Risk of cardiac conduction disorders, and pacemaker implantations among individuals tested for serum Borrelia burgdorferi antibodies, a nationwide, matched, population-based cohort study.

OBJECTIVES: To investigate the short- and long-term risks of atrioventricular block and other cardiac conduction disorders associated with being tested for Borrelia burgdorferi (Bb) antibodies or Bb seropositivity as measures of confounding by indication and Bb infection, respectively. METHODS: We performed a nationwide population-based matched cohort study (Denmark, 1993-2021). We included 52 200 Bb-seropositive individuals (stratified as only Bb-IgM-seropositive [n = 26 103], only Bb-IgG-seropositive [n = 18 698], and Bb-IgM-and-IgG-seropositive [n = 7399]) and two age- and sex-matched comparison cohorts: 104 400 Bb-seronegative individuals and 261 000 population controls. We investigated the risk associated with being tested for serum Bb antibodies and being Bb seropositive. Outcomes were atrioventricular block and other conduction disorders. We calculated short-term odds ratios (aOR) (within 1 month), and long-term hazard ratios (aHR) (after 1 month) adjusted for age, sex, diabetes, chronic heart failure, and kidney disease with 95% CI. RESULTS: Compared with population controls, individuals tested for Bb antibodies had increased short- and long-term risks of atrioventricular block (aOR 47.9, 95% CI: 30.0-76.7, aHR 1.3, 95% CI:1.2-1.3), and other conduction disorders (aOR 18.2, 95% CI: 10.1-32.8, aHR 1.2, 95% CI: 1.1-1.4). Compared with Bb-seronegative individuals, only Bb-IgM-and-IgG-seropositive individuals had increased short-term risk of atrioventricular block (aOR: 2.1, 95% CI: 1.5-3.1). DISCUSSION: The results suggest that Bb antibody testing is included in the diagnostic work-up of conduction disorders. Finally, that Bb seropositivity is not associated with other conduction disorders than atrioventricular block or with increased long-term risk of conduction disorders.

Outer surface protein E (OspE) mediates Borrelia burgdorferi sensu stricto strain-specific complement evasion in the eastern fence lizard, Sceloporus undulatus.

In North America, Lyme disease is primarily caused by the spirochetal bacterium Borrelia burgdorferi sensu stricto (Bb), which is transmitted between multiple vertebrate hosts and ixodid ticks, and is a model commonly used to study host-pathogen interactions. While Bb is consistently observed in its mammalian and avian reservoirs, the bacterium is rarely isolated from North American reptiles. Two closely related lizard species, the eastern fence lizard (Sceloporus undulatus) and the western fence lizard (Sceloporus occidentalis), are examples of reptiles parasitized by Ixodes ticks. Vertebrates are known to generate complement as an innate defense mechanism, which can be activated before Bb disseminate to distal tissues. Complement from western fence lizards has proven lethal against one Bb strain, implying the role of complement in making those lizards unable to serve as hosts to Bb. However, Bb DNA is occasionally identified in distal tissues of field-collected eastern fence lizards, suggesting some Bb strains may overcome complement-mediated clearance in these lizards. These findings raise questions regarding the role of complement and its impact on Bb interactions with North American lizards. In this study, we found Bb seropositivity in a small population of wild-caught eastern fence lizards and observed Bb strain-specific survivability in lizard sera. We also found that a Bb outer surface protein, OspE, from Bb strains viable in sera, promotes lizard serum survivability and binds to a complement inhibitor, factor H, from eastern fence lizards. Our data thus identify bacterial and host determinants of eastern fence lizard complement evasion, providing insights into the role of complement influencing Bb interactions with North American lizards.

VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force.

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.

Lipoproteome screening of the Lyme disease agent identifies inhibitors of antibody-mediated complement killing.

Spirochetal pathogens, such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria, such as Proteobacteria and Firmicutes, few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least 80 surface-exposed lipoproteins throughout its tick vector­vertebrate host lifecycle. Several B. burgdorferi lipoproteins have been shown to serve roles in cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-initiated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ElpB and ElpQ (also termed ErpB and ErpQ, respectively). Using biochemical, microbiological, and biophysical approaches, we demonstrate that ElpB and ElpQ bind the activated forms of the C1 proteases, C1r and C1s, and represent a distinct mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing. In addition to identifying a mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host­pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.

Structure-Based Design of Diagnostics and Vaccines for Lyme Disease.

Changes in climate have increased the geographical range of insect vectors responsible for the transmission of several diseases. Lyme disease, caused by the bacterial pathogen Borrelia burgdorferi, has become recognized as the most prevalent arthropod-borne infection in the USA. It is transmitted to humans through the bite of infected blacklegged ticks. As yet, there are no commercial vaccines available that effectively provide protection against Lyme disease. Vaccination strategies involving use of subunit vaccines developed in many laboratories have been found to be less efficient in protecting against the disease. Hence, there is a need to develop powerful vaccines that provide robust protection against Borrelia. Recently, using the principle of structure-based design, we designed and developed novel diagnostics and vaccine candidates that protected against Lyme disease in animal models. This chapter describes design and development of peptides based on the principle of structure-based design for use in diagnostics and vaccines to protect against Lyme disease in an animal model.

Persistent Anti-Borrelia IgM Antibodies without Lyme Borreliosis in the Clinical and Immunological Context.

The aim of the study was to investigate the etiology of persistent IgM antibodies against Borrelia burgdorferi sensu lato (sl) and to analyze their association with nonspecific symptoms. The study group comprised individuals with persistent IgM antibodies in the absence of IgG. The relation between ELISA values and time elapsed since past erythema migrans (EM) was analyzed. Previous antibiotic treatments were assessed. The association between persistent IgM and nonspecific symptoms was evaluated statistically. Specificity of IgM antibodies for outer surface protein C (OspC) of B. burgdorferi sl was examined by immunoblotting. Further, we investigated the cross-reactivity with Borrelia-unrelated proteins. Fifty-nine patients (46 women; 78%) were included in the study group. The mean IgM-ELISA values did not change significantly during follow-up (median 6.2 months). The mean ELISA value in the study group was dependent on time elapsed since past EM. Nonspecific symptoms improved significantly more often in patients with lower IgM ELISA results. Persistent IgM antibodies were specific for the C-terminal PKKP motif of OspC. Cross-reacting C-terminal PKKP antigens from both human and prokaryotic origins were identified. We demonstrate that the C-terminal PKKP motif plays a main role for the reactivity of persistent Borrelia IgM toward OspC. However, cross-reactivity to other eukaryotic and/or prokaryotic antigens may hamper the specificity of OspC in the serological diagnosis of Lyme borreliosis. Lack of improvement of nonspecific symptoms was associated with higher IgM ELISA values. IMPORTANCE The reactivity of human IgM with the outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is frequently used to detect Borrelia specific IgM in commercial immunoassays, and such antibodies usually occur in the early phase of the infection. We identified a group of individuals with persistent Borrelia IgM without symptoms of Lyme borreliosis. We used their sera to demonstrate that the C-terminal epitope of OspC binds the IgM. Strikingly, we found that the same epitope occurs also in certain proteins of human and environmental origin; the latter include other bacteria and food plants. Our experimental data show that these Borrelia-unrelated proteins cross-react with the OpsC-specific IgM. This knowledge is important for the development of serologic assays for Lyme borreliosis and provides a cross-reactive explanation for the persistence of Borrelia-IgM.

Seroprevalence of antibodies against Borrelia burgdorferi sensu lato in healthy blood donors in Romania: an update.

BACKGROUND: The Borrelia burgdorferi sensu lato (s.l.) genogroup is the causative agent responsible for Lyme borreliosis, a common tick-borne infectious disease in some temperate regions of the Northern Hemisphere. In humans, the clinical manifestations of Lyme borreliosis vary from dermatological infection to severe systemic manifestations. In Romania, data on the seroprevalence of Lyme borreliosis and associated risk factors are scarce and outdated, as the only seroprevalence study with a large dataset was published more than 20 years ago. Therefore, the aim of the present study was to evaluate the seroprevalence for Borrelia burgdorferi s.l. in healthy blood donors from six Romanian counties and identify the associated risk factors. METHODS: The study was conducted among 1200 healthy blood donors aged between 18 and 65 years during November 2019 and September 2020 from six counties in the northwestern and central parts of Romania. A two-tiered testing strategy was applied. Positive and equivocal immunoenzymatic test results for IgG and IgM antibodies were further confirmed by Western blot. RESULTS: Serum samples from 20% of the blood donors had positive or equivocal IgG and IgM ELISA index values. In total, 2.3% of the serum samples for IgG and 1.8% for IgM were positive by Western blot. The seroprevalence for both antibodies varied between 1.5% (Satu-Mare) and 6.5% (Bistrița-Nasaud) in the six counties investigated. The highest seroprevalence was observed in men (4.7%), in blood donors performing their professional activities outdoors (4.2%), and in those aged ≥ 56 years (8%). CONCLUSIONS: These findings confirm the presence of specific IgG and IgM antibodies to B. burgdorferi s.l. among healthy blood donors from Romania. Furthermore, potential risk factors, such as gender, age, and behavior, associated with the presence of positive B. burgdorferi s.l. antibodies among healthy blood donors were identified.

A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes.

Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography-coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1') and reveals a surprising role for C1r B loop-interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway-specific inhibition by a human pathogen.

Optimizing use of multi-antibody assays for Lyme disease diagnosis: A bioinformatic approach.

Multiple different recombinant and peptide antigens are now available for serodiagnosis of Lyme disease (LD), but optimizing test utilization remains challenging. Since 1995 the Centers for Disease Control and Prevention (CDC) has recommended a 2-tiered serologic approach consisting of a first-tier whole-cell enzyme immunoassay (EIA) for polyvalent antibodies to Borrelia burgdorferi followed by confirmation of positive or equivocal results by IgG and IgM immunoblots [standard 2-tiered (STT) approach]. Newer modified 2-tiered (MTT) approaches employ a second-tier EIA to detect antibodies to B. burgdorferi rather than immunoblotting. We applied modern bioinformatic techniques to a large public database of recombinant and peptide antigen-based immunoassays to improve testing strategy. A retrospective CDC collection of 280 LD samples and 559 controls had been tested using the STT approach as well as kinetic-EIAs for VlsE1-IgG, C6-IgG, VlsE1-IgM, and pepC10-IgM antibodies. When used individually, the cutoff for each kinetic-EIA was set to generate 99% specificity. Utilizing logistic-likelihood regression analysis and receiver operating characteristic (ROC) techniques we determined that VlsE1-IgG, C6-IgG, and pepC10-IgM antibodies each contributed significant diagnostic information; a single-tier diagnostic score (DS) was generated for each sample using a weighted linear combination of antibody levels to these 3 antigens. DS performance was then compared to the STT and to MTT models employing different combinations of kinetic-EIAs. After setting the DS cutoff to match STT specificity (99%), the DS was 22.5% more sensitive than the STT for early-acute-phase disease (95% CI: 11.8% to 32.2%), 16.0% more sensitive for early-convalescent-phase disease (95% CI: 7.2% to 24.7%), and equivalent for detection of disseminated infection. The DS was also significantly more sensitive for early-acute-phase LD than MTT models whose specificity met or exceeded 99%. Prospective validation of this single-tier diagnostic score for Lyme disease will require larger studies using a broader range of potential cross-reacting conditions.

Lyme arthritis: linking infection, inflammation and autoimmunity.

Infectious agents can trigger autoimmune responses in a number of chronic inflammatory diseases. Lyme arthritis, which is caused by the tick-transmitted spirochaete Borrelia burgdorferi, is effectively treated in most patients with antibiotic therapy; however, in a subset of patients, arthritis can persist and worsen after the spirochaete has been killed (known as post-infectious Lyme arthritis). This Review details the current understanding of the pathogenetic events in Lyme arthritis, from initial infection in the skin, through infection of the joints, to post-infectious chronic inflammatory arthritis. The central feature of post-infectious Lyme arthritis is an excessive, dysregulated pro-inflammatory immune response during the infection phase that persists into the post-infectious period. This response is characterized by high amounts of IFNγ and inadequate amounts of the anti-inflammatory cytokine IL-10. The consequences of this dysregulated pro-inflammatory response in the synovium include impaired tissue repair, vascular damage, autoimmune and cytotoxic processes, and fibroblast proliferation and fibrosis. These synovial characteristics are similar to those in other chronic inflammatory arthritides, including rheumatoid arthritis. Thus, post-infectious Lyme arthritis provides a model for other chronic autoimmune or autoinflammatory arthritides in which complex immune responses can be triggered and shaped by an infectious agent in concert with host genetic factors.

Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.

Pathogens possess the ability to adapt and survive in some host species but not in others-an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.

Comparative analysis of antibody responses to outer surface protein (Osp)A and OspC in dogs vaccinated with Lyme disease vaccines.

Lyme disease (LD), the most common tick-borne disease of canines and humans in N. America, is caused by the spirochete Borreliella burgdorferi. Subunit and bacterin vaccines are available for the prevention of LD in dogs. LD bacterin vaccines, which are comprised of cell lysates of two strains of B. burgdorferi, contain over 1000 different proteins and cellular constituents. In contrast, subunit vaccines are defined in composition and consist of either outer surface protein (Osp)A or OspA and an OspC chimeritope. In this study, we comparatively assessed antibody responses to OspA and OspC induced by vaccination with all canine bacterin and subunit LD vaccines that are commercially available in North America. Dogs were administered a two-dose series of the vaccine to which they were assigned (3 weeks apart): Subunit-AC, Subunit-A, Bacterin-1, and Bacterin-2. Antibody titers to OspA and OspC were determined by ELISA and the ability of each vaccine to elicit antibodies that recognize diverse OspC proteins (referred to as OspC types) assessed by immunoblot. While all of the vaccines elicited similar OspA antibody responses, only Subunit-AC triggered a robust and broadly cross-reactive antibody response to divergent OspC proteins. The data presented within provide new information regarding vaccination-induced antibody responses to key tick and mammalian phase antigens by both subunit and bacterin LD canine vaccine formulations.

Documentation of a false positive Lyme disease serologic test in a patient with untreated Babesia microti infection carries implications for accurately determining the frequency of Lyme disease coinfections.

Although many infections may potentially cause a false positive Borrelia burgdorferi antibody test, particularly for IgM antibody, babesiosis is not regarded as an infection that does this. A patient with untreated babesiosis is described here who developed transient IgM seropositivity for antibodies to B. burgdorferi. Thus, the diagnosis of Lyme disease coinfection in patients with active babesiosis, as in patients with human granulocytic anaplasmosis, is more convincingly accomplished if an objective clinical manifestation of Lyme disease is present, such as an erythema migrans skin lesion.

Host Specialisation, Immune Cross-Reaction and the Composition of Communities of Co-circulating Borrelia Strains.

We use mathematical modelling to examine how microbial strain communities are structured by the host specialisation traits and antigenic relationships of their members. The model is quite general and broadly applicable, but we focus on Borrelia burgdorferi, the Lyme disease bacterium, transmitted by ticks to mice and birds. In this system, host specialisation driven by the evasion of innate immunity has been linked to multiple niche polymorphism, while antigenic differentiation driven by the evasion of adaptive immunity has been linked to negative frequency dependence. Our model is composed of two host species, one vector, and multiple co-circulating pathogen strains that vary in their host specificity and their antigenic distances from one another. We explore the conditions required to maintain pathogen diversity. We show that the combination of host specificity and antigenic differentiation creates an intricate niche structure. Unequivocal rules that relate the stability of a strain community directly to the trait composition of its members are elusive. However, broad patterns are evident. When antigenic differentiation is weak, stable communities are typically composed entirely of generalists that can exploit either host species equally well. As antigenic differentiation increases, more diverse stable communities emerge, typically around trait compositions of generalists, generalists and very similar specialists, and specialists roughly balanced between the two host species.

Evaluation of factors influencing tick bites and tick-borne infections: a longitudinal study.

BACKGROUND: Various tick-borne infections like borreliosis and rickettsiosis pose a health risk to humans in many parts of the world. We investigated seroprevalence of and seroconversion to Borrelia burgdorferi and Rickettsia spp. and relation to tick-bites, weather and clinical manifestations in Denmark. METHODS: Blood donors were enrolled at the Hospital of Southern Jutland in June-July with follow-up November-February of 2018 and 2019. Blood samples were collected, and a questionnaire regarding tick bites, potential exposures and symptoms was completed at each visit. Samples were tested for presence of IgM and IgG antibodies directed against B. burgdorferi and Rickettsia spp. using R. helvetica and R. felis as antigens. Data were examined for correlation between tick bites, serological results, potential exposures and symptoms. RESULTS: Two-hundred and fourteen (93 follow-ups) and 130 (38 follow-ups) blood donors were included in 2018 and 2019, respectively. The total borrelia seroconversion rate was 6.3% (CI 2.1-10.5), while the prevalence of IgM and IgG antibodies was 7.8% (CI 4.9-10.6) and 6.7% (CI 4-9.3), respectively. Seroconversion to Rickettsia spp. was detected in one participant. Tick bites and seroconversion were not significantly associated with the reported unspecific symptoms, but unspecific symptoms were common in the study population. There was no significant difference in number of tick bites or seroconversion/prevalence between seasons with highly alternating weather. CONCLUSIONS: Results suggest that weather conditions in an individual year have a limited impact. Anti-Borrelia-antibodies do not seem to persist in serum for several years. Rickettsiosis is of limited concern in Denmark.

Tick-human interactions: from allergic klendusity to the α-Gal syndrome.

Ticks and the pathogens they transmit, including bacteria, viruses, protozoa, and helminths, constitute a growing burden for human and animal health worldwide. The ability of some animal species to acquire resistance to blood-feeding by ticks after a single or repeated infestation is known as acquired tick resistance (ATR). This resistance has been associated to tick-specific IgE response, the generation of skin-resident memory CD4+ T cells, basophil recruitment, histamine release, and epidermal hyperplasia. ATR has also been associated with protection to tick-borne tularemia through allergic klendusity, a disease-escaping ability produced by the development of hypersensitivity to an allergen. In addition to pathogen transmission, tick infestation in humans is associated with the α-Gal syndrome (AGS), a type of allergy characterized by an IgE response against the carbohydrate Galα1-3Gal (α-Gal). This glycan is present in tick salivary proteins and on the surface of tick-borne pathogens such as Borrelia burgdorferi and Anaplasma phagocytophilum, the causative agents of Lyme disease and granulocytic anaplasmosis. Most α-Gal-sensitized individuals develop IgE specific against this glycan, but only a small fraction develop the AGS. This review summarizes our current understanding of ATR and its impact on the continuum α-Gal sensitization, allergy, and the AGS. We propose that the α-Gal-specific IgE response in humans is an evolutionary adaptation associated with ATR and allergic klendusity with the trade-off of developing AGS.

Quantitative multiplexed strategies for human Lyme disease serological testing.

Lyme disease, which is primarily caused by infection with the bacterium Borrelia burgdorferi in the United States or other Borrelia species internationally, presents an ongoing challenge for diagnostics. Serological testing is the primary means of diagnosis but testing approaches differ widely, with varying degrees of sensitivity and specificity. Moreover, there is currently no reliable test to determine disease resolution following treatment. A distinct challenge in Lyme disease diagnostics is the variable patterns of human immune response to a plurality of antigens presented by Borrelia spp. during the infection. Thus, multiplexed testing approaches that capture these patterns and detect serological response against multiple antigens may be the key to prompt, accurate Lyme disease diagnosis. In this review, current state-of-the-art multiplexed diagnostic approaches are presented and compared with respect to their diagnostic accuracy and their potential for monitoring response to treatment.

Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease.

The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.

Blocking Borrelia burgdorferi transmission from infected ticks to nonhuman primates with a human monoclonal antibody.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.