Tick borne relapsing fever - a systematic review and analysis of the literature.

Tick borne relapsing fever (TBRF) is a zoonosis caused by various Borrelia species transmitted to humans by both soft-bodied and (more recently recognized) hard-bodied ticks. In recent years, molecular diagnostic techniques have allowed to extend our knowledge on the global epidemiological picture of this neglected disease. Nevertheless, due to the patchy occurrence of the disease and the lack of large clinical studies, the knowledge on several clinical aspects of the disease remains limited. In order to shed light on some of these aspects, we have systematically reviewed the literature on TBRF and summarized the existing data on epidemiology and clinical aspects of the disease. Publications were identified by using a predefined search strategy on electronic databases and a subsequent review of the reference lists of the obtained publications. All publications reporting patients with a confirmed diagnosis of TBRF published in English, French, Italian, German, and Hungarian were included. Maps showing the epidemiogeographic mosaic of the different TBRF Borrelia species were compiled and data on clinical aspects of TBRF were analysed. The epidemiogeographic mosaic of TBRF is complex and still continues to evolve. Ticks harbouring TBRF Borrelia have been reported worldwide, with the exception of Antarctica and Australia. Although only molecular diagnostic methods allow for species identification, microscopy remains the diagnostic gold standard in most clinical settings. The most suggestive symptom in TBRF is the eponymous relapsing fever (present in 100% of the cases). Thrombocytopenia is the most suggestive laboratory finding in TBRF. Neurological complications are frequent in TBRF. Treatment is with beta-lactams, tetracyclines or macrolids. The risk of Jarisch-Herxheimer reaction (JHR) appears to be lower in TBRF (19.3%) compared to louse-borne relapsing fever (LBRF) (55.8%). The overall case fatality rate of TBRF (6.5%) and LBRF (4-10.2%) appears to not differ. Unlike LBRF, where perinatal fatalities are primarily attributable to abortion, TBRF-related perinatal fatalities appear to primarily affect newborns.

Bactericidal activity of avian complement: a contribution to understand avian-host tropism of Lyme borreliae.

Complement has been considered as an important factor impacting the host-pathogen association of spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and may play a role in the spirochete's ecology. Birds are known to be important hosts for ticks and in the maintenance of borreliae. Recent field surveys and laboratory transmission studies indicated that certain avian species act as reservoir hosts for different Borrelia species. Nevertheless, our current understanding of the molecular mechanisms determining host tropism of Borrelia is still in its fledgling stage. Concerning the role of complement in avian-host tropism, only a few bird species and Borrelia species have been analysed so far. Here, we performed in vitro serum bactericidal assays with serum samples collected from four bird species including the European robin Erithacus rubecula, the great tit Parus major, the Eurasian blackbird Turdus merula, and the racing pigeon Columba livia, as well as four Borrelia species (B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi sensu stricto). From July to September 2019, juvenile wild birds were caught using mist nets in Portugal. Racing pigeons were sampled in a loft in October 2019. Independent of the bird species analysed, all Borrelia species displayed an intermediate serum-resistant or serum-resistant phenotype except for B. afzelii challenged with serum from blackbirds. This genospecies was efficiently killed by avian complement, suggesting that blackbirds served as dead-end hosts for B. afzelii. In summary, these findings suggest that complement contributes in the avian-spirochete-tick infection cycle and in Borrelia-host tropism.

Impact of four local anaesthetics on growth and viability of in vitro cultured Borrelia burgdorferi sensu stricto, Borrelia bavariensis and Borrelia afzelii.

Many local anaesthetics, including lidocaine, procaine and ropivacaine inhibit bacterial growth. This study investigates potential effects of these local anaesthetics on growth of Borrelia burgdorferi sensu stricto (Bbss), Borrelia bavariensis (Bbav) and Borrelia afzelii (Ba). For this purpose, Borrelia spp. organisms were either continuously or temporarily exposed to one of four local anaesthetics preparations: 20 mg/ml procaine hydrochloride (P); 10 mg/ml ropivacaine hydrochloride (R); 20 mg/ml lidocaine hydrochloride (L1, L2). L2 also contained the preservatives methyl-benzoate and propyl-benzoate, whereas P, R and L1 did not. All four local anaesthetic preparations inhibited in vitro growth of Borrelia spp. depending on concentration and exposure time. There are differences in sensitivity among the Borrelia spp. with Bbav being more susceptible to growth inhibition than Bbss and Ba. When comparing the different local anaesthetic preparations with their regard to inhibition of growth of Borrelia spp. organisms, P showed the lowest impact. It cannot be completely excluded that preservatives present in L2, methyl-benzoate and propyl-benzoate, may be a reason for further inhibition of Borrelia spp. organisms. Concentrations of local anaesthetics used in these experiments may also be present in the skin of patients during regular medical procedures. These are preliminary findings and further experiments, preferably in vivo, are necessary. To minimize the risk to produce false negative results with cultures, we recommend using procaine in a preparation without preservatives for local anaesthesia prior to skin sampling.

Louse-borne relapsing fever-A systematic review and analysis of the literature: Part 2-Mortality, Jarisch-Herxheimer reaction, impact on pregnancy.

Louse-borne relapsing fever (LBRF) is a classical epidemic disease, which in the past was associated with war, famine, poverty, forced migration, and crowding under poor hygienic conditions around the world. The disease's causative pathogen, the spirochete bacterium Borrelia recurrentis, is confined to humans and transmitted by a single vector, the human body louse Pediculus humanus corporis. Since the disease was at its peak before the days of modern medicine, many of its aspects have never been formally studied and to date remain incompletely understood. In order to shed light on some of these aspects, we have systematically reviewed the accessible literature on LBRF since the recognition of its mode of transmission in 1907, and summarized the existing data on mortality, Jarisch-Herxheimer reaction (JHR), and impact on pregnancy. Publications were identified by using a predefined search strategy of electronic databases and a subsequent review of the reference lists of the obtained publications. All publications reporting patients with a confirmed diagnosis of LBRF published in English, French, German, and Spanish since 1907 were included. Data extraction followed a predefined protocol and included a grading system to judge the certainty of the diagnosis of reported cases. The high mortality rates often found in literature are confined to extreme scenarios. The case fatality rate (CFR) of untreated cases is on average significantly lower than frequently assumed. In recent years, a rise in the overall CFRs is documented, for which reasons remain unknown. Lacking standardized criteria, a clear diagnostic threshold defining antibiotic treatment-induced JHR does not exist. This explains the wide range of occurrence rates found in literature. Pre-antibiotic era data suggest the existence of a JHR-like reaction also in cases treated with arsenicals and even in untreated cases. LBRF-related adverse outcomes are observed in 3 out of 4 pregnancies.

Labdanum and Labdanes of Cistus creticus and C. ladanifer: Anti-Borrelia activity and its phytochemical profiling✰.

BACKGROUND: Intrigued by testimonies of Saxon borreliosis self-help groups concerning considerabl improvements of their symptoms by ingestion of Cistus creticus L. (Cistaceae) leaf preparations, we recently reported on the growth inhibiting activity of extracts with different polarities and its volatile oil against Borrelia burgdorferi sensu stricto (Bbss) in vitro, determined by a bioassay guided procedure. The most active volatile oil (only about 0.10% in leaves) was found to be dominated by labdane-type manoyloxides as well as carvacrol, determined via GC-MS. HYPOTHESIS: These terpenes are major active constituents of the old pharmaceutical oleoresin labdanum, which is secreted from the leaf surface of C. creticus and traditionally harvested, e.g., on Crete by brushing the shrubs. METHODS: In order to elucidate the definite anti-Borrelia active principles of C. creticus, preparative scale separation of the diethyl­ether soluble fraction of Cretan labdanum was achieved by combined silica gel 60-and RP-18 CC and analysed by novel TLC-Extractor/ES-MS as well as by 1d/2d-1H/13C-NMR data. For the antispirochaetal activity tests against Bbss in vitro, all samples were solubilised in water with addition of polysorbate 80, the effect of which on bacterial growth was examined and found to be negligible. RESULTS: This led to isolation and identification of the monoterpene carvacrol as well as of the four major manoyloxides manoyloxide (A), 3-acetoxy-manoyloxide (B), 3­hydroxy-manoyloxide (C), and epi­manoyloxide (D). Additionally, 2-keto-manoyloxide (E) and sclareol (F) were identified via GC/EI-MS. In subsequent microbiological tests of the isolated compounds, epi­manoyloxide (D) exhibited by far the strongest individual antispirochaetal effect, equal to the positive control amoxicilline. Furthermore, manoyloxide (A), carvacrol, and the diethyl­ether soluble fraction of labdanum as a whole contribute to the strong antispirochaetal activity, while the other labdanes were less active. Isolated manoyloxides were further used as external standards for a GC-MS screening of labdanum samples from different origins, revealing exceptionally high contents of all analysed manoyloxides in the samples of Cretan labdanum from C. creticus, while their contents in other commercial available labdanum samples were lower by several orders of magnitude. Especially in Spanish labdanum samples, declared as Cistus ladanifer L., mainly simple alkanes and at most traces of epi­manoyloxide (D) and of manoyloxide (A) could be detected. CONCLUSION: The application of C. creticus preparations by Lyme disease self-help groups may be considered as a reasonable therapy approach. For the first time, isolated epi­manoyloxide and carvacrol could be evaluated as most promising candidates for drug development and labdanum based phytomedicine development, respectively. They should serve as vital active markers for quality assessments of C. creticus preparations.

Anti-borreliae efficacy of selected organic oils and fatty acids.

BACKGROUND: Borrelia sp. is a causative pathogen of Lyme disease which has become a worldwide health concern. Non-toxic approaches especially directed toward latent persistent forms of this pathogen are desired. Lipids in the form of volatile and non-volatile oils, and fatty acids with proven anti-borreliae efficacy could become an additional support or an alternative for consideration in treatment approaches. METHODS: In this study we investigated 47 lipids (30 volatile and non-volatile oils, and 17 fatty acids) of plant and animal origin against typical motile, knob/round-shaped persisters, and biofilm-like aggregates of Borrelia burgdorferi s.s. and Borrelia garinii, which are identified as pathogenic factors of Lyme disease in the USA and Europe, using direct microscopic counting and spectrofluorometric measurements. RESULTS: Out of all examined lipids, 5 oils (Bay leaf oil, Birch oil, Cassia oil, Chamomile oil German, and Thyme oil) at or below 0.25%, and 3 fatty acids (13Z,16Z Docosadienoic acid, erucic acid, and petroselinic acid) at or below 0.75 mg/ml, showed bactericidal activity against typical motile spirochetes and knob/round-shaped persisters. Only Bay leaf oil and Cassia oil, including their major constituents, eugenol and cinnamaldehyde, showed to target biofilm-like aggregates of both tested Borrelia spp. at the same concentration, although with 20-30% eradication mark. CONCLUSION: Based on obtained results, volatile oils were more potent than non-volatile oils, and unsaturated fatty acids were more effective than saturated fatty acids. Among all tested oils, Bay leaf oil and Cassia oil, with their major components eugenol and cinnamaldehyde, seem to have the highest anti-borreliae efficacy.

Lyme Neuroborreliosis.

BACKGROUND: The new German S3 guideline on Lyme neuroborreliosis is intended to provide physicians with scientifically based information and recommendations on the diagnosis and treatment of this disease. METHODS: The scientific literature was systematically searched and the retrieved publications were assessed at the German Cochrane Center (Deutsches Cochrane Zentrum) in Freiburg in the 12 months beginning in March 2014. In addition to the main search terms "Lyme disease," "neuroborreliosis," "Borrelia," and "Bannwarth," 28 further terms relating to neurological manifestations of the disease were used for the search in the Medline and Embase databases and in the Cochrane Central Register of Controlled Trials. RESULTS: In the treatment of early Lyme neuroborreliosis, orally administered doxycycline is well tolerated, and its efficacy is equivalent to that of intravenously administered beta-lactam antibiotics (penicillin G, ceftriaxone, and cefotaxime) (relative risk [RR]: 0.98, 95% confidence interval [CI]: [0.68; 1.42], P = 0.93). 14 days of treatment suffice for early Lyme neuroborreliosis, and 14-21 days of treatment usually suffice for late (chronic) Lyme neuroborreliosis. CONCLUSION: Lyme neuroborreliosis has a favorable prognosis if treated early. The long-term administration of antibiotics over many weeks or even months for putative chronic Lyme neuroborreliosis with nonspecific symptoms yields no additional benefit and carries the risk of serious adverse effects.

In Vitro Antimicrobial Susceptibility of Clinical Isolates of Borrelia miyamotoi.

Borrelia miyamotoi is an emerging relapsing fever (RF) Borrelia species that is reported to cause human disease in regions in which Lyme borreliosis is endemic. We recently showed that B. miyamotoi tick isolates are resistant to amoxicillin in vitro; however, clinical isolates have not been studied. Therefore, our aim was to show the antimicrobial susceptibility of recently obtained clinical isolates of B. miyamotoi A dilution series of various antibiotics was made in modified Kelly-Pettenkofer medium with 10% fetal calf serum. The susceptibilities of different B. miyamotoi clinical, B. miyamotoi tick, RF Borrelia, and Borrelia burgdorferisensu lato isolates were tested by measuring MICs through colorimetric changes and by counting motile spirochetes by dark-field microscopy after 72 h of incubation. The ceftriaxone and azithromycin MIC ranges of the six B. miyamotoi clinical isolates tested were 0.03 to 0.06 mg/liter and 0.0016 to 0.0032 mg/liter, respectively. These values are similar to MICs for RF Borrelia strains and B. miyamotoi tick isolates. All tested RF Borrelia strains were susceptible to doxycycline (microscopic MIC range, 0.0625 to 0.25 mg/liter). In contrast to the MICs of the tested B. burgdorferi sensu lato strains and in line with our previous findings, the amoxicillin MICs (range, 8 to 32 mg/liter) of all RF Borrelia strains, including B. miyamotoi clinical isolates, were above the clinical breakpoint for resistance (≤4 mg/liter). Clinical isolates of B. miyamotoi are highly susceptible to doxycycline, azithromycin, and ceftriaxone in vitro Interestingly, as described previously for tick isolates, amoxicillin shows poor in vitro activity against B. miyamotoi clinical isolates.

In Vitro Susceptibility of the Relapsing-Fever Spirochete Borrelia miyamotoi to Antimicrobial Agents.

Hard-tick-borne relapsing fever (HTBRF) is an emerging infectious disease throughout the temperate zone caused by the relapsing-fever spirochete Borrelia miyamotoi Antibiotic treatment of HTBRF is empirically based on the treatment of Lyme borreliosis; however, the antibiotic susceptibility of B. miyamotoi has not been studied to date. Thus, we set out to determine the in vitro antimicrobial susceptibility of B.miyamotoi A microdilution method with 96-well microtiter plates was used to determine the antibiotic susceptibilities of two B.miyamotoi strains isolated on two different continents (Asia and North America), two Borrelia burgdorferisensu lato strains, and one Borrelia hermsii isolate for purposes of comparison. The MIC and minimal bactericidal concentration (MBC) were determined by both microscopy and colorimetric assays. We were able to show that relative to the B. burgdorferi sensu lato isolates, both B.miyamotoi strains and B. hermsii demonstrated greater susceptibility to doxycycline and azithromycin, equal susceptibility to ceftriaxone, and resistance to amoxicillin in vitro The MIC and MBC of amoxicillin for B. miyamotoi evaluated by microscopy were 16 to 32 mg/liter and 32 to 128 mg/liter, respectively. Since B. miyamotoi is susceptible to doxycycline, azithromycin, and ceftriaxone in vitro, our data suggest that these antibiotics can be used for the treatment of HTBRF. Oral amoxicillin is currently used as an alternative for the treatment of HTBRF; however, since we found that the B. miyamotoi strains tested were resistant to amoxicillin in vitro, this issue warrants further study.

Reciprocal cooperation of phytochemicals and micronutrients against typical and atypical forms of Borrelia sp.

AIMS: Borrelia sp., a causative pathogenic factor of Lyme disease (LD), has become a major public health threat. Current treatments based on antibiotics often lead to relapse after their withdrawal. Naturally derived substances that could work synergistically to display higher efficacy compared with the individual components may serve as a resource for the development of novel approaches to combat both active and latent forms of Borrelia sp. METHODS AND RESULTS: Using checkerboard assay, we investigated the anti-borreliae reciprocal cooperation of phytochemicals and micronutrients against two species of Borrelia selected as prevalent causes of LD in the United States and Europe. We tested 28 combinations of phytochemicals such as polyphenols (baicalein, luteolin, rosmarinic acids), fatty acids (monolaurin, cis-2-decenoic acid) and micronutrients (ascorbic acid, cholecalciferol and iodine). The results showed that the combinations of baicalein with luteolin as well as monolaurin with cis-2-decenoic acid expressed synergistic anti-spirochetal effects. Moreover, baicalein and luteolin, when combined with rosmarinic acid or iodine, produced additive bacteriostatic and bactericidal effects against typical corkscrew motile spirochaetes and persistent knob/round-shaped forms, respectively. An additive anti-biofilm effect was noticed between baicalein with luteolin and monolaurin with cis-2-decenoic acid. Finally, application of the combination of baicalein with luteolin increased cytoplasmic permeability of Borrelia sp. but did not cause DNA damage. CONCLUSIONS: These results show that a specific combination of flavones might play a supporting role in combating Borrelia sp. through either synergistic or additive anti-borreliae effects. SIGNIFICANCE AND IMPACT OF THE STUDY: Presented here in vitro results might help advancing our knowledge and improving the approach to target Borrelia sp.

[Neuroborreliosis - Diagnostics, treatment and course]. / Neuroborreliose ­ Diagnostik, Therapie und Verlauf.

Lyme neuroborreliosis is a tick-borne infectious disease caused by the spirochete bacterium Borrelia burgdorferi sensu lato. Clinical manifestations are classified as early and late Lyme neuroborreliosis. Early manifestations are much more common than late manifestations. Serological testing should only be performed when typical neurological symptoms are present because false positive results are common due to a high seroprevalence in the population. Cerebrospinal fluid (CSF) analysis should be performed if Lyme neuroborreliosis is suspected. A systematic review found similar effects of beta-lactam antibiotics and doxycycline regarding the outcome of neurological symptoms and adverse effects. The prognosis after antibiotic treatment is usually favorable and residual symptoms can rarely persist. Impairments in quality of life, fatigue, depression and cognitive impairment are not more frequent in patients after treatment of Lyme neuroborreliosis than in the normal healthy population.

[Challenge of neuroborreliosis]. / Herausforderung Neuroborreliose.

Lyme borreliosis is the most frequent tick-borne infection in Europe. It is a multisystemic disease affecting the skin, joints, heart, in rare cases the eyes and regularly the nervous system. Taking current clinical and microbiological guidelines into account, neuroborreliosis can in general be diagnosed and treated successfully. An appropriate guideline-conform antibiotic treatment is effective and in most cases recovery from acute neuroborreliosis is complete. Nevertheless, the evidence base regarding pharmacological treatment needs reform and improvement. Contrary to this scientifically based medical opinion, divergent opinions presented in the media cause uncertainty and confusion among patients and also some physicians. The currently available scientific data on epidemiology, treatment and performance of microbiological testing reveals gaps and therefore a large scope for interpretation. In clinical practice, diagnostics and therapeutic methods that do not fulfill the criteria of evidence-based medicine are widely used due to the uncertain data situation. The Clinical Network Neuroborreliosis (KNN) is a publicly funded network of clinicians and clinical laboratory physicians with the goal to improve knowledge for disease-oriented evaluation of diagnostic and therapeutic methods. In addition, the Robert Koch Institute (RKI) and the KNN are performing an epidemiological study for collation and estimation of the number of neuroborreliosis cases in Germany (aNBorD study).

Cooperation of Doxycycline with Phytochemicals and Micronutrients Against Active and Persistent Forms of Borrelia sp.

Phytochemicals and micronutrients represent a growing theme in antimicrobial defense; however, little is known about their anti-borreliae effects of reciprocal cooperation with antibiotics. A better understanding of this aspect could advance our knowledge and help improve the efficacy of current approaches towards Borrelia sp. In this study, phytochemicals and micronutrients such as baicalein, luteolin, 10-HAD, iodine, rosmarinic acid, and monolaurin, as well as, vitamins D3 and C were tested in a combinations with doxycycline for their in vitro effectiveness against vegetative (spirochetes) and latent (rounded bodies, biofilm) forms of Borrelia burgdorferi and Borrelia garinii. Anti-borreliae effects were evaluated according to checkerboard assays and supported by statistical analysis. The results showed that combination of doxycycline with flavones such as baicalein and luteolin exhibited additive effects against all morphological forms of studied Borrelia sp. Doxycycline combined with iodine demonstrated additive effects against spirochetes and biofilm, whereas with fatty acids such as monolaurin and 10-HAD it produced FICIs of indifference. Additive anti-spirochetal effects were also observed when doxycycline was used with rosmarinic acid and both vitamins D3 and C. Antagonism was not observed in any of the cases. This data revealed the intrinsic anti-borreliae activity of doxycycline with tested phytochemicals and micronutrients indicating that their addition may enhance efficacy of this antibiotic in combating Borrelia sp. Especially the addition of flavones balcalein and luteolin to a doxycycline regimen could be explored further in defining more effective treatments against these bacteria.

Louse-borne relapsing fever - report of four cases in Switzerland, June-December 2015.

BACKGROUND: Louse-borne relapsing fever (LBRF) is a neglected disease that has been restricted to East Africa for many decades. Several cases in refugees from the Horn of Africa have been recently diagnosed in four European countries. CASE PRESENTATION: We report four additional cases of LBRF in asylum seekers from Somalia and Eritrea who presented with fever shortly after arriving in Switzerland during a seven-month period. Multiple spirochetes were visualized on stained blood films which were identified as Borrelia recurrentis by 16S rRNA gene sequencing. All patients recovered after antibiotic treatment with ceftriaxone and/or doxycycline. Concurrent infections (malaria and tuberculosis) were diagnosed in half of our patients. Possible modes of transmission and preventive measures are discussed. CONCLUSIONS: These reported cases highlight the ongoing transmission of LBRF in migrants from East Africa. Diagnosis of LBRF cases and prevention of autochthonous transmission in asylum seeker camps are important steps for the near future.

A case of canine borreliosis in Iran caused by Borrelia persica.

Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by Borrelia persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489 bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with B. persica sequences derived from clinical cases or from O. tholozani ticks. Our IGS sequence also showed 99% similarity over 414 bp (85% coverage) with a strain from a domestic dog, and 96% over 328 bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbor minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks.

Lyme disease: a rigorous review of diagnostic criteria and treatment.

Lyme disease was originally identified in Lyme, Connecticut, based upon an unusual cluster of what appeared to be patients with juvenile rheumatoid arthritis. It was subsequently identified as a new clinical entity originally called Lyme arthritis based on the observation that arthritis was a major clinical feature. However, Lyme arthritis is now called Lyme disease based upon the understanding that the clinical features include not only arthritis, but also potential cardiac, dermatologic and neurologic findings. Lyme disease typically begins with an erythematous rash called erythema migrans (EM). Approximately 4-8% of patients develop cardiac, 11% develop neurologic and 45-60% of patients manifest arthritis. The disease is transmitted following exposure to a tick bite containing a spirochete in a genetically susceptible host. There is considerable data on spirochetes, including Borrelia burgdorferi (Bb), the original bacteria identified in this disease. Lyme disease, if an organism had not been identified, would be considered as a classic autoimmune disease and indeed the effector mechanisms are similar to many human diseases manifest as loss of tolerance. The clinical diagnosis is highly likely based upon appropriate serology and clinical manifestations. However, the serologic features are often misinterpreted and may have false positives if confirmatory laboratory testing is not performed. Antibiotics are routinely and typically used to treat patients with Lyme disease, but there is no evidence that prolonged or recurrent treatment with antibiotics change the natural history of Lyme disease. Although there are animal models of Lyme disease, there is no system that faithfully recapitulates the human disease. Further research on the effector mechanisms that lead to pathology in some individuals should be further explored to develop more specific therapy.

Design and development of a novel vaccine for protection against Lyme borreliosis.

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.