Klemen Strle

Timely and accurate diagnosis of Lyme disease (LD) remains limited by complicated serological testing, which requires specialized laboratory equipment and has a multi-day turnaround, thereby limiting its utility in point-of-care (POC) settings. We have previously demonstrated a grating-coupled fluorescent plasmonic (GC-FP) biosensor in a microfluidics-based format (MF) for serological detection of LD. In this work, we demonstrate translation of an MF to a simplified dipstick assay. The assay utilizes miniaturized 4×4 mm gold-coated chips with a nanoscale grating that enhances fluorescence through surface plasmon-coupled emission (SPCE). The unique dipstick-based (DS) workflow eliminates the need for microfluidic pumps and complex instrumentation and has a total assay time of 24 min. The dipstick format integrates both tiers of the standard two-tiered diagnostic algorithm on a single chip, enabling simultaneous detection of first-tier screening antigen C6 and second-tier confirmatory antigens VlsE, DbpA, and DbpB. Validation was performed using thirty human serum samples tested in DS GC-FP chips, MF GC-FP chips, and a commercially available FDA-cleared LD diagnostic assay (ZEUS Borrelia MTTT®). All thirty human samples were correctly classified across all platforms, yielding 100% agreement in final diagnostic outcomes. The dipstick format substantially reduces equipment requirements, material costs, and operational complexity while preserving diagnostic accuracy, making it well-suited for deployment in urgent care centers, physician offices, and resource-limited settings.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a zoonosis caused by several species of Borrelia burgdorferi sensu lato and transmitted by the bite of infected ticks of the Ixodes ricinus complex. Lyme borreliosis in North America and Europe differs in certain respects, likely reflecting the different Borrelia species that cause human disease in these locations. The earliest manifestation of Lyme borreliosis is the skin lesion erythema migrans, which develops at the tick bite site, typically 7-14 days after the bite. Some untreated patients will then (within the first few weeks or months after onset of the infection) develop additional erythema migrans skin lesions or other clinical manifestations such as borrelial lymphocytoma, nervous system involvement or carditis. Several months or even years after infection onset, Lyme arthritis or acrodermatitis chronica atrophicans may develop. The diagnosis of typical erythema migrans is clinical, whereas for all other manifestations the diagnosis is supported via serological testing. Treatment with an appropriate antibiotic will result in resolution of clinical symptoms in most patients; however, some patients experience prolonged subjective symptoms, which usually improve over time. Repeated courses of antimicrobials are not beneficial except in rare cases when there is objective evidence of treatment failure.

Lyme disease is caused by an infection with the spirochete Borrelia burgdorferi, and is the most common vector-borne disease in North America. B. burgdorferi isolates harbor extensive genomic and proteomic variability and further comparison of isolates is key to understanding the infectivity of the spirochetes and biological impacts of identified sequence variants. Here, we applied both transcriptome analysis and mass spectrometry-based proteomics to assemble peptide datasets of B. burgdorferi laboratory isolates B31, MM1, and the infective isolate B31-5A4, to provide a publicly available Borrelia PeptideAtlas. Included are total proteome, secretome, and membrane proteome identifications of the individual isolates. Proteomic data collected from 35 different experiment datasets, totaling 386 mass spectrometry runs, have identified 81,967 distinct peptides, which map to 1,113 proteins. The Borrelia PeptideAtlas covers 86% of the total B31 proteome of 1,291 protein sequences. The Borrelia PeptideAtlas is an extensible comprehensive peptide repository with proteomic information from B. burgdorferi isolates useful for Lyme disease research.

Background: Postinfectious Lyme arthritis (LA) is associated with dysregulated immunity and autoreactive T- and B-cell responses in joints. Here we explored the role of host genetic variation in this outcome.Methods: The frequency of 253 702 single-nucleotide polymorphisms (SNPs) was determined in 147 patients with LA (87 with postinfectious LA and 60 with antibiotic-responsive LA), and for comparison in 90 patients with erythema migrans or the general population (n = 2504). Functional outcome of candidate SNPs was assessed by evaluating their impact on clinical outcome and on immune responses in blood and synovial fluid in patients with LA.Results: Six SNPs associated with late cornified envelope (LCE3) genes were present at greater frequency in patients with postinfectious LA compared to those with antibiotic-responsive LA (70% vs 30%; odds ratio, 2; P < .01). These SNPs were associated with heightened levels of inflammatory Th17 cytokines in serum but lower levels of interleukin 27, a regulatory cytokine, implying that they may contribute to dysregulated Th17 immunity in blood. Moreover, in patients with postinfectious LA, the levels of these Th17 mediators correlated directly with autoantibody responses in synovial fluid, providing a possible link between LCE3 SNPs, maladaptive systemic Th17 immunity, and autoreactive responses in joints.Conclusions: Variation in the LCE3 locus, a known genetic risk factor in psoriasis and psoriatic arthritis, is associated with dysregulated systemic Th17 immunity and heightened autoantibody responses in joints. These findings underscore the importance of host genetic predisposition and systemic Th17 immunity in the pathogenesis of postinfectious (antibiotic-refractory) Lyme arthritis.

Background: HLA-DR-expressing fibroblast-like synoviocytes (FLS) are a prominent cell type in synovial tissue in chronic inflammatory forms of arthritis. We recently showed that peptides from several extracellular matrix (ECM) proteins, including fibronectin-1 (FN1), contained immunogenic CD4+ T cell epitopes in patients with postinfectious Lyme arthritis (LA). However, the role of FLS in presentation of these T cell epitopes remains uncertain. Methods: Primary LA FLS and primary murine FLS stimulated with interferon gamma (IFNγ), Borrelia burgdorferi , and/or B. burgdorferi peptidoglycan (PG) were assessed for properties associated with antigen presentation. HLA-DR-presented peptides from stimulated LA FLS were identified by immunopeptidomics analysis. OT-II T cells were cocultured with stimulated murine FLS in the presence of cognate ovalbumin antigen to determine the potential of FLS to act as inducible antigen presenting cells (APC). Results: FLS expressed HLA-DR molecules within inflamed synovial tissue and tendons from patients with post-infectious LA patients in situ. MHC class II and costimulatory molecules were expressed by FLS following in vitro stimulation with IFNγ and B. burgdorferi and presented both foreign and self MHC-II peptides, including T cell epitopes derived from two Lyme autoantigens fibronectin-1 (FN1) and endothelial cell growth factor (ECGF). Stimulated murine FLS induced proliferation of naïve OT-II CD4+ T cells, particularly when FLS were stimulated with both IFNγ and PG. Conclusions: MHC-II+ FLS are inducible APCs that can induce CD4+ T cell activation and can present Lyme autoantigens derived from ECM proteins, thereby amplifying tissue-localized autoimmune CD4+ T cell responses in LA. AUTHORS’ SUMMARY: This study demonstrates that IFNγ-activated MHC-II+ fibroblast-like synoviocytes (FLS) stimulated with Borrelia burgdorferi present foreign and self MHC-II antigens, including Lyme autoantigens. Furthermore, IFNγ-activated MHC-II+ FLS stimulated with B. burgdorferi peptidoglycan can induce activation and proliferation of naïve CD4+ T cells in an MHC-II antigen-dependent manner, demonstrating that activated MHC-II+ FLS are inducible antigen presenting cells.

BACKGROUND. Autoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR–presented peptides, and therefore the reasons for these associations, has frequently remained elusive. METHODS. Using immunopeptidomics to detect HLA-DR–presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi–mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins. RESULTS. Of 24 postinfectious LA patients, 58% had CD4⁺ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4⁺ T cells in synovial fluid were T-bet–expressing Th1 cells, a small percentage were RoRγt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Tregs. CONCLUSION. Autoreactive, proinflammatory CD4⁺ T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity. FUNDING. Supported by National Institute of Allergy and Infectious Diseases R01-AI101175, R01-AI144365, and F32-AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01-AR062098 and T32-AR007258; NIH grants P41-GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital.

Lyme disease, caused by an infection with the spirochete Borrelia burgdorferi , is the most common vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and proteomic variability and further comparison is key to understanding the spirochetes infectivity and biological impacts of identified sequence variants. To achieve this goal, both transcript and mass spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public datasets, to provide a publicly available Borrelia PeptideAtlas ( http://www.peptideatlas.org/builds/borrelia/ ). Included is information on total proteome, secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The diverse proteomic information from multiple isolates with credible data presented by the Borrelia PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective isolates and may be key in the infection process.

This form of Lyme neuroborreliosis is a distinct clinical entity with specific signs and symptoms.