Patients and controls
Samples were obtained from nineteen adult patients from an endemic area enrolled in ongoing Lyme disease studies at the Lyme Disease Center at the State University New York at Stony Brook, NY and at New York Medical College, Valhalla, NY. Sixteen of these patients presented with early localized infection typified by the presence of well defined erythema migrans (EM). They had no other signs of disseminated infection. None of these patients developed Lyme arthritis. Seven patients were evaluated within 1 month of EM presentation (acute) and nine different patients at 3 months post EM presentation (convalescent). Also, three patients presenting with Lyme disease symptoms (EM or arthralgia, mialgia) that had been previously vaccinated with recombinant OspA were tested. All patients were treated with suitable antibiotics at the time of diagnosis. We did not test antibiotic resistant Lyme arthritis patients because we didn't have any in that category during the course of the study. We don't see those patients often in either Long Island or Westchester County, highly endemic areas for Lyme disease. Twenty-one healthy adults from the same endemic area, with no prior history of Lyme disease and no prior serologic screening, were recruited as controls. Whole blood samples were drawn from each enrolled patient into a heparinized tube. The blood was kept at room temperature and used within 24 h after it was obtained.
Antigens
Two synthetic, twenty amino acid peptides, B. burgdorferi OspAB31 (aa164-183), GYVLEGTLTAEKTTLVVKEG and hLFAαL (aa326-345), acetyl-ELQKKIYVIEGTSKQDLTSF, were custom contracted through the Small Scale Peptide Synthesis laboratory at the HHMI Biopolymer / Keck Foundation Biotechnology Resource Laboratory (Yale University School of Medicine, CT). Suitably protected peptide chains were constructed in an automated, stepwise solid-phase protocol using FMOC N-protection for N-alpha protection at each cycle. The peptides were all synthesized on substituted benzhydrylamine-resins to yield amidated C-termini. The amide group is a better approximation of peptide bonds than are free carboxyl termini.
Since T cell receptors recognize structures rather than amino acid sequences and both peptides of 20 residues are about 70% different we did not use an unrelated peptide as control. Borrelia whole cell sonicate derived from B. afzelii, strain Pko, was developed by CSL Biosciences (Melbourne, Australia) in collaboration with CDC (Fort Collins, Colorado, USA), as a stimulating antigen for the T cell Lyme assay. This antigen was found to have a higher stimulation rate in blood from Lyme disease patients than that of B. burgdorferi sonicate used in [8]. This antigen was kindly provided by Dr. Roland Martin (CSL Biosciences) and was used as an internal control to check the performance of the assay.
T-Cell assay
We used the QuantiFERON®-CMI test (CSL Biosciences, Melbourne, Australia). This assay allows detection of cytokine production, which may occur in the absence of proliferation. In persons exposed to B. burgdorferi, an early, vigorous, and sustained specific T-cell response develops that precedes a measurable antibody response [9]. The production of IFNγ 16–24 hours after antigen stimulation is dependent on the presence of T-cells capable of rapid response kinetics (antigen-primed effector and/or antigen-primed memory population). These T-cell populations are short lived and are strictly dependent on the presence of antigen [10]. This assay is based on the release of gamma-interferon (IFNγ) from sensitized lymphocytes during a 16 to 24 hour incubation with antigens specific for the pathogenic agent and subsequently quantification of IFNγ levels by a single step ELISA. The ratio of IFNγ response to antigen versus the response to negative control is quantified to determine if IFNγ production is due to prior exposure. The assay is simple to perform and does not require the isolation of lymphocytes [8]. Incorporation of the positive control mitogen in the test allows for the detection of immunocompromised individuals (eg. due to AIDS) and thereby removes the risk of detection of false negatives.
The test was carried out as instructed by the manufacturer. A healthy individual is expected to have 0.8–1.2 × 106 Th cells/ml of whole blood. First, heparinized whole blood was aliquoted at 1 ml/per well (about 106 T helper cells/ml) in a 24 well cell culture cluster (Costar) and stimulated with 120 μl of the following antigens and controls: sonicated borrelia or WCS at 2.5 μg/ml, peptide hLFAαL (aa326-345) at 2.5 μg/ml, peptide OspAB31 (aa164-183) at 10 μg/ml, mitogen (PHA) and negative (PBS) controls.
After overnight incubation (16–20 h) at 37°C in a humid chamber, the plasma was removed and the amount of IFNγ was determined by a rapid single step ELISA provided with the test kit, as per protocol. The single step ELISA was run against a standard curve of human IFNγ standards. The OD results from patient blood stimulated with B. burgdorferi antigen (corrected for the saline control) were converted into International Units per ml (IU/ml) from a standard curve of the human IFNγ standards. The IU/ml IFNγ values were used to calculate the % specific response to the antigen for each sample. The % specific response is the ratio of IFNγ response to test antigen (Ag) minus negative control (N) versus the response to mitogen (M) minus negative control (N), times 100. We determined the cutoff at 10% specific response.
Statistical analysis
The comparison between the response to OspAB31 (aa164-183) and hLFAαL (aa326-345) was done using the McNemar's exact test for correlated proportions [11] because the same population of patients was tested in a paired-sample design. P values of < 0.05 were considered statistically significant.