Antibiotic Treatment-Resistant Lyme Arthritis (TRLA) An Autoimmune Disease? NIH 2/3/04

Clinical Features Arthritic knee Late Lyme Disease (Single joint) Ixodes tick Borrelia burgdorferi (Bb) Erythema migrans Erythema migrans (localized and multiple) Flu-like illness Early Lyme Disease Early Disseminated Lyme Disease Neurologic – cranial neuropathy, meningitis, radiculoneuropathy Joint – Acute, inflammatory large joint arthritis Carditis Late Lyme Disease Neurologic – peripheral neuropathy, encephalopathy Chronic arthritis LD progresses thru distinct clinical phases – early lyme disease (EM and flu like illness)  early dissemnated lyme disease (systemic toxicity like fever, chills, secondary skin lesions, cardiac, neurological and joint involvement) late lyme diseasethis is just in genetically susceptible indiv. (chronic inflammation of jts, as well as nervous system)

Acute Inflammatory vs. Chronic Arthritis Develops after a prolonged period of latency/minimally symptomatic disease Defined as one year or more of persistent joint inflammation, usually the knee Does not subside in response to antibiotic therapy Correlates with the development of a strong immune response to OspA (Outer Surface Protein A) Early attacks - within weeks to a few months after disease onset Sudden pain, swelling with massive effusions Mostly intermittent Remits after a few days/weeks sometimes without antibiotic therapy Experience recurrent attacks in same joint. 3 or fewer joints involved overall. In minority of patients, infection can be controlled by host immune response. Majority are cured by antibiotics. But in a minority of patients, the illness becomes chronic Treatment Resistant Lyme Arthritis (TRLA)

Osp Expression during Transmission 1. unfed tick 2. feeding tick, from gut to salivary gland 3. recently infected host A+/C- A-/C- A-/C+ A+/C+

Evidence for OspA Upregulation In Vivo Bb transmitted to the host express little or no OspA. Later in the course of infection, patients develop strong spiking titers of OspA antibodies coinciding with periods of arthritis (Kalish 1995). If these responses indicate OspA upregulation, then are inflammatory cues stimulating OspA expression by Bb?

Western Blot Analysis of Osp Expression In vitro vs. In vivo vs. In vivo-Inflamed Bb, in vivo chamber-grown, zymosan-induced inflammation Bb, in vitro chamber-grown Bb, in vivo chamber-grown Flagellin, 41 kD OspA, 31 kD OspC, 21 kD

Prolonged Bb infection for the development of TRLA Antibiotic treatment in the first couple of weeks after exposure eliminates development of TRLA Antibiotic treatment later on has no effect => Initial exposure to spirochetes required

Autoimmune basis for TRLA Synovial samples are PCR-negative for B.burgdorferi DNA after antibiotic treatment1 Increased frequency of HLA-DR4 alleles: HLA-DRB1*0401, 0404 and 0101, 0102 in the affected population2 Bb rarely cultured from SF/tissue, DNA readily detected. Oral/iv/both antibiotics for a median of 8 weeks. Median of 7 mo from therapy to surgery. All patients had some kind of knee swelling. Pcrs done on both tissue and fluid. Can detect 1-10 bb in spiked samples, but not in real ones. [OspA & OspB: plasmid encoded; Flagellar protein: chromosome encoded] 1. Joint inflammation may persist after the apparent eradication of spirochetes with therapy 2. Within a HLA-DR4 population, Osp-reactive patients had a significantly longer duration of arthritis, post-treatment, than Osp non-reactive patients 3. Esp in dr4 patients. So a dr4 patient who’s seropositive for ospA has a significantly ihgher arthritis duration than a sero-neg patient of same haplo. Say – historically, the first correlation with the shift was made wrt to dev of the response 1.Carlson et al., Arthritis & Rheumatism 42(12) 1999 2.Steere et al., New Engl Journal of Med.161 1990

Molecular mimicry with OspA? Strong T-cell response to OspA: 1. OspA-reactive T-cells in synovial fluid1 2. Human LFA-1 candidate T-cell autoantigen2 1.Meyer et al. PNAS 97(21), 2000 2. Gross et al. Science 281, 1998 A Shift in Paradigm: 1. Linked T-B recognition of autoantigen (GPI) – RA mouse model 2. Ectopic germinal centers in RA synovium 1.Matsumoto et al.Science 286,1999 2. Kim et al. J Immunol. 162(5), 1999 Strong B-cell response to OspA

Generation of a strong OspA response Pathogenic Antibodies Model Generation of a strong OspA response Antibiotics B T OspA Structurally related -migration  development of a strong innate response to Bb, the adaptive response picks up  spirochetal clearance. OspA response develops late in the course of disease. Most people treated with antibiotics will go on to clear disease. However, subset of patients, the arthritis persists after apparent clearance of the pathogen. We hypo. That b-T cells that got activated activated during the original inflammatory insult persist in the lesions, receive inappropriate activatation signals from autoantigen. Favorite model is the linked B-T recogntion of a molecular mimic of OspA  pathogenic antibodies  mediate joint damage Tihs model would be the closest to the mathis model; also would explain both the ospA ab titers as well as the hla-dr restirction of trla Molecular Mimicry B T Pathogenic Antibodies Chronic arthritis

Antibodies in Lyme Arthritis 1. Increased Titers of OspA antibodies IgG response to OspA develops later in the course of infection High titers of OspA antibodies coincide with periods of maximal arthritis Mark the transition from episodic to chronic arthritis Persist after treatment 2. Germinal centers in inflamed synovium 2222222 Tightly intermixed B-& T-cells Follicular dendritic cells Activated germinal center B-cells Plasma cells In LD lit., historically, antibody reactivity to various bb proteins during the disease and attempted to make correlations with the clinical course and outcome. Historically, most of the early studies followed antibody reactivity to various bb proteins during the disease and attempted to make correlations with the clinical course and outcome. Observation that Hla-dr specificity same as RA and the prevailing view that chronic arthritides are mainly t-cell mediated. Most work, esp in lab focussed on charac t-cell responses to ospA and linkage with autoimmunity, molecularly mimicry etc. at the t-cell level. Like RA, trla is a chronic arthritic disease - C1q binding used as marker for active disease – essentially a read-out for levels of complement fixing immune complexes. Gradual shift/accumulation of this activity in SF, as arth more chronic – makes sense given that bb has tropism for the joints and initiates a local immune response Kalish et al., Infection and Immunity 61(7), 1993 Akin et al., Infection and Immunity, 67(1),1998 Steere et al., Arthritis and Rheumatism, .31 (4), 1988

Isolation of Ig from single B cells Stepwise introduction of somatic mutations

Patient ID: DC Sample ID: 3-1 Rearranged kappa light chain Common CDR3 Common CDR3

Patient ID: DC Sample ID: 2-1 Rearranged gamma chain

Patient ID: DC Sample ID: 2-1 Rearranged kappa light chain SS subs close to cdr2 and QT sus close to cdr3

OspA vaccine Is it a problem?

Modification of Main OspA T cell Epitope: Minimize Binding to HLA-DRB1 Protein Sequence DR binding B.b. B31-OspA 165-173 YVLEGTLTA + 6.5 hLFA-1a L332-340 YVIEGTSKQ + 7.3 B. afzelii OspA 165-173 FTLEGKVAN - 1.3 FTK-OspA 165-173 FTLEGKLTA + 0.2 position 1 2 3 4 5 6 7 8 9

Allen C. Steere B. David Stollar Jenifer Coburn Acknowledgements Theresa Willett Helena Crowley Srimoyee Ghosh Allen C. Steere B. David Stollar Jenifer Coburn