathophysiology is under debate. The LymeProspect study has been designed to investigate the prevalence, severity, and a wide range of hypotheses on the etiology of persistent symptoms among patients treated for Lyme borreliosis in the Netherlands. METHODS: LymeProspect is a prospective, observational cohort study among adults with proven or probable Lyme borreliosis, either erythema migrans or disseminated manifestations, included at the start of antibiotic treatment. During one year of follow-up, participants are subjected to questionnaires every three months and blood is collected repeatedly during the first three months. The primary outcome is the prevalence of persistent symptoms after treatment, assessed by questionnaires online focusing on fatigue (CIS, subscale fatigue severity), pain (SF-36, subscale pain) and neurocognitive dysfunction (CFQ). Potential microbiological, immunological, genetic, epidemiological and cognitive-behavioral determinants for persistent symptoms are secondary outcome measures. Control cohorts include patients with long-lasting symptoms and unconfirmed Lyme disease, population controls, and subjects having reported a tick bite not followed by Lyme borreliosis. DISCUSSION: This article describes the background and design of the LymeProspect study protocol. This study is characterized by a prospective, explorative and multifaceted design. The results of this study will provide insights into the prevalence and determinants of persistent symptoms after treatment for Lyme borreliosis, and may provide a rationale for preventive and treatment recommendations. TRIAL REGISTRATION: NTR4998 (Netherlands Trial Register). Date of registration: 13 February 2015.

The Ixodes ricinus salivary gland proteome during feeding and B. Afzelii infection: New avenues for an anti-tick vaccine.

INTRODUCTION: Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. METHOD: Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. RESULTS: We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. CONCLUSION: Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine.

Urokinase receptor is necessary for bacterial defense against pneumonia-derived septic melioidosis by facilitating phagocytosis.

Urokinase receptor (urokinase-type plasminogen activator receptor [uPAR], CD87), a GPI-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in Southeast Asia. In this study, we investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. uPAR mRNA and surface expression was increased in patients with septic melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced growth and dissemination of B. pseudomallei when compared with wild-type mice, corresponding with increased pulmonary and hepatic inflammation. uPAR knockout mice demonstrated significantly reduced neutrophil migration toward the pulmonary compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPAR-deficient macrophages and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency did not influence hemostatic and fibrinolytic responses during severe melioidosis. These data suggest that uPAR is crucially involved in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils toward the primary site of infection and subsequently facilitating the phagocytosis of B. pseudomallei.

The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi.

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1beta mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization.

Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC) functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi-induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK)-dependent decrease of IL-6 and TNF-alpha mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor-induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.

Evaluation of Disease Causality of Rare Ixodes ricinus-Borne Infections in Europe.

In Europe, Ixodes ricinus ticks transmit pathogens such as Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV). In addition, there is evidence for transmission to humans from I. ricinus of Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Babesia venatorum, Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia helvetica and Rickettsia monacensis. However, whether infection with these potential tick-borne pathogens results in human disease has not been fully demonstrated for all of these tick-borne microorganisms. To evaluate the available evidence for a causative relation between infection and disease, the current study analyses European case reports published from 2008 to 2018, supplemented with information derived from epidemiological and experimental studies. The evidence for human disease causality in Europe found in this review appeared to be strongest for A. phagocytophilum and B. divergens. Nonetheless, some knowledge gaps still exist. Importantly, comprehensive evidence for pathogenicity is lacking for the remaining tick-borne microorganisms. Such evidence could be gathered best through prospective studies, for example, studies enrolling patients with a fever after a tick bite, the development of specific new serological tools, isolation of these microorganisms from ticks and patients and propagation in vitro, and through experimental studies.

A combination of antibodies against Bm86 and Subolesin inhibits engorgement of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae in vitro.

BACKGROUND: Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro. RESULTS: Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium. CONCLUSIONS: Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.

The tick salivary protein Salp15 inhibits the killing of serum-sensitive Borrelia burgdorferi sensu lato isolates.

Borrelia burgdorferi, the agent of Lyme disease, is transmitted by ticks. During transmission from the tick to the host, spirochetes are delivered with tick saliva, which contains the salivary protein Salp15. Salp15 has been shown to protect spirochetes against B. burgdorferi-specific antibodies. We now show that Salp15 from both Ixodes ricinus and Ixodes scapularis protects serum-sensitive isolates of Borrelia burgdorferi sensu lato against complement-mediated killing. I. ricinus Salp15 showed strong protective effects compared to those of I. scapularis Salp15. Deposition of terminal C5b to C9 (one molecule each of C5b, C6, C7, and C8 and one or more molecules of C9) complement complexes, part of the membrane attack complex, on the surface of B. burgdorferi was inhibited in the presence of Salp15. In the presence of normal human serum, serum-sensitive Borrelia burgdorferi requires protection against complement-mediated killing, which is provided, at least in part, by the binding to the tick salivary protein Salp15.

Investigating Human Dendritic Cell Immune Responses to Borrelia burgdorferi.

Dendritic cells (DCs) are professional antigen-presenting cells that recognize and phagocytose pathogens, and help to orchestrate adaptive immune responses to combat them. DCs are abundant in the skin where Borrelia burgdorferi first enters the body during a tick bite, and are thus critical in determining the initial stages of the innate and adaptive immune responses against Borrelia. Here, we describe two methods to study the response of DCs to Borrelia; an in vitro approach using monocyte-derived DCs (moDCs) and an ex vivo approach using a human skin model.

MRP8/14 does not contribute to dissemination or inflammation in a murine model of Lyme borreliosis.

Myeloid-related protein (MRP)8 and MRP14 form a complex (MRP8/14) that is released by activated neutrophils and monocytes during infection. MRP8/14 has been shown to have bacteriostatic activity in vitro against Borrelia burgdorferi, the spirochete that causes Lyme borreliosis. Furthermore, levels of MRP8/14 have been shown to be elevated in the joints of patients with Lyme arthritis. We hypothesized that MRP8/14 has a protective effect during B. burgdorferi infection. To determine the role of MRP8/14 in the immune response to B. burgdorferi, we studied the course of B. burgdorferi infection in wildtype (wt) and mrp14-/- mice. In addition, we studied the response of leukocytes from mice lacking MRP8/14 to B. burgdorferi ex vivo. We demonstrated similar levels of B. burgdorferi dissemination, cytokine and immunoglobulin production in infected wt and mrp14-/- mice after 21 days. Neutrophils and monocytes lacking MRP8/14 were undiminished in their ability to become activated or phagocytose B. burgdorferi. In conclusion, we did not find a central role of MRP8/14 in the immune response against B. burgdorferi. As the levels of MRP8/14 in the serum of infected mice were low, we speculate that MRP8/14 is not released in levels great enough to influence the course of B. burgdorferi infection.

Tick-host-pathogen interactions in Lyme borreliosis.

Borrelia burgdorferi, the spirochetal agent of Lyme borreliosis, is predominantly transmitted by Ixodes ticks. Spirochetes have developed many strategies to adapt to the different environments that are present in the arthropod vector and the vertebrate host. This review focuses on B. burgdorferi genes that are preferentially expressed in the tick and the vertebrate host, and describes how selected gene products facilitate spirochete survival throughout the enzootic life cycle. Interestingly, B. burgdorferi also enhances expression of specific Ixodes scapularis genes, such as TROSPA and salp15. The importance of these genes and their products for B. burgdorferi survival within the tick, and during the transmission process, will also be reviewed. Moreover, we discuss how such vector molecules could be used to develop vector-antigen-based vaccines to prevent the transmission of B. burgdorferi and, potentially, other arthropod-borne microbes.

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis.

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.

Correction: Lyme borreliosis.

This corrects the article DOI: 10.1038/nrdp.2016.90.

Serological and molecular evidence for spotted fever group Rickettsia and Borrelia burgdorferi sensu lato co-infections in The Netherlands.

Only a few reported cases indicate that Rickettsia helvetica and Rickettsia monacensis can cause disease in humans. Exposure to these two spotted fever group (SFG) rickettsiae occurs through bites of Ixodes ricinus, also the primary vector of Lyme borreliosis in Europe. To date, it is unclear how often exposure to these two microorganisms results in infection or disease. We show that of all the Borrelia burgdorferi s.l.-positive ticks, 25% were co-infected with rickettsiae. Predominantly R. helvetica was detected while R. monacensis was only found in approximately 2% of the ticks. In addition, exposure to tick-borne pathogens was compared by serology in healthy blood donors, erythema migrans (EM)-patients, and patients suspected of Lyme neuroborreliosis (LNB). As could be expected, seroreactivity against B. burgdorferi sensu lato was lower in blood donors (6%) compared to EM patients (34%) and suspected LNB cases (64%). Interestingly, seroreactivity against SFG Rickettsia antigens was not detected in serum samples from blood donors (0%), but 6% of the EM patients and 21% of the LNB suspects showed anti-rickettsial antibodies. Finally, the presence of B. burgdorferi s.l. and Rickettsia spp. in cerebrospinal fluid samples of a large cohort of patients suspected of LNB (n=208) was investigated by PCR. DNA of B. burgdorferi s.l., R. helvetica and R. monacensis was detected in seventeen, four and one patient, respectively. In conclusion, our data show that B. burgdorferi s.l. and SFG rickettsiae co-infection occurs in Dutch I. ricinus and that Lyme borreliosis patients, or patients suspected of Lyme borreliosis, are indeed exposed to both tick-borne pathogens. Whether SFG rickettsiae actually cause disease, and whether co-infections alter the clinical course of Lyme borreliosis, is not clear from our data, and warrants further investigation.

Surface display of a borrelial lipoprotein on meningococcal outer membrane vesicles.

Outer Membrane Vesicles (OMVs) are gaining attention as vaccine candidates. The successful expression of heterologous antigens in OMVs, with the OMV functioning both as adjuvant and delivery vehicle, has greatly enhanced their vaccine potential. Since there are indications that surface exposed antigens might induce a superior immune response, targeting of heterologous antigens to the OMV surface is of special interest. Several systems for surface display of heterologous antigens on OMVs have been developed. However, these systems have not been used to display lipidated membrane-associated proteins known as lipoproteins, which are emerging as key targets for protective immunity. We were therefore interested to see whether we could express a foreign lipoprotein on the outer surface of OMVs. When outer surface protein A (OspA), a borrelial surface-exposed lipoprotein, was expressed in meningococci, it was found that although OspA was present in OMVs, it was no longer surface-exposed. Therefore, a set of fusions of OspA to different regions of factor H binding protein (fHbp), a meningococcal surface-exposed lipoprotein, were designed and tested for their surface-exposure. An N-terminal part of fHbp was found to be necessary for the successful surface display of OspA on meningococcal OMVs. When mice were immunized with this set of OMVs, an OspA-specific antibody response was only elicited by OMVs with clearly surface-exposed OspA, strengthening the idea that the exact positioning of an antigen in the OMV affects the immune response. This method for the surface display of heterologous lipoproteins on OMVs is a step forward in the development of OMVs as a vaccine platform.

Lyme borreliosis.

Lyme borreliosis is a tick-borne disease that predominantly occurs in temperate regions of the northern hemisphere and is primarily caused by the bacterium Borrelia burgdorferi in North America and Borrelia afzelii or Borrelia garinii in Europe and Asia. Infection usually begins with an expanding skin lesion, known as erythema migrans (referred to as stage 1), which, if untreated, can be followed by early disseminated infection, particularly neurological abnormalities (stage 2), and by late infection, especially arthritis in North America or acrodermatitis chronica atrophicans in Europe (stage 3). However, the disease can present with any of these manifestations. During infection, the bacteria migrate through the host tissues, adhere to certain cells and can evade immune clearance. Yet, these organisms are eventually killed by both innate and adaptive immune responses and most inflammatory manifestations of the infection resolve. Except for patients with erythema migrans, Lyme borreliosis is diagnosed based on a characteristic clinical constellation of signs and symptoms with serological confirmation of infection. All manifestations of the infection can usually be treated with appropriate antibiotic regimens, but the disease can be followed by post-infectious sequelae in some patients. Prevention of Lyme borreliosis primarily involves the avoidance of tick bites by personal protective measures.