Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme？) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.
Steere, A.C. Lyme disease. N. Engl. J. Med. 2001, 345, 115–125, doi:10.1056/NEJM200107123450207.
[3]
Steere, A.C.; Coburn, J.; Glickstein, L. The emergence of Lyme disease. J. Clin. Invest. 2004, 113, 1093–1101.
[4]
Centers for Disease Control and Prevention. Reported Cases of Lyme Disease by Year, United States, 2002–2011. Available online: http://www.cdc.gov/lyme/stats/chartstables/casesbyyear.html (accessed on 1 July 2013).
[5]
Young, J.D. Underreporting of Lyme disease. N. Engl. J. Med. 1998, 338, 1629, doi:10.1056/NEJM199805283382216.
[6]
Gross, D.M.; Huber, B.T. Cellular and molecular aspects of Lyme arthritis. Cell. Mol. Life Sci. 2000, 57, 1562–1569, doi:10.1007/PL00000641.
Chandra, A.; Wormser, G.P.; Klempner, M.S.; Trevino, R.P.; Crow, M.K.; Latov, N.; Alaedini, A. Anti-neural antibody reactivity in patients with a history of Lyme borreliosis and persistent symptoms. Brain Behav. Immun. 2010, 24, 1018–1024, doi:10.1016/j.bbi.2010.03.002.
[9]
Ramesh, G.; Benge, S.; Pahar, B.; Philipp, M.T. A possible role for inflammation in mediating apoptosis of oligodendrocytes as induced by the Lyme disease spirochete Borrelia burgdorferi. J. Neuroinflammation 2012, 9, 72, doi:10.1186/1742-2094-9-72.
[10]
Aucott, J.; Morrison, C.; Munoz, B.; Rowe, P.C.; Schwarzwalder, A.; West, S.K. Diagnostic challenges of early Lyme disease: lessons from a community case series. BMC. Infect. Dis. 2009, 9, 79, doi:10.1186/1471-2334-9-79.
[11]
Dattwyler, R.J.; Volkman, D.J.; Halperin, J.J.; Luft, B.J.; Thomas, J.; Golightly, M.G. Specific immune responses in Lyme borreliosis. Characterization of T cell and B cell responses to Borrelia burgdorferi. Ann. N. Y. Acad. Sci. 1988, 539, 93–102, doi:10.1111/j.1749-6632.1988.tb31842.x.
[12]
Krause, A.; Burmester, G.R.; Rensing, A.; Schoerner, C.; Schaible, U.E.; Simon, M.M.; Herzer, P.; Kramer, M.D.; Wallich, R. Cellular immune reactivity to recombinant OspA and flagellin from Borrelia burgdorferi in patients with Lyme borreliosis. Complexity of humoral and cellular immune responses. J. Clin. Invest. 1992, 90, 1077–1084, doi:10.1172/JCI115923.
Seriburi, V.; Ndukwe, N.; Chang, Z.; Cox, M.E.; Wormser, G.P. High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice. Clin. Microbiol. Infect. 2012, 18, 1236–1240.
[15]
Brown, S.L.; Hansen, S.L.; Langone, J.J. Role of serology in the diagnosis of Lyme disease. JAMA 1999, 282, 62–66, doi:10.1001/jama.282.1.62.
[16]
Brunner, M. New method for detection of Borrelia burgdorferi antigen complexed to antibody in seronegative Lyme disease. J. Immunol. Methods. 2001, 249, 185–190, doi:10.1016/S0022-1759(00)00356-2.
[17]
Tylewska-Wierzbanowska, S.; Chmielewski, T. Limitation of serological testing for Lyme borreliosis: evaluation of ELISA and western blot in comparison with PCR and culture methods. Wien. Klin. Wochenschr. 2002, 114, 601–605.
[18]
Forsberg, P.; Ernerudh, J.; Ekerfelt, C.; Roberg, M.; Vrethem, M.; Bergstrom, S. The outer surface proteins of Lyme disease borrelia spirochetes stimulate T cells to secrete interferon-gamma (IFN-gamma): Diagnostic and pathogenic implications. Clin. Exp. Immunol. 1995, 101, 453–460.
[19]
Horowitz, H.W.; Pavia, C.S.; Bittker, S.; Forseter, G.; Cooper, D.; Nadelman, R.B.; Byrne, D.; Johnson, R.C.; Wormser, G.P. Sustained cellular immune responses to Borrelia burgdorferi: Lack of correlation with clinical presentation and serology. Clin. Diagn. Lab. Immunol. 1994, 1, 373–378.
[20]
Nordberg, M.; Forsberg, P.; Nyman, D.; Skogman, B.H.; Nyberg, C.; Ernerudh, J.; Eliasson, I.; Ekerfelt, C. Can ELISPOT be applied to a clinical setting as a diagnostic utility for Neuroborreliosis? Cells 2012, 1, 153–167, doi:10.3390/cells1020153.
[21]
Aguero-Rosenfeld, M.E.; Nowakowski, J.; Bittker, S.; Cooper, D.; Nadelman, R.B.; Wormser, G.P. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. J. Clin. Microbiol. 1996, 34, 1–9.
[22]
Dressler, F.; Yoshinari, N.H.; Steere, A.C. The T-cell proliferative assay in the diagnosis of Lyme disease. Ann. Intern. Med. 1991, 115, 533–539, doi:10.7326/0003-4819-115-7-533.
[23]
Lastavica, C.C.; Snydman, D.R.; Schenkein, D.P.; Berardi, V.P.; Pariser, K.M. Demonstration of Borrelia burgdorferi in a patient with chronic Lyme arthritis. Zentralbl. Bakteriol. Mikrobiol. Hyg. A 1986, 263, 288.
[24]
Dattwyler, R.J.; Volkman, D.J.; Luft, B.J.; Halperin, J.J.; Thomas, J.; Golightly, M.G. Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi. N. Engl. J. Med. 1988, 319, 1441–1446, doi:10.1056/NEJM198812013192203.
[25]
Hilton, E.; Tramontano, A.; DeVoti, J.; Sood, S.K. Temporal study of immunoglobin M seroreactivity to Borrelia burgdorferi in patients treated for Lyme borreliosis. J. Clin. Microbiol. 1997, 35, 774–776.
[26]
Kalish, R.A.; McHugh, G.; Granquist, J.; Shea, B.; Ruthazer, R.; Steere, A.C. Persistence of immunoglobulin M or immunoglobulin G antibody responses to Borrelia burgdorferi 10–20 years after active Lyme disease. Clin. Infect. Dis. 2001, 33, 780–785, doi:10.1086/322669.
[27]
Hagen, J.; Houchins, J.P.; Kalyuzhny, A.E. Combining ELISPOT and ELISA to measure amounts of cytokines secreted by a single cell. In Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, 2nd ed.; Kalyuzhny, A., Ed.; Springer Science+Business Media, LLC: New York, NY, USA, 2012; Volume 792, pp. 115–122.
[28]
Lehmann, P.V.; Zhang, W. Unique Strengths of ELISPOT for T Cell Diagnostics. In Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, 2nd ed.; Kalyuzhny, A., Ed.; Springer Science+Business Media, LLC: New York, NY, USA, 2012; Volume 792, pp. 3–23.
[29]
Tary-Lehmann, M.; Hamm, C.D.; Lehmann, P.V. Validating Reference Samples for Comparison in a Regulated ELISPOT Assay. In Validation of Cell-Based Assays in the GLP Setting: A Practical Guide, 1st ed.; Prabhakar, U., Kelley, M., Eds.; John Wiley & Sons, Ltd.: West Sussex, UK, 2008; pp. 127–146.
[30]
Lalvani, A.; Pathan, A.A.; Durkan, H.; Wilkinson, K.A.; Whelan, A.; Deeks, J.J.; Reece, W.H.; Latif, M.; Pasvol, G.; Hill, A.V. Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet 2001, 357, 2017–2021, doi:10.1016/S0140-6736(00)05115-1.
[31]
Lalvani, A. Counting antigen-specific T cells: A new approach for monitoring response to tuberculosis treatment? Clin. Infect. Dis. 2004, 38, 757–759, doi:10.1086/381763.
[32]
Centers for Disease Control and Prevention. Signs and Symptoms of Lyme Disease, United States. 2013. Available online: http://www.cdc.gov/lyme/signs_symptoms/index.html (accessed on 1 July 2013).
[33]
Wormser, G.P.; Dattwyler, R.J.; Shapiro, E.D.; Halperin, J.J.; Steere, A.C.; Klempner, M.S.; Krause, P.J.; Bakken, J.S.; Strle, F.; Stanek, G.; et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: Clinical practice guidelines by the Infectious Diseases Society of America. Clin. Infect. Dis. 2006, 43, 1089–1134.
[34]
Gross, D.M.; Forsthuber, T.; Tary-Lehmann, M.; Etling, C.; Ito, K.; Nagy, Z.A.; Field, J.A.; Steere, A.C.; Huber, B.T. Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 1998, 281, 703–706, doi:10.1126/science.281.5377.703.
[35]
Widhe, M.; Jarefors, S.; Ekerfelt, C.; Vrethem, M.; Bergstrom, S.; Forsberg, P.; Ernerudh, J. Borrelia-Specific interferon-gamma and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome. J. Infect. Dis. 2004, 189, 1881–1891, doi:10.1086/382893.
[36]
Widhe, M.; Skogman, B.H.; Jarefors, S.; Eknefelt, M.; Enestrom, G.; Nordwall, M.; Ekerfelt, C.; Croner, S.; Bergstrom, S.; Forsberg, P.; et al. Up-regulation of Borrelia-specific IL-4- and IFN-gamma-secreting cells in cerebrospinal fluid from children with Lyme neuroborreliosis. Int. Immunol. 2005, 17, 1283–1291, doi:10.1093/intimm/dxh304.
[37]
Zhang, W.; Tary-Lehmann, M. Objective, User-Independent ELISPOT Data Analysis Based on Scientifically Validated Principles. In Validation of Cell-Based Assays in the GLP Setting: A Practical Guide, 1st ed.; Prabhakar, Uma, Kelley, Marian, Eds.; John Wiley & Sons, Ltd.: West Sussex, UK, 2008; pp. 126–147.
[38]
Ekerfelt, C.; Forsberg, P.; Svenvik, M.; Roberg, M.; Bergstrom, S.; Ernerudh, J. Asymptomatic Borrelia-seropositive individuals display the same incidence of Borrelia-specific interferon-gamma (IFN-gamma)-secreting cells in blood as patients with clinical Borrelia infection. Clin. Exp. Immunol. 1999, 115, 498–502, doi:10.1046/j.1365-2249.1999.00840.x.
[39]
Zhang, W.; Caspell, R.; Karulin, A.Y.; Ahmad, M.; Haicheur, N.; Abdelsalam, A.; Johannesen, K.; Vignard, V.; Dudzik, P.; Georgakopoulou, K.; et al. LISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators. J. Immunotoxicol. 2009, 6, 227–234, doi:10.3109/15476910903317546.
[40]
Hesse, M.D.; Karulin, A.Y.; Boehm, B.O.; Lehmann, P.V.; Tary-Lehmann, M. A T cell clone's avidity is a function of its activation state. J. Immunol. 2001, 167, 1353–1361.
[41]
Fardy, J. Evaluation of Diagnositc Tests. In Methods of Molecular Biology, Clinical Epidemiology; Parfrey, P., Barrett, B., Eds.; Humana Press: Totowa, NJ, USA, 2009; Volume 473, pp. 127–136.
