Search

VMP-like sequence (vls) antigenic variation systems are present in every Lyme disease Borrelia strain with complete genome sequences. The linear plasmidencoded vls system consists of a single expression site (vlsE) and contiguous array(s) of silent cassettes that have ~90% identity with the central cassette region of the cognate vlsE gene; antigenic variation occurs through random, segmental, and unidirectional recombination of vls silent cassette sequences into the vlsE expression site. Automated annotation programs do not accurately recognize vls silent cassette sequences, so these regions are not correctly annotated in most genomic sequences. In this study, the vls sequences were re-analyzed in the genomic sequences of 31 available Lyme disease Borrelia and one relapsing fever Borrelia organisms, and this information was utilized to systematically compare the vls systems in different species and strains. In general, the results confirm the conservation of the overall architecture of the vls system, such as the head-to-head arrangement of vlsE and a contiguous series of vlsS silent cassette sequences and presence of inverted repeat sequences between the two regions. However, the data also provide evidence for the divergence of the vls silent cassette arrays through point mutations, short indels, duplication events, and rearrangements. The probable occurrence of convergent evolution toward a vls system-like locus is exemplified by Borrelia turcica, a variable large protein (Vlp) expressing organism that is a member of the relapsing fever Borrelia group. [ABSTRACT FROM AUTHOR]

VMP-like sequence (vls) antigenic variation systems are present in every Lyme disease Borrelia strain with complete genome sequences. The linear plasmid-encoded vls system consists of a single expression site (vlsE) and contiguous array(s) of silent cassettes that have ~90% identity with the central cassette region of the cognate vlsE gene; antigenic variation occurs through random, segmental, and unidirectional recombination of vls silent cassette sequences into the vlsE expression site. Automated annotation programs do not accurately recognize vls silent cassette sequences, so these regions are not correctly annotated in most genomic sequences. In this study, the vls sequences were re-analyzed in the genomic sequences of 31 available Lyme disease Borrelia and one relapsing fever Borrelia organisms, and this information was utilized to systematically compare the vls systems in different species and strains. In general, the results confirm the conservation of the overall architecture of the vls system, such as the head-to-head arrangement of vlsE and a contiguous series of vlsS silent cassette sequences and presence of inverted repeat sequences between the two regions. However, the data also provide evidence for the divergence of the vls silent cassette arrays through point mutations, short indels, duplication events, and rearrangements. The probable occurrence of convergent evolution toward a vls system-like locus is exemplified by Borrelia turcica, a variable large protein (Vlp) expressing organism that is a member of the relapsing fever Borrelia group.

ABSTRACT Borrelia burgdorferi, the agent of Lyme disease, is estimated to cause >400,000 annual infections in the United States. Serology is the primary laboratory method to support the diagnosis of Lyme disease, but current methods have intrinsic limitations that require alternative approaches or targets. We used a high-density peptide array that contains >90,000 short overlapping peptides to catalog immunoreactive linear epitopes from >60 primary antigens of B. burgdorferi. We then pursued a machine learning approach to identify immunoreactive peptide panels that provide optimal Lyme disease serodiagnosis and can differentiate antibody responses at various stages of disease. We examined 226 serum samples from the Lyme Biobank and the National Institutes of Health, which included sera from 110 individuals diagnosed with Lyme disease, 31 probable cases from symptomatic individuals, and 85 healthy controls. Cases were grouped based on disease stage and presentation and included individuals with early localized, early disseminated, and late Lyme disease. We identified a peptide panel originating from 14 different epitopes that differentiated cases versus controls, whereas another peptide panel built from 12 unique epitopes differentiated subjects with various disease manifestations. Our method demonstrated an improvement in B. burgdorferi antibody detection over the current two-tiered testing approach and confirmed the key diagnostic role of VlsE and FlaB antigens at all stages of Lyme disease. We also uncovered epitopes that triggered a temporal antibody response that was useful for differentiation of early and late disease. Our findings can be used to streamline serologic targets and improve antibody-based diagnosis of Lyme disease.IMPORTANCESerology is the primary method of Lyme disease diagnosis, but this approach has limitations, particularly early in disease. Currently employed antibody detection assays can be improved by the identification of alternative immunodominant epitopes and the selection of optimal diagnostic targets. We employed high-density peptide arrays that enabled precise epitope mapping for a wide range of B. burgdorferi antigens. In combination with machine learning, this approach facilitated the selection of serologic targets early in disease and the identification of serological indicators associated with different manifestations of Lyme disease. This study provides insights into differential antibody responses during infection and outlines a new approach for improved serologic diagnosis of Lyme disease.

Objective: The diagnosis of Lyme borreliosis is based on two-tier testing using an ELISA and Western blot. About 5–10% of patients report persistent symptoms of unknown etiology after treatment, resulting in substantial difficulties in further diagnostic workup. This paper presents a study aimed at determining whether serology can differentiate between patients with persistent symptoms attributed to Lyme and other patients with Lyme borreliosis. Methods: A retrospective cohort study included 162 samples from four subgroups: patients with persistent symptoms of Lyme (PSL), early Lyme borreliosis with erythema migrans (EM), patients tested in a general practitioner setting (GP), and healthy controls (HC). ELISA, Western blots, and multiplex assays from different manufacturers were used to determine inter-test variations in PSL and to compare reactivity against Borrelia-specific antigens among the groups. Results: In comparing the IgG and IgM reactivity by Western blot, IgG was more often positive in the PSL group than in the GP group. The individual antigen reactivity was similar between the PSL and EM or GP groups. Inter-test agreement among the manufacturers was variable, and agreement was higher for IgG testing compared to IgM. Conclusions: Serological testing is unable to define the subgroup of patients with persistent symptoms attributed to Lyme borreliosis. Additionally, the current two-tier testing protocol shows a large variance among different manufacturers in these patients. [ABSTRACT FROM AUTHOR]

ABSTRACT VlsE (variable major protein-like sequence, expressed) is an outer surface protein of the Lyme disease pathogen (Borreliella species) responsible for its within-host antigenic variation and a key diagnostic biomarker of Lyme disease. However, the high sequence variability of VlsE poses a challenge to the development of consistent VlsE-based diagnostics and therapeutics. In addition, the standard diagnostic protocols detect immunoglobins elicited by the Lyme pathogen, not the presence of the pathogen or its derived antigens. Here, we described the development of recombinant monoclonal antibodies (rMAbs) that bound specifically to conserved epitopes on VlsE. We first quantified amino-acid sequence variability encoded by the vls genes from 13 B. burgdorferi genomes by evolutionary analyses. We showed broad inconsistencies of the sequence phylogeny with the genome phylogeny, indicating rapid gene duplications, losses, and recombination at the vls locus. To identify conserved epitopes, we synthesized peptides representing five long conserved invariant regions (IRs) on VlsE. We tested the antigenicity of these five IR peptides using sera from three mammalian host species including human patients, the natural reservoir white-footed mouse (Peromyscus leucopus), and VlsE-immunized New Zealand rabbits (Oryctolagus cuniculus). The IR4 and IR6 peptides emerged as the most antigenic and reacted strongly with both the human and rabbit sera, while all IR peptides reacted poorly with sera from natural hosts. Four rMAbs binding specifically to the IR4 and IR6 peptides were identified, cloned, and purified. Given their specific recognition of the conserved epitopes on VlsE, these IR-specific rMAbs are potential novel diagnostic and research agents for direct detection of Lyme disease pathogens regardless of strain heterogeneity. IMPORTANCE Current diagnostic protocols of Lyme disease indirectly detect the presence of antibodies produced by the patient upon infection by the bacterial pathogen, not the pathogen itself. These diagnostic tests tend to underestimate early-stage bacterial infections before the patients develop robust immune responses. Further, the indirect tests do not distinguish between active or past infections by the Lyme disease bacteria in a patient sample. Here, we described novel monoclonal antibodies that have the potential to become the basis of direct and definitive diagnostic detection of the Lyme disease pathogen, regardless of its genetic heterogeneity.

The Lyme disease spirochete Borrelia burgdorferi , encodes an elaborate antigenic variation system that promotes the ongoing variation of a major surface lipoprotein, VlsE. Changes in VlsE are continual and always one step ahead of the host acquired immune system, which requires 1–2 weeks to generate specific antibodies. By the time this happens, new VlsE variants have arisen that escape immunosurveillance, providing an avenue for persistent infection. This antigenic variation system is driven by segmental gene conversion events that transfer information from a series of silent cassettes (vls2-16) to the expression locus, vlsE. The molecular details of this process remain elusive. Recombinational switching at vlsE is RecA-independent and the only required factor identified to date is the RuvAB branch migrase. In this work we have used next generation long-read sequencing to analyze the effect of several DNA replication/recombination/repair gene disruptions on the frequency of gene conversions at vlsE and report a requirement for the mismatch repair protein MutL. Site directed mutagenesis of mutL suggests that the putative MutL endonuclease activity is required for recombinational switching at vlsE. This is the first report of an unexpected essential role for MutL in a bacterial recombination system and expands the known function of this protein as well as our knowledge of the details of the novel recombinational switching mechanism for vlsE variation. [ABSTRACT FROM AUTHOR]

The Lyme disease spirochete Borrelia burgdorferi, encodes an elaborate antigenic variation system that promotes the ongoing variation of a major surface lipoprotein, VlsE. Changes in VlsE are continual and always one step ahead of the host acquired immune system, which requires 1–2 weeks to generate specific antibodies. By the time this happens, new VlsE variants have arisen that escape immunosurveillance, providing an avenue for persistent infection. This antigenic variation system is driven by segmental gene conversion events that transfer information from a series of silent cassettes (vls2-16) to the expression locus, vlsE. The molecular details of this process remain elusive. Recombinational switching at vlsE is RecA-independent and the only required factor identified to date is the RuvAB branch migrase. In this work we have used next generation long-read sequencing to analyze the effect of several DNA replication/recombination/repair gene disruptions on the frequency of gene conversions at vlsE and report a requirement for the mismatch repair protein MutL. Site directed mutagenesis of mutL suggests that the putative MutL endonuclease activity is required for recombinational switching at vlsE. This is the first report of an unexpected essential role for MutL in a bacterial recombination system and expands the known function of this protein as well as our knowledge of the details of the novel recombinational switching mechanism for vlsE variation.

This retrospective case-control study assesses the sensitivity, specificity, and area under the curve of the ZEUS Borrelia VlsE1/pepC10 assay in comparison with the C6-ELISA in European patients with Lyme borreliosis, healthy blood donors, and potentially cross-reactive controls. We included a convenience series of 161 sera from patients with physician-confirmed early localized or disseminated Lyme borreliosis (n = 143), 400 sera from healthy blood donors and 44 sera with potentially cross-reactive antibodies, on which we performed the aforementioned serological assays and the recomLine immunoblot. Diagnostic parameters were compared in various single-tier and two-tier algorithms. The specificities of the C6-ELISA and the ZEUS Borrelia VlsE1/pepC10 were comparable in healthy blood donors (e.g., single-tier permissive: C6: 362/400, 90.5% [87.2–93.2]; VlsE1/pepC10: 361/400, 90.3% [86.9–93.0]). The C6-ELISA had an apparently higher sensitivity in EM sera (e.g., both time points combined: C6: 61/76, 80.3% [69.5–88.5]; VlsE1/pepC10: 54/76, 71.1% [59.5–80.9]), but these differences were all not-significant. Interestingly, the VlsE1/pepC10 assay had a significantly higher specificity in sera with potentially cross-reactive antibodies (e.g., single-tier permissive: C6: 34/44, 77.3% [62.2–88.5]; VlsE1/pepC10: 40/44, 90.9% [78.3–97.5]; p = 0.031). While the areas under the curve for both assays were excellent, that of the C6-ELISA exceeded that of the VlsE1/pepC10 (C6: AUC = 0.925; VlsE1/pepC10: AUC = 0.878; p = 0.003). The novel ZEUS Borrelia VlsE1/pepC10 assay has generally comparable diagnostic parameters to the C6-ELISA with potentially improved specificity in cross-reactive sera. Thus, it is a useful tool for the serodiagnosis of Lyme borreliosis in Europe. [ABSTRACT FROM AUTHOR]

The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions.

Summary The Lyme disease spirochete, Borrelia burgdorferi , uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE , and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions. [ABSTRACT FROM AUTHOR]

Background. The conventional 2-tiered serologic testing protocol for Lyme disease (LD), an enzyme immunoassay (EIA) followed by immunoglobulin M and immunoglobulin G Western blots, performs well in late-stage LD but is insensitive in patients with erythema migrans (EM), the most common manifestation of the illness. Western blots are also complex, difficult to interpret, and relatively expensive. In an effort to improve test performance and simplify testing in early LD, we evaluated several modified 2-tiered testing (MTTT) protocols, which use 2 assays designed as first-tier tests sequentially, without the need of Western blots. Methods. The MTTT protocols included (1) a whole-cell sonicate (WCS) EIA followed by a C6 EIA; (2) a WCS EIA followed by a VlsE chemiluminescence immunoassay (CLIA); and (3) a variable major protein-like sequence, expressed (VlsE) CLIA followed by a C6 EIA. Sensitivity was determined using serum from 55 patients with erythema migrans; specificity was determined using serum from 50 patients with other illnesses and 1227 healthy subjects. Results. Sensitivity of the various MTTT protocols in patients with acute erythema migrans ranged from 36% (95% confidence interval [CI], 25%-50%) to 54% (95% CI, 42%-67%), compared with 25% (95% CI, 16%-38%) using the conventional protocol (P = .003-0.3). Among control subjects, the 3 MTTT protocols were similarly specific (99.3%-99.5%) compared with conventional 2-tiered testing (99.5% specificity; P = .6-1.0). Conclusions. Although there were minor differences in sensitivity and specificity among MTTT protocols, each provides comparable or greater sensitivity in acute EM, and similar specificity compared with conventional 2-tiered testing, obviating the need for Western blots. [ABSTRACT FROM AUTHOR]

Many parasites have evolved antigenic variation systems that alter surface proteins in order to evade recognition by presently expressed antibodies and subsequent death. Although the amino acid positions in antigens to which antibodies most commonly target are expected to be the most variable, this assumption has not been investigated. Using the vls antigenic variation system of Borrelia burgdorferi as a model, we first investigated this assumption computationally and then developed a sensitive immunoassay to experimentally validate the computational results. There was a strong correlation between variability at an amino acid position and each of the computational metrics associated with antibody reactivity. However, empirical measures of antibody reactivity were not consistently greater at the variable amino acid positions than at the invariant amino acid positions. The inconsistent experimental support for this hypothesis suggests that the biological effect of variability at an amino acid position is obfuscated by other factors. [ABSTRACT FROM AUTHOR]

Changing environmental conditions present an evolutionary challenge for all organisms. The environment of microbial pathogens, including the adaptive immune responses of the infected host, changes rapidly and is lethal to the pathogen lineages that cannot quickly adapt. The dynamic immune environment creates strong selective pressures favoring microbial pathogen lineages with antigenic variation systems that maximize the antigenic divergence among expressed antigenic variants. However, divergence among expressed antigens may be constrained by other molecular features such as the efficient expression of functional proteins. We computationally examined potential conflicting selection pressures on antigenic variation systems using the vls antigenic variation system in Borrelia burgdorferi as a model system. The vls system alters the sequence of the expressed antigen by recombining gene fragments from unexpressed but divergent ‘cassettes’ into the expression site, vlsE. The in silico analysis of natural and altered cassettes from seven lineages in the B. burgdorferi sensu lato species complex revealed that sites that are polymorphic among unexpressed cassettes, as well as the insertion/deletion mutations, are organized to maximize divergence among the expressed antigens within the constraints of translational ability and high translational efficiency. This study provides empirical evidence that conflicting selection pressures on antigenic variation systems can limit the potential antigenic divergence in order to maintain proper molecular function. [ABSTRACT FROM AUTHOR]

For the diagnosis of Lyme borreliosis, a C6 enzyme-linked immunosorbent assay used as a stand-alone test or in the second tier of a 2-tiered algorithm performed as well as conventional 2-tiered testing using European assays.Background. Physicians in the United States sometimes need to evaluate a patient for suspected Lyme borreliosis (LB) who may have acquired the infection in Europe. Using serum samples from European LB patients, we compared the performance of European and US serodiagnostic tests, including newer-generation assays containing Vmp-like sequence, expressed or its C6 peptide.Methods. The sensitivity of each assay was determined using 64 serum samples from LB patients with early or late disease manifestations who acquired the infection in Europe. Specificity was measured using 100 sera from healthy subjects from a nonendemic area.Results. For the detection of European-acquired infection, conventional 2-tiered testing (enzyme-linked immunosorbent assay [ELISA] followed by immunoblotting) using US assays had an overall sensitivity and specificity of 52% and 100%, compared with 81% (P = .0007) and 99% (P = 1.00) using analogous European tests. The sensitivity of a US C6 ELISA used as a stand-alone test (88% overall) was statistically comparable to that of conventional 2-tiered testing using European tests (P = .47) and was 100% specific. Similarly, an alternative 2-tiered algorithm using a standard US ELISA followed by a C6 ELISA was comparably sensitive (84% overall) compared with conventional 2-tiered testing using European assays (P = .82), and specificity remained 100%.Conclusions. European assays outperformed analogous US assays in a conventional 2-tiered testing algorithm. However, a C6 ELISA used as a stand-alone test or in the second tier of a 2-tiered algorithm performed comparably to conventional 2-tiered testing using European assays, and can be used for evaluation of any patient, regardless of travel history. [ABSTRACT FROM AUTHOR]

Abstract: The VlsE lipoprotein of Borrelia burgdorferi elicits a strong immune response during the course of Lyme disease. The present study was aimed at characterization of the epitopes of VlsE targeted by the antibody response in patients with post-Lyme disease syndrome, a condition characterized by persisting symptoms of pain, fatigue, and/or neurocognitive impairment despite antibiotic treatment of B. burgdorferi infection. Epitope mapping was carried out using microarrays that contained synthesized overlapping peptides covering the full sequence of VlsE from B. burgdorferi B31. In addition to the previously characterized IR6 region in the variable domain, specific sequences in the N- and C-terminal invariable domains of VlsE were found to be major B cell epitopes in affected patients. The crystal structure of VlsE indicated that the newly described epitopes form a contiguous region in the surface-exposed membrane-proximal part of the monomeric form of the protein. [Copyright &y& Elsevier]

Abstract: A total of 161 positive sera of the IgM class against Borrelia burgdorferi s.l. and 149 positive sera of the IgG class obtained from screening tests with Polish forestry workers (ELISA recombinant Borrelia IgM and IgG, Biomedica, Austria) were tested with IgM and IgG immunoblot -Borrelia LINE (Genzyme Virotech GmbH, Germany). Of the IgM ELISA-positive sera, 72% were positive using Borrelia LINE IgM. IgM antibodies against particular antigens in Borrelia LINE tests were as follows: OspC (70%), VlsE (1.8%), P39 (40%), EBV (0.6%). All sera which were positive for IgG using ELISA were also found to be positive for IgG in Borrelia LINE IgG. IgG antibodies against individual antigens using Borrelia Line tests were as follows: VlsE (100%), p39 (95%), p83 (67.5%), iv1-BBA36 (42.5%), iv2-BBO323 (72.5%), iv3-Crasp3 (50%), iv4-p4 (12.5%). Antibodies against VlsE in the IgM class undergo quick seroconversion to the IgG class (demonstrated by Borrelia LINE tests) in an ongoing infection despite a persistingly positive result for the IgM class obtained using ELISA with recombinant antigens. The presence of numerous antibodies indicates the varied immunological response against B. burgdorferi antigens. A broad range of ‘in vivo’ IgG class antigens may be very helpful to confirm B. burgdorferi infection using Borrelia LINE tests. [Copyright &y& Elsevier]

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection. [ABSTRACT FROM AUTHOR]

• This is the first profiling of Borreliella burgdorferi specific maternal antibody in pups. • Sera from dam and pups had similar immunoreactivity patterns and were bactericidal. • Abs against diagnostic antigens (VlsE, OspF) persisted longer than Abs to other proteins. • Antibodies in the pups were derived from colostrum and not an active infection. • Vaccination triggered an immune memory response to diverse OspC types. Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered. Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog. [ABSTRACT FROM AUTHOR]

The spirochetes that cause Lyme disease have an elaborate antigenic variation system that produces millions of variants, thus evading the immune response. Verhey et al. have applied next-generation sequencing and computational analysis to gain new insights into how these bacteria keep ‘one step ahead’ of elimination by the host. [ABSTRACT FROM AUTHOR]

Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes ( Canis latrans ) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum , respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively. IMPORTANCE The incidence of Lyme disease ( Borreliella burgdorferi ) and anaplasmosis ( Anaplasma phagocytophilum ) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals., (Copyright © 2020 Izac et al.)

Arp is an immunogenic protein of the Lyme disease spirochete Borrelia burgdorferi and contributes to joint inflammation during infection. Despite Arp eliciting a strong humoral response, antibodies fail to clear the infection. Given previous evidence of immune avoidance mediated by the antigenically variable lipoprotein of B. burgdorferi, VlsE, we use passive immunization assays to examine whether VlsE protects the pathogen from anti-Arp antibodies. The results show that spirochetes are only able to successfully infect passively immunized mice when VlsE is expressed. Subsequent immunofluorescence assays reveal that VlsE prevents binding of Arp-specific antibodies, thereby providing an explanation for the failure of Arp antisera to clear the infection. The results also show that the shielding effect of VlsE is not universal for all B. burgdorferi cell-surface antigens. The findings reported here represent a direct demonstration of VlsE-mediated protection of a specific B. burgdorferi surface antigen through a possible epitope-shielding mechanism., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused by Borreliella burgdorferi No vaccine is available for humans. Dogmatically, B. burgdorferi can establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that the B. burgdorferi spirochete is effectively cleared by anti- B. burgdorferi antibodies of New Zealand White rabbits, despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologous B. burgdorferi spirochetes and significantly reduce the pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify the specificities of the protective rabbit antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved the use of random peptide phage display libraries coupled with next-generation sequencing and our computational algorithms, repertoires of nonprotective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the protective rabbit sera were mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine., (Copyright © 2019 American Society for Microbiology.)

Borrelia burgdorferi is a tick-borne bacterium responsible for approximately 300,000 annual cases of Lyme disease (LD) in the United States, with increasing incidences in other parts of the world. The debilitating nature of LD is mainly attributed to the ability of B. burgdorferi to persist in patients for many years despite strong anti- Borrelia antibody responses. Antimicrobial treatment of persistent infection is challenging. Similar to infection of humans, B. burgdorferi establishes long-term infection in various experimental animal models except for New Zealand White (NZW) rabbits, which clear the spirochete within 4 to 12 weeks. LD spirochetes have a highly evolved antigenic variation vls system, on the lp28-1 plasmid, where gene conversion results in surface expression of the antigenically variable VlsE protein. VlsE is required for B. burgdorferi to establish persistent infection by continually evading otherwise potent antibodies. Since the clearance of B. burgdorferi is mediated by humoral immunity in NZW rabbits, the previously reported results that LD spirochetes lose lp28-1 during rabbit infection could potentially explain the failure of B. burgdorferi to persist. However, the present study unequivocally disproves that previous finding by demonstrating that LD spirochetes retain the vls system. However, despite the vls system being fully functional, the spirochete fails to evade anti- Borrelia antibodies of NZW rabbits. In addition to being protective against homologous and heterologous challenges, the rabbit antibodies significantly ameliorate LD-induced arthritis in persistently infected mice. Overall, the current data indicate that NZW rabbits develop a protective antibody repertoire, whose specificities, once defined, will identify potential candidates for a much-anticipated LD vaccine., (Copyright © 2019 American Society for Microbiology.)

The tick-borne pathogen Borrelia burgdorferi is responsible for approximately 300,000 Lyme disease (LD) cases per year in the United States. Recent increases in the number of LD cases, in addition to the spread of the tick vector and a lack of a vaccine, highlight an urgent need for designing and developing an efficacious LD vaccine. Identification of protective epitopes that could be used to develop a second-generation (subunit) vaccine is therefore imperative. Despite the antigenicity of several lipoproteins and integral outer membrane proteins (OMPs) on the B. burgdorferi surface, the spirochetes successfully evade antibodies primarily due to the VlsE-mediated antigenic variation. VlsE is thought to sterically block antibody access to protective epitopes of B. burgdorferi However, it is highly unlikely that VlsE shields the entire surface epitome. Thus, identification of subdominant epitope targets that induce protection when they are made dominant is necessary to generate an efficacious vaccine. Toward the identification, we repeatedly immunized immunocompetent mice with live-attenuated VlsE-deleted B. burgdorferi and then challenged the animals with the VlsE-expressing (host-adapted) wild type. Passive immunization and Western blotting data suggested that the protection of 50% of repeatedly immunized animals against the highly immune-evasive B. burgdorferi was antibody mediated. Comparison of serum antibody repertoires identified in protected and nonprotected animals permitted the identification of several putative epitopes significantly associated with the protection. Most linear putative epitopes were conserved between the main pathogenic Borrelia genospecies and found within known subdominant regions of OMPs. Currently, we are performing immunization studies to test whether the identified protection-associated epitopes are protective for mice., (Copyright © 2018 American Society for Microbiology.)

Lyme disease (LD), the most prevalent tick-borne illness in North America, is caused by Borrelia burgdorferi The long-term survival of B. burgdorferi spirochetes in the mammalian host is achieved though VlsE-mediated antigenic variation. It is mathematically predicted that a highly variable surface antigen prolongs bacterial infection sufficiently to exhaust the immune response directed toward invariant surface antigens. If the prediction is correct, it is expected that the antibody response to B. burgdorferi invariant antigens will become nonprotective as B. burgdorferi infection progresses. To test this assumption, changes in the protective efficacy of the immune response to B. burgdorferi surface antigens were monitored via a superinfection model over the course of 70 days. B. burgdorferi-infected mice were subjected to secondary challenge by heterologous B. burgdorferi at different time points postinfection (p.i.). When the infected mice were superinfected with a VlsE-deficient clone (ΔVlsE) at day 28 p.i., the active anti-B. burgdorferi immune response did not prevent ΔVlsE-induced spirochetemia. In contrast, most mice blocked culture-detectable spirochetemia induced by wild-type B. burgdorferi (WT), indicating that VlsE was likely the primary target of the antibody response. As the B. burgdorferi infection further progressed, however, reversed outcomes were observed. At day 70 p.i. the host immune response to non-VlsE antigens became sufficiently potent to clear spirochetemia induced by ΔVlsE and yet failed to prevent WT-induced spirochetemia. To test if any significant changes in the anti-B. burgdorferi antibody repertoire accounted for the observed outcomes, global profiles of antibody specificities were determined. However, comparison of mimotopes revealed no major difference between day 28 and day 70 antibody repertoires., (Copyright © 2016 American Society for Microbiology.)

Borrelia burgdorferi is the causative bacterial agent of Lyme disease, the most prevalent tick-borne infection in North America. The ability of B. burgdorferi to cause disease is highly dependent on its capacity to evade the immune response during infection of the mammalian host. One of the ways in which B. burgdorferi is known to evade the immune response is antigenic variation of the variable major protein (VMP)-like sequence (Vls) E lipoprotein. Past research involving the B. burgdorferi antigenic variation system has implicated a gene-conversion mechanism for vlsE recombination, analyzed the long-term dynamic changes occurring within VlsE, and established the critical importance of antigenic variation for persistent infection of the mammalian host. However, a role for the VlsE protein other than providing an antigenic disguise is currently unknown, but it has been proposed that the protein may function in other forms of immune evasion. Although a substantial number of additional proteins reside on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may use a VlsE-mediated system for immune avoidance of its surface antigens. Several recent experimental studies involving host reinfection, superinfection, and the importance of VlsE antigenic variation during the pathogen's enzootic cycle have been used to address this question. Here, the cumulative results from these studies are reviewed, and the knowledge gaps that remain regarding the role of VlsE for immune avoidance are discussed.