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Lyme disease, caused by Borrelia burgdorferi (Bb), can progress to Lyme arthritis (LA). While most patients with LA respond successfully to antibiotic therapy, a small percentage fail to improve, a condition known as antibiotic-refractory Lyme arthritis (ARLA). While T cell responses are known to drive ARLA, molecular mechanisms for ARLA remain unknown. In this issue of the JCI, Dirks et al. isolated disease-specific Th cells from patients with ARLA residing in Germany. A distinct TCR-β motif distinguished ARLA from other rheumatic diseases. Notably, the TCR-β motif was linked predominantly to HLA-DRB1*11 or 13 alleles, which differed from alleles in patients from North America. It also mapped primarily to T peripheral helper (Tph) cells, as opposed to classical Th1 cells. These findings provide a roadmap explaining how T cell responses necessary for control of an infection can, despite antibiotic therapy, drive a disadvantageous T cell response, resulting in a postinfectious, inflammatory arthritis.

Background: Rising Lyme borreliosis incidence rates, potential for severe outcomes, and limitations in accurate and timely diagnosis for treatment initiation suggest the need for a preventive vaccine; however, no vaccine is currently available for human use. We performed two studies in adults to optimise the dose level and vaccination schedule for VLA15, an investigational Lyme borreliosis vaccine targeting outer surface protein A (OspA) serotypes 1-6, which are associated with the most common pathogenic Borrelia species in Europe and North America., Methods: Both randomised, observer-blind, placebo-controlled, multicentre phase 2 studies included participants aged 18-65 years without recent history of Lyme borreliosis or tick bites. Study one was conducted at nine clinical research and study centre sites in the USA (n=6), Germany (n=2), and Belgium (n=1); study two was conducted at five of the study one US sites. Based on a randomisation list created by an unmasked statistician for each study, participants were randomly assigned via an electronic case report form randomisation module to receive 90 μg (study one only), 135 μg, or 180 μg VLA15 or placebo by intramuscular injection at months 0, 1, and 2 (study one) or 0, 2, and 6 (study two). Study one began with a run-in phase to confirm safety, after which the Data Safety Monitoring Board recommended the removal of the 90 μg group and continuation of the study. In the study one run-in phase, randomisation was stratified by study site, whereas in the study one main phase and in study two, randomisation was stratified by study site, age group, and baseline B burgdorferi (sensu lato) serostatus. All individuals were masked, other than staff involved in randomisation, vaccine preparation or administration, or safety data monitoring. The primary endpoint for both studies was OspA-specific IgG geometric mean titres (GMTs) at 1 month after the third vaccination and was evaluated in the per-protocol population. Safety endpoints were evaluated in the safety population: all participants who received at least one vaccination. Both studies are registered at ClinicalTrials.gov (study one NCT03769194 and study two NCT03970733) and are completed., Findings: For study one, 573 participants were screened and randomly assigned to treatment groups between Dec 21, 2018, and Sept, 26, 2019. For study two, 248 participants were screened and randomly assigned between June 26 and Sept 3, 2019. In study one, 29 participants were assigned to receive 90 μg VLA15, 215 to 135 μg, 205 to 180 μg, and 124 to placebo. In study two, 97 participants were assigned to receive 135 μg VLA15, 100 to 180 μg, and 51 to placebo. At 1 month after the third vaccination (ie, month 3), OspA-specific IgG GMTs in study one ranged from 74·3 (serotype 1; 95% CI 46·4-119·0) to 267·4 units per mL (serotype 3; 194·8-367·1) for 90 μg VLA15, 101·9 (serotype 1; 87·1-119·4) to 283·2 units per mL (serotype 3; 248·2-323·1) for 135 μg, and 115·8 (serotype 1; 98·8-135·7) to 308·6 units per mL (serotype 3; 266·8-356·8) for 180 μg. In study two, ranges at 1 month after the third vaccination (ie, month 7) were 278·5 (serotype 1; 214·9-361·0) to 545·2 units per mL (serotype 2; 431·8-688·4) for 135 μg VLA15 and 274·7 (serotype 1; 209·4-360·4) to 596·8 units per mL (serotype 3; 471·9-754·8) for 180 μg. Relative to placebo, the VLA15 groups had more frequent reports of solicited local adverse events (study one: 94%, 95% CI 91-96 vs 26%, 19-34; study two: 96%, 93-98 vs 35%, 24-49 after any vaccination) and solicited systemic adverse events (study one: 69%, 65-73 vs 43%, 34-52; study two: 74%, 67-80 vs 51%, 38-64); most were mild or moderate. In study one, unsolicited adverse events were reported by 52% (48-57) of participants in the VLA15 groups and 52% (43-60) of those in the placebo groups; for study two these were 65% (58-71) and 69% (55-80), respectively. Percentages of participants reporting serious unsolicited adverse events (study one: 2%, 1-4; study two: 4%, 2-7) and adverse events of special interest (study one: 1%, 0-2; study two: 1%, 0-3) were low across all groups. A single severe, possibly related unsolicited adverse event was reported (worsening of pre-existing ventricular extrasystoles, which resolved after change of relevant concomitant medication); no related serious adverse events or deaths were reported., Interpretation: VLA15 was safe, well tolerated, and elicited robust antibody responses to all six OspA serotypes. These findings support further clinical development of VLA15 using the 180 μg dose and 0-2-6-month schedule, which was associated with the greatest immune responses., Funding: Valneva., Competing Interests: Declaration of interests NB, LW, VK, MO, NW, RH, MS, KD, SE-L, and WB are current or former employees of Valneva and may hold stock or stock options. UD, AK, and JL-S are paid consultants for Valneva., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Tick-borne infections are increasing in the United States and around the world. The most common tick-borne disease in the United States is Lyme disease caused by infection with the spirochete Borrelia burgdorferi ( Bb ), and pathogenesis varies from subclinical to severe. Bb infection is transmitted by Ixodes ticks, which can carry multiple other microbial pathogens, including Ehrlichia species. To address how the simultaneous inoculation of a distinct pathogen impacted the course of Bb -induced disease, we used C57BL/6 (B6) mice which are susceptible to Bb infection but develop only mild joint pathology. While infection of B6 mice with Bb alone resulted in minimal inflammatory responses, mice co-infected with both Bb and the obligate intracellular pathogen Ehrlichia muris ( Em ) displayed hematologic changes, inflammatory cytokine production, and emergency myelopoiesis similar to what was observed in mice infected only with Em . Moreover, infection of B6 mice with Bb alone resulted in no detectable joint inflammation, whereas mice co-infected with both Em and Bb exhibited significant inflammation of the ankle joint. Our findings support the concept that co-infection with Ehrlichia can exacerbate inflammation, resulting in more severe Bb -induced disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bonin, Torres, Marcinkiewicz, Duhamel, Yang, Pal, DiSpirito, Nowak, Lin and MacNamara.)

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC's LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance., (© 2024. The Author(s).)

Background: Modified 2-tiered testing (MTTT) for Lyme disease utilizes automatable, high throughput immunoassays (AHTIs) in both tiers without involving western immunoblots, offering performance and practical advantages over standard 2-tiered testing (STTT; first-tier AHTI followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) western immunoblots). For MTTT, Centers for Disease Control and Prevention recommends using AHTI test kits that have been cleared by Food and Drug Administration (FDA) specifically for this intended use. We evaluated performance of FDA-cleared MTTT commercial test kits from 3 manufacturers by comparing with STTT results., Methods: We performed MTTT (total antibody AHTI with reflex to separate IgM and IgG AHTIs) using test kits from Diasorin, Gold Standard Diagnostics (GSD), and Zeus Scientific on 382 excess serum samples submitted to the clinical laboratory for routine Lyme disease serologic testing in July 2018, measuring agreement between MTTT and STTT using the κ statistic., Results: Overall agreement with STTT was 0.87 (95% confidence interval [CI], .77-.97) using Diasorin assays (almost perfect agreement), 0.80 (95% CI, .68-.93) using GSD assays (substantial agreement) and 0.79 (95% CI, .68-.90) using Zeus assays (substantial agreement). For detection of IgM reactivity, agreement between MTTT and STTT was 0.70 (.51-.90; substantial), 0.63 (95% CI, .44-.82; substantial) and 0.56 (95% CI, .38-.73; moderate), respectively. For detection of IgG reactivity, MTTT/STTT agreement was 0.73 (95% CI,.58-.88), 0.78 (95% CI, .62-.94), and 0.75 (95% CI, .60-.90), respectively (substantial agreement in all cases)., Conclusions: MTTT results obtained using commercial test kits from 3 different manufacturers had substantial to almost perfect agreement with STTT results overall and moderate to substantial agreement for IgM and IgG detection independently. Commercial MTTT tests can be used broadly for the diagnosis of Lyme disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Lyme arthritis (LA) was recognized as a separate entity in 1975 because of geographic clustering of children often diagnosed with juvenile rheumatoid arthritis in Lyme, Connecticut. After identification of erythema migrans as a common early feature of the illness, a prospective study of such patients implicated Ixodes scapularis ticks in disease transmission. In 1982, the causative agent, now called Borrelia burgdorferi, was cultured from these ticks and from Lyme disease patients. Subsequently, it was shown that LA could usually be treated successfully with oral antibiotics but sometimes required intravenous antibiotics. Yet, a small percentage of patients developed a dysregulated, proinflammatory immune response leading to persistent postinfectious synovitis with vascular damage, cytotoxic and autoimmune responses, and fibroblast proliferation, a lesion similar to that of rheumatoid arthritis. The message from postinfectious LA for other autoimmune arthritides is that a complex immune response with autoimmune features can begin with a microbial infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: During infection with the Lyme arthritis (LA) pathogen Borrelia burgdorferi, T-cell responses to both host and pathogen are dysregulated, resulting in chronic infection and frequent development of autoimmunity., Methods: To assess CD4+ T-cell epitopes presented during development of LA, we used an unbiased, immunopeptidomics approach to characterize the major histocompatibility complex (MHC) class II immunopeptidome in B burgdorferi-infected C57BL/6 (B6) mice, which develop mild, self-limiting LA, and infected B6 Il10-/- mice, which develop severe, persistent LA at 0, 4, and 16 weeks postinfection (22-23 mice per group)., Results: Peptides derived from proteins involved in adaptive T- and B-cell responses and cholesterol metabolism, including human Lyme autoantigen apolipoprotein B-100 (apoB-100), were enriched in infected Il10-/- mice; whereas peptides derived from proteins involved in neutrophil extracellular net formation were enriched in infected B6 mice. Presentation of apoB-100 peptides showed evidence of epitope expansion during infection. Of several identified B burgdorferi peptides, only 1, a methyl-accepting chemotaxis protein peptide Mcp4442-462, was immunogenic., Conclusions: ApoB-100, a human Lyme autoantigen, undergoes marked epitope expansion during LA development. The paucity of immunogenic B burgdorferi epitopes supports previous findings suggesting CD4+ T-cell responses are suppressed in murine LA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Lyme disease is caused by the spirochete, Borrelia burgdorferi, which is transmitted by Ixodes spp ticks. The rise in Lyme disease cases since its discovery in the 1970s has reinforced the need for a vaccine. A vaccine based on B burgdorferi outer surface protein A (OspA) was approved by the Food and Drug Administration (FDA) several decades ago, but was pulled from the market a few years later, reportedly due to poor sales, despite multiple organizations concluding that it was safe and effective. Newer OspA-based vaccines are being developed and are likely to be available in the coming years. More recently, there has been a push to develop vaccines that target the tick vector instead of the pathogen to inhibit tick feeding and thus prevent transmission of tick-borne pathogens to humans and wildlife reservoirs. This review outlines the history of Lyme disease vaccines and this movement to anti-tick vaccine approaches., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH 15-20  and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1., (© 2024. The Author(s).)

The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn's disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi , is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.

BACKGROUNDAntibiotic-Refractory Lyme Arthritis (ARLA) involves a complex interplay of T cell responses targeting Borrelia burgdorferi antigens progressing toward autoantigens by epitope spreading. However, the precise molecular mechanisms driving the pathogenic T cell response in ARLA remain unclear. Our aim was to elucidate the molecular program of disease-specific Th cells.METHODSUsing flow cytometry, high-throughput T cell receptor (TCR) sequencing, and scRNA-Seq of CD4+ Th cells isolated from the joints of patients with ARLA living in Europe, we aimed to infer antigen specificity through unbiased analysis of TCR repertoire patterns, identifying surrogate markers for disease-specific TCRs, and connecting TCR specificity to transcriptional patterns.RESULTSPD-1hiHLA-DR+CD4+ effector T cells were clonally expanded within the inflamed joints and persisted throughout disease course. Among these cells, we identified a distinct TCR-β motif restricted to HLA-DRB1*11 or *13 alleles. These alleles, being underrepresented in patients with ARLA living in North America, were unexpectedly prevalent in our European cohort. The identified TCR-β motif served as surrogate marker for a convergent TCR response specific to ARLA, distinguishing it from other rheumatic diseases. In the scRNA-Seq data set, the TCR-β motif particularly mapped to peripheral T helper (TPH) cells displaying signs of sustained proliferation, continuous TCR signaling, and expressing CXCL13 and IFN-γ.CONCLUSIONBy inferring disease-specific TCRs from synovial T cells we identified a convergent TCR response in the joints of patients with ARLA that continuously fueled the expansion of TPH cells expressing a pathogenic cytokine effector program. The identified TCRs will aid in uncovering the major antigen targets of the maladaptive immune response.FUNDINGSupported by the German Research Foundation (DFG) MO 2160/4-1; the Federal Ministry of Education and Research (BMBF; Advanced Clinician Scientist-Program INTERACT; 01EO2108) embedded in the Interdisciplinary Center for Clinical Research (IZKF) of the University Hospital Würzburg; the German Center for Infection Research (DZIF; Clinical Leave Program; TI07.001_007) and the Interdisciplinary Center for Clinical Research (IZKF) Würzburg (Clinician Scientist Program, Z-2/CSP-30).

Background: Lyme borreliosis is a tick-borne disease caused by the bacterium Borrelia burgdorferi (Bb) sensu lato complex. Previous studies have suggested an association between Lyme borreliosis and heart failure, which have been suggested to be a possible manifestation of Lyme carditis. We aimed to investigate the risk of heart failure among individuals tested for serum Bb antibodies, and serum Bb seropositive individuals., Methods: We performed a matched nationwide cohort study (Denmark, 1993-2020) and included 52,200 Bb seropositive individuals, and two age- and sex-matched comparison cohorts: 1) 104,400 Bb seronegative comparison cohort members, and 2) 261,000 population controls. We investigated the risk associated with 1) being tested for serum Bb antibodies, and 2) being Bb seropositive. Outcomes were: 1) a composite of heart failure, cardiomyopathy, and/or myocarditis diagnosis, and 2) redemption of cardiovascular medicine used for treatment of heart failure. We calculated short-term odds ratios (aOR) (within 1 month) and long-term hazard rates (aHR) (after 1 month) adjusted for age, sex, diabetes, pre-existing heart failure, and kidney disease., Results: Compared with the population controls, individuals tested for Bb antibodies, regardless of the test result, had increased short-term risk of heart failure, cardiomyopathy, and myocarditis (aOR 8.3, 95 %CI: 6.7-10.2), and both increased short- and long-term risk of redemption of cardiovascular medicine (aOR 4.3, 95 %CI: 3.8-4.8, aHR 1.13, 95 % CI: 1.11-1.15). The Bb seropositive individuals had no increased short- or long-term risk of any outcome compared with Bb seronegative comparison cohort members., Conclusions: In conclusion, Bb antibody tests seemed to be performed in the diagnostic work-up of heart failure, but Bb seropositivity was not associated with heart failure., Competing Interests: Conflict of interest MMT has received travel grants outside this work from GlaxoSmithKline Pharma A/S. RBD has advisory board activity with Pfizer. JHS reports institutional research grant from Medtronic (outside this study), speakers’ honorarium from Medtronic and membership of an advisory board in Medtronic. AML reports speakers’ honorarium/travel grants/advisory board activity and unrestricted grant from Gilead, speakers honorarium /travel grants from GSK, speaker's honorarium/advisory board activity from Pfizer outside this work. None of the authors report any conflict of interests in connection with this article., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most common tick-borne illness in the United States. Despite the rise in Lyme disease incidence, there is no vaccine against B. burgdorferi approved for human use. Little is known about the immune correlates of protection needed to prevent Lyme disease. In this work, a mouse model was used to characterize the immune response and compare the protection provided by two USDA-approved vaccines for use in canines: Duramune (bacterin vaccine) and Vanguard crLyme (subunit vaccine composed of two outer surface proteins, OspA and OspC). C3H/HeNCrl mice were immunized with two doses of either Duramune or Vanguard, and immune responses and protection against B. burgdorferi were assessed in short (35 days) and long-term (120 days) studies. Flow cytometry, ELISPOT detection of antibody-producing cells, and antibody affinity studies were performed to identify correlates of vaccine-mediated protection. Both vaccines induced humoral responses, with high IgG titers against B. burgdorferi. However, the levels of anti-B. burgdorferi antibodies decayed over time in Vanguard-vaccinated mice. While both vaccines triggered the production of antibodies against both OspA and OspC, antibody levels against these proteins were also lower in Vanguard-vaccinated mice 120 days post-vaccination. Both vaccines only provided partial protection against B. burgdorferi at the dose used in this model. The protection provided by Duramune was superior to Vanguard 120 days post-vaccination, and was characterized by higher antibody titers, higher abundance of long-lived plasma cells, and higher avidity antibodies than Vanguard. Overall, these studies provide insights into the importance of the humoral memory response to veterinary vaccines against Lyme disease and will help inform the development of future human vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lyme arthritis can present similarly to other causes of joint pain and swelling including septic arthritis and other acute and chronic arthropathies of childhood. Septic arthritis, although rare, constitutes an orthopedic emergency and requires early surgical intervention to reduce the risk of permanent joint damage. Currently, results of standard serologic tests to diagnose Lyme disease take days to weeks, which is unhelpful in acute clinical decision-making. Thus, some children with Lyme arthritis are treated empirically for septic arthritis undergoing unnecessary invasive procedures and hospital admission while on inappropriate antibiotic therapy. We retrospectively validated the Quidel Sofia Lyme Fluorescent Immunoassay, a rapid serologic assay that can detect IgG and/or IgM antibodies to Borrelia burgdorferi in 10 minutes, in residual serum samples collected from 51 children who had Lyme arthritis and 55 children with musculoskeletal presentations who were Lyme negative. The sensitivity and specificity of the Sofia IgG to identify cases of Lyme arthritis in children were 100% (95% confidence interval [CI] of 93.0%-100%) and 96.4% (95% CI: 87.5%-99.6%), respectively. The positive likelihood ratio (LR) was 27.5 (95% CI 7-107), and the negative LR was 0.00 (95% LR 0.00-0.15). We propose that the Sofia IgG, a rapid method for identifying Lyme arthritis, may be useful in differentiating Lyme arthritis from other forms of arthritis. Used in conjunction with readily available clinical and laboratory variables, it could help to rapidly identify children who are at low risk of septic arthritis in Lyme-endemic regions., Importance: Lyme arthritis is a common manifestation of Lyme disease in children, with clinical features overlapping with other causes of acute and chronic joint pain/swelling in children. We have demonstrated that the Sofia IgG is a reliable test to rule in and rule out the diagnosis of Lyme arthritis in children with musculoskeletal presentations in a Lyme-endemic region. When used in conjunction with clinical and laboratory variables routinely considered when differentiating Lyme arthritis from other diagnoses, the Sofia IgG has the potential to fill an important gap in care, especially when acute decision-making is necessary. The Sofia IgG should be included in prospective research studies examining clinical prediction tools to identify children at low risk of septic arthritis., Competing Interests: The authors declare no conflict of interest.

Two patients, recently treated with the B-cell-depleting monoclonal antibody, rituximab, had 2-3 months of progressive systemic symptoms; comprehensive investigations did not clarify the diagnosis. Transient radicular pain at disease onset had suggested neuroborreliosis, but seronegativity and an atypical clinical course made this unlikely. However, PCR identified Borrelia burgdorferi DNA in cerebrospinal fluid, establishing the diagnosis of neuroborreliosis. Both the clinical picture and the laboratory findings can be atypical in people with neuroborreliosis who have recently been treated with rituximab. In B-cell depleted patients living in endemic areas, one should suspect neuroborreliosis even when the typical symptoms are drowned out by more atypical symptoms; PCR should be used as a diagnostic supplement when the serological response is uncertain or absent., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Multiple sclerosis (MS) is a chronic, demyelinating disease of the central nervous system that affects mainly young people. It is believed that the autoimmune process observed in the pathogenesis of MS is influenced by a complex interaction between genetic and environmental factors, including infectious agents. The results of this study suggest the protective role of Toxoplasma gondii infections in MS. Interestingly, high Toxoplasma IgM seropositivity in MS patients receiving immunomodulatory drugs (IMDs) was identified. On the other hand, Borrelia infections seem to be positively associated with MS. Although the interpretation of our results is limited by the retrospective nature of the studies, the results strongly indicate that further experimental and clinical studies are needed to explain the role of infectious agents in the development and pathophysiological mechanisms of MS., (© 2024. The Author(s).)

The incidence of Lyme borreliosis has risen, accompanied by persistent symptoms. The innate immune system and related cytokines are crucial in the host response and symptom development. We characterized cytokine production capacity before and after antibiotic treatment in 1,060 Lyme borreliosis patients. We observed a negative correlation between antibody production and IL-10 responses, as well as increased IL-1Ra responses in patients with disseminated disease. Genome-wide mapping the cytokine production allowed us to identify 34 cytokine quantitative trait loci (cQTLs), with 31 novel ones. We pinpointed the causal variant at the TLR1-6-10 locus and validated the regulation of IL-1Ra responses at transcritpome level using an independent cohort. We found that cQTLs contribute to Lyme borreliosis susceptibility and are relevant to other immune-mediated diseases. Our findings improve the understanding of cytokine responses in Lyme borreliosis and provide a genetic map of immune function as an expanded resource., (© 2024. The Author(s).)

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2024. Published by Elsevier Ltd.)

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is the most common tickborne disease. Its neuronal form, neuroborreliosis, comprises 3 to 38% of borreliosis cases in Europe. Borrelia outer surface proteins and virulence factors, OspE and BBK32, have been previously reported to help cause infection by promoting attachment to human host epithelial cells and evading complement attack. We assessed the serological responses to BBK32 and OspE in 19 individuals diagnosed with neuroborreliosis to see whether antibodies that could both target the bacteria and neutralize the virulence mechanisms on the microbial surface emerge. Results evaluate levels of total protein, IgG and the chemokine CXCL13, a determinant for B-cell recruitment during neuroinflammation, in patients' cerebrospinal fluid samples. Antibody levels against BBK32 and OspE correlated with those against VlsE, a well-characterized diagnostic serological marker of the disease. A dual serological profile of the patients was observed. K-means clustering split the cohort into two discrete groups presenting distinct serological and CNS responses. One group contained young patients with low levels of anti-BBK32 and OspE antibodies. The other group showed stronger responses, possibly following prolonged infections or reinfections. Additionally, we assessed anti-ganglioside antibodies that could cause autoimmunity or complement dysregulation but observed that they did not correlate with neuroborreliosis in our patient cohort. The dual nature of antibody responses against the virulence factors BBK32 and OspE in neuroborreliosis patients may suggest the necessity of repeated exposures for efficient immune responses. Better protection could be achieved if the virulence factors were formulated into vaccines., (© 2024 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA β-strands 19, 20, and 21, and the loops between β-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022. Published by Elsevier GmbH.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 vector–vertebrate 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 host–pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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-Năsăud) 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., (© 2021. The Author(s).)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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., Competing Interests: Richard Porwancher holds patents for bioinformatic methods for Lyme disease diagnosis (US 8,005,627 B2; US 8,694,265 B2; EP 2 084 535 B9), received previous grant support from the Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Fort Collins, CO, USA and from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Bethesda, MD, USA, and has served as a consultant to the New Jersey Board of Medical Examiners, Trenton, NJ, USA. Richard Porwancher was previously employed by and Lisa Landsberg had been a consultant to Infectious Disease Consultants, PC, 1245 Whitehorse-Mercerville Rd., Suite 411, Mercerville, NJ 08619. The above interests do not alter our adherence to PLOS ONE policies on sharing data and materials.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lyme disease is the most common tick-transmitted disease in the northern hemisphere and is caused by the spirochete Borrelia burgdorferi and related Borrelia species. The constellation of symptoms attributable to this malady results from vascular dissemination of B. burgdorferi throughout the body to invade various tissue types. However, little is known about the mechanism by which the spirochetes can breach the blood vessel wall to reach distant tissues. We have studied this process by direct observation of spirochetes in the microvasculature of living mice using multi-laser spinning-disk intravital microscopy. Our results show that in our experimental system, instead of phagocytizing B. burgdorferi, host neutrophils are involved in the production of specific cytokines that activate the endothelium and potentiate B. burgdorferi escape into the surrounding tissue. Spirochete escape is not induced by paracellular permeability and appears to occur via a transcellular pathway. Neutrophil repurposing to promote bacterial extravasation represents a new and innovative pathogenic strategy., (© 2021 John Wiley & Sons Ltd.)

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., (© 2021. Springer Nature Limited.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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.

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.

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., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

Ixodes scapularis ticks transmit multiple pathogens, including Borrelia burgdorferi sensu stricto, and encode many proteins harboring epidermal growth factor (EGF)-like domains. We show that I. scapularis produces multiple orthologs for Bm86, a widely studied tick gut protein considered as a target of an anti-tick vaccine, herein termed as Is86. We show that Is86 antigens feature at least three identifiable regions harboring EGF-like domains (termed as EGF-1, EGF-2, and EGF-3) and are differentially upregulated during B. burgdorferi infection. Although the RNA interference-mediated knockdown of Is86 genes did not show any influences on tick engorgement or B. burgdorferi sensu stricto persistence, the immunization of murine hosts with specific recombinant EGF antigens marginally reduced spirochete loads in the skin, in addition to affecting tick blood meal engorgement and molting. However, given the borderline impact of EGF immunization on tick engorgement and pathogen survival in the vector, it is unlikely that these antigens, at least in their current forms, could be developed as potential vaccines. Further investigations of the biological significance of Is86 (and other tick antigens) would enrich our knowledge of the intricate biology of ticks, including their interactions with resident pathogens, and contribute to the development of anti-tick measures to combat tick-borne illnesses.

Background: Lyme disease, caused by Borrelia burgdorferi, is a spirochetal disease. Lyme disease-related ocular findings may also provide important clues. Ocular involvement is most commonly seen as uveitis, chorioretinitis, conjunctivitis, keratitis, episcleritis, papillitis, panuveitis, ischemic optic neuropathy, papilledema, and retinal vasculitis., Case: A 27-year-old male patient was admitted with a history of fatigue, malaise, and sudden loss of vision in his left eye for 3 days. The best visual acuity was found 20/20 in the right eye and 20/400 in the left eye. Fluorescein fundus angiography showed no pathological findings in the right eye; but hyperfluorescence that was compatible with choroiditis foci was seen in the left eye. Optical coherence tomography (OCT) showed choroidal thickening in the left eye compared to the right eye. Lyme IgM antibody was found to be positive, explaining choroiditis etiology, while IgG values were found to be negative. Western blot verification test was positive. The patient was treated with 2 × 100 mg doxycycline (21 days) with a diagnosis of Lyme disease, prednol 1 mg/kg/day (10 days) for choroiditis. Omeprazole tablets were given 1 × 1 during the period of cortisone intake. On the third day of treatment, visual acuity increased to 20/200 and continued to increase until reaching 20/20 in the second week., Conclusions: Lyme disease is rare, but must be kept in mind when investigating the etiology of chorioretinitis and retinal vasculitis. The patient reported here is, to our knowledge, the second case reported in literature that shows atypical clinic for Lyme disease with unilateral chorioretinitis without Erythema chronicum migrans (ECM).

BB0405 is a surface exposed Borrelia burgdorferi protein and its vaccination protected mice against B. burgdorferi infection. As BB0405 is highly conserved across different B. burgdorferi sensu lato species, we investigated whether vaccination with recombinant BB0405 or through intradermal bb0405 DNA tattoo vaccination could provide protection against different Borrelia species, specifically against Borrelia afzelii, the predominant B. burgdorferi sensu lato genospecies causing Lyme borreliosis across Eurasia. We immunized C3H/HeN mice with recombinant BB0405 or with a codon-optimized bb0405 DNA vaccine using the pVAC plasmid and immunized corresponding control groups mice with only adjuvant or empty vectors. We subsequently subjected these immunized mice to a tick challenge with B. afzelii CB43-infected Ixodes ricinus nymphs. Upon vaccination, recombinant BB0405 induced a high total IgG response, but bb0405 DNA vaccination did not elicit antibody responses. Both vaccine formulations did not provide protection against Borrelia afzelii strain CB43 after tick challenge. In an attempt to understand the lack of protection of the recombinant vaccine, we determined expression of BB0405 and showed that B. afzelii CB43 spirochetes significantly and drastically downregulate the expression of BB0405 protein at 37 °C compared to 33 °C, where as in B. burgdorferi B31 spirochetes expression levels remain unaltered. Vaccination with recombinant BB0405 was previously shown to protect against B. burgdorferi sensu stricto. Here we show that vaccination with either recombinant BB0405 (or non-immunogenic bb0405 DNA), despite being highly conserved among B. burgdorferi sl genospecies, does not provide cross-protection against B. afzelii, mostly likely due to downregulation of this protein in B. afzelii in the mammalian host.

Introduction: Borrelia burgdorferi sensu lato (sl) is the causative agent of Lyme borreliosis. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines. DNA tattoo vaccination with B. afzelii strain PKo OspC in mice has proven to be fully protective against B. afzelii syringe challenge and induces a favorable humoral immunity compared to recombinant protein vaccination. Alternatively, several recombinant protein vaccines based on tick proteins have shown promising effect in tick-bite infection models. In this study, we evaluated the efficacy of DNA vaccines against Borrelia OspC or tick antigens in a tick-bite infection model., Method: We vaccinated C3H/HeN mice with OspC using a codon-optimized DNA vaccine or with recombinant protein. We challenged these mice with B. burgdorferi sensu stricto (ss)-infected Ixodes scapularis nymphs. Subsequently, we vaccinated C3H/HeN mice with DNA vaccines coding for tick proteins for which recombinant protein vaccines have previously resulted in interference with tick feeding and/or Borrelia transmission: Salp15, tHRF, TSLPI, and Tix-5. These mice were also challenged with B. burgdorferi ss infected Ixodes scapularis nymphs., Results: DNA tattoo and recombinant OspC vaccination both induced total IgG responses. Borrelia cultures and DNA loads of skin and bladder remained negative in the mice vaccinated with OspC DNA vaccination, except for one culture. DNA vaccines against tick antigens Salp15 and Tix-5 induced IgG responses, while those against tHRF and TSLPI barely induced any IgG response. In addition, Borrelia cultures, and DNA loads from mice tattooed with DNA vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 were all positive., Conclusion: A DNA tattoo vaccine against OspC induced high specific IgG titers and provided near total protection against B. burgdorferi ss infection by tick challenge. In contrast, DNA tattoo vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 induced low to moderate IgG titers and did not provide protection. Therefore, DNA tattoo vaccination does not seem a suitable vaccine strategy to identify, or screen for, tick antigens for anti-tick vaccines. However, DNA tattoo vaccination is a straightforward and effective vaccination platform to assess novel B. burgdorferi sl antigen candidates in a relevant tick challenge model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Klouwens, Trentelman, Wagemakers, Ersoz, Bins and Hovius.)

Objective: Cerebral microbleeds (CMBs) is a subtype of cerebral small vessel disease. Their underlying pathogenesis remains unclear. The aim of this study was to investigate the association between infectious burden (IB) and CMBs., Methods: Seven hundred and seventy-three consecutive patients who were hospitalized in the Department of Neurology in General Hospital of Western Theater Command without severe neurological symptoms were recruited and selected in this pilot cross-sectional study. CMBs were assessed using the susceptibility-weighted imaging sequence of magnetic resonance imaging. Immunoglobulin G antibodies against common pathogens, including herpes simplex virus (HSV)-1, HSV-2, cytomegalovirus (CMV), Chlamydia pneumoniae (C. pneumoniae), Mycoplasma pneumoniae (M. pneumoniae), Epstein-Barr virus (EBV), Helicobacter pylori (HP), and Borrelia burgdorferi (B. burgdorferi), were measured by commercial ELISA assays. IB was defined as a composite serologic measure of exposure to these common pathogens., Results: Patients with and without CMBs were defined as the CMBs group (n = 76) and the non-CMBs group (n = 81), respectively. IB was significantly different between the CMBs and non-CMBs groups. After adjusted for other risk factors, the increased IB was independently associated with the presence of CMBs (P = 0.031, OR = 3.00, 95% CI [1.11-8.15]). IB was significantly positively associated with the number of CMBs (Spearman ρ = 0.653, P < 0.001). The levels of serum inflammatory markers were significantly different between the CMBs and non-CMBs groups and among the categories of IB., Interpretation: IB consisting of HSV-1, HSV-2, CMV, C. pneumoniae, M. pneumoniae, EBV, HP, and B. burgdorferi was associated with CMBs. All the findings suggested that pathogen infection could be involved in the pathogenesis of CMBs., (© 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Background: Dogs in the US are commonly infected with vector-borne pathogens, including heartworm and tick-borne disease agents. The geographic distribution of both arthropod vectors and the pathogens they transmit continues to expand., Methods: To describe the current geographic distribution and prevalence of antigen of Dirofilaria immitis and antibody to Borrelia burgdorferi, Ehrlichia spp., and Anaplasma spp. in dogs, we summarized over 144 million test results from 2013 to 2019, inclusive, by county, state, and region. Canine seroprevalence by state was compared to population-adjusted human reports of tick-borne diseases., Results: Results varied regionally, with D. immitis antigen and Ehrlichia spp. antibodies more frequently detected in the Southeast (2.6% and 5.2%, respectively) and antibody to B. burgdorferi and Anaplasma spp. most common in the Northeast (12.1% and 7.3%, respectively). Overall, percent positive test results to D. immitis decreased in the Southeast by 33.3% when compared to earlier summaries using the same strategy (from 3.9 to 2.6%). Geographic expansion of areas where dogs commonly test positive for Ehrlichia spp. was evident, likely because of a change in the test made in 2012 to allow detection of antibodies to E. ewingii concomitant with expansion of vector tick populations. Percent positive test results to Ehrlichia spp. increased in every region; this shift was particularly pronounced in the Southeast, where percent positive test results increased fourfold (from 1.3 to 5.2%). Continued geographic expansion of B. burgdorferi and A. phagocytophilum was apparent in the Northeast, Midwest, and Upper South, although canine seroprevalence of antibody to B. burgdorferi was much lower than prior surveys in many Lyme-endemic areas. Annual reports of human cases of Lyme disease, ehrlichiosis, and anaplasmosis were associated with percent positive canine results by state for the three tick-borne disease agents (R 2  = 0.812, 0.521, and 0.546, respectively). Within endemic areas, percent positive test results for all three tick-borne agents demonstrated evidence of geographic expansion., Conclusions: Large scale analysis of results from screening dogs in practice for evidence of vector-borne infections, including those with zoonotic importance, continues to be a valuable strategy for understanding geographic trends in infection risk over time.

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo., Competing Interests: The authors have declared that no competing interests exist.