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Infection and Immunity, January 1999, p. 173-181, Vol. 67, No. 1
Divisions of Rheumatology/Immunology and
Pediatric Rheumatology1 and
Division of
Rheumatology/Immunology,2 Tufts University
School of Medicine, New England Medical Center, Tupper Research
Institute, Boston, Massachusetts, and
Howard Hughes Medical
Institute3 and
Division of
Rheumatology,4 Yale University School of
Medicine, New Haven, Connecticut
Received 17 July 1998/Accepted 23 September 1998
In an effort to implicate immune responses to specific
Borrelia burgdorferi proteins that may have a role in
chronic Lyme arthritis, we studied the natural history of the antibody
response to B. burgdorferi in serial serum samples from 25 patients monitored throughout the course of Lyme disease. In these
patients, the immunoglobulin G (IgM) and IgG antibody responses to 10 recombinant B. burgdorferi proteins, determined during
early infection, early arthritis, and maximal arthritis, were
correlated with the severity and duration of maximal arthritis. The
earliest responses were usually to outer surface protein C (OspC), P35,
P37, and P41; reactivity with OspE, OspF, P39, and P93 often developed
weeks later; and months to years later, 64% of patients had responses to OspA and OspB. During early infection and early arthritis, the
levels of IgG antibody to P35 correlated inversely with the subsequent
severity or duration of maximal arthritis. In contrast, during periods
of maximal arthritis, the levels of IgG antibody to OspA and OspB,
especially to a C-terminal epitope of OspA, correlated directly with
the severity and duration of arthritis. Thus, the higher the IgG
antibody response to P35 earlier in the infection, the milder and
briefer the subsequent arthritis, whereas during maximal arthritis, the
higher the IgG response to OspA and OspB, the more severe and prolonged
the arthritis.
Lyme disease, which is caused by the
tick-borne spirochete Borrelia burgdorferi, usually begins
with localized infection of the skin, erythema migrans, followed days
to weeks later by dissemination of the spirochete to multiple sites,
including joints (28, 40). Weeks to months later, brief
attacks of arthritis or arthralgia, lasting days or weeks, often occur
in a few large joints. Months later, more prolonged episodes of
arthritis, lasting months, may develop, and about 10% of patients with
arthritis have continuous joint inflammation for 1 year or longer, a
condition which we have termed chronic Lyme arthritis (44).
The synovial histology in these patients is similar to that seen in
other forms of chronic inflammatory arthritis, including rheumatoid
arthritis (22, 41).
B. burgdorferi contains at least 30 immunogenic proteins
(2, 9, 11, 12, 27, 33, 39) with as many as 10 cell membrane
or outer membrane proteins, including outer surface lipoproteins (Osp)A
through -F (3-6, 19, 21, 25, 30, 31). The spirochete expresses different proteins at different times in its life cycle, and
this may be critical in the spirochete's homing to and survival in
various tissues (1, 10, 14, 37). We have been interested in
the expression of B. burgdorferi in the joints of patients with Lyme arthritis and in immune responses that influence this phase
of the illness. In two previous studies, we used a unique set of serial
serum samples from untreated patients monitored throughout the course
of Lyme disease in the late 1970s prior to the use of antibiotic
therapy for this illness (23, 24). Only with this set of
serum samples is it possible to determine how the antibody responses to
B. burgdorferi develop and change during the various stages
of the illness.
In the initial study, 11 of the 15 patients (73%) monitored throughout
the illness developed strong immunoglobulin G (IgG) responses to OspA
and OspB near the beginning of prolonged episodes of arthritis, from 7 months to 5 years after disease onset (23). Moreover, the
combination of the HLA-DR4 specificity and OspA or OspB reactivity was
associated with chronic arthritis and lack of response to antibiotic
therapy. However, other recombinant proteins were not yet available to
test the specificity of these associations.
In the second study (24), OspA epitope mapping was done in
10 patients monitored throughout the illness. In these patients, an
early IgM response was often found to epitopes throughout the protein.
Of the 10 patients, 7 who developed arthritis of moderate or prolonged
duration did not have IgG responses to OspA early in the illness, but
they had strong responses to this protein near the beginning of
prolonged arthritis. In contrast, two of the three patients who had
only brief attacks of arthritis had weak IgG responses to OspA early in
the illness and during periods of arthritis. Thus, patients who had
difficulty with IgG isotype switching to OspA early in the illness
seemed more likely to develop prolonged arthritis. However, the number
of patients tested was too small for meaningful statistical comparisons.
Our goal in this study was to address two major questions raised by the
previous studies. First, in an effort to compare initial responses
according to the severity and duration of subsequent arthritis, we
increased the sample size to include all 25 untreated patients in whom
serial serum samples in our archival collection were available at
appropriate time points. Second, to determine the specificity of the
OspA and OspB associations with prolonged arthritis, we tested the
serum samples for reactivity with 10 recombinant spirochetal proteins
that are now available. The ultimate purpose of these studies was to
implicate immune responses to specific B. burgdorferi
proteins that may have a role in the pathogenesis of chronic Lyme arthritis.
Patients.
During the late 1970s, all patients were monitored
by one of us in the Lyme disease clinic at the Yale University School
of Medicine. Clinical data were recorded in patients' charts, and blood samples from each visit were stored at
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Immunoglobulin (IgG) Antibody Response to OspA and OspB
Correlates with Severe and Prolonged Lyme Arthritis and the IgG
Response to P35 Correlates with Mild and Brief Arthritis
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ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
70°C. At the time of
this study, serial serum samples were available from 25 untreated patients during early infection when erythema migrans was present, during early episodes of arthritis or arthralgia, and during periods of
maximal arthritis. All 25 patients met Centers for Disease Control and
Prevention (CDC) criteria for the diagnosis of Lyme disease
(7): they had erythema migrans followed by oligoarticular arthritis with positive IgG responses to B. burgdorferi as
interpreted by the CDC/ASTPHLD criteria (8). Their ages
ranged from 3 to 59 years (median, 32 years); 13 were male and 12 were
female. Joint involvement in these individuals was representative of
the range of severity and duration of Lyme arthritis (44).
Recombinant B. burgdorferi antigens. Full-length, unlipidated OspA (amino acids [aa] 16 to 273), the fragments OspA1 (aa 16 to 108), OspA2 (aa 105 to 201), and OspA3 (aa 168 to 273), and full-length, unlipidated OspB and OspC were generated as recombinant fusion proteins with Escherichia coli maltose binding protein (MBP) as previously described (23). The plasmid vectors pTRH44 and pTRH46, containing OspA and OspB from B. burgdorferi B31, respectively, were kindly provided by Alan Barbour (21), whereas the DNA used for the OspC construct was obtained from B. burgdorferi 297 (19). Restriction fragments containing these gene segments were inserted at the 3' end of the E. coli malE gene, which encodes MBP. During the logarithmic growth phase of E. coli, protein production was induced, and the bacteria were lysed by passage through a French pressure cell. The supernatant was then passed over a cross-linked amylose column by using buffer containing maltose, and the MBP fusion proteins were eluted. Purified unlipidated OspE, OspF, P35, and P37 were generated at the Yale University School of Medicine by using similar techniques. E. coli carrying the appropriate plasmids encoding these B. burgdorferi proteins and the fusion partner glutathione transferase were lysed by sonication, and the lysates were passed over a glutathione column to elute the purified recombinant proteins (13, 25). P39, P41, and P93 were a kind gift from John M. Robinson, Abbott Laboratories, Abbott Park, Ill. (33).
ELISA.
The serum samples, which had been stored at
70°C,
were tested by enzyme-linked immunosorbent assay (ELISA) for IgM and
IgG antibodies to various recombinant B. burgdorferi
antigens, using modifications of previously described methods
(24). Ninety-six-well Immulon plates (Dynatech Inc.,
Kensington, Md.) were coated with each of the antigens at a
concentration of 1 µg/well. These concentrations were shown to
be in antigen excess by using checkerboard dilutions of each
recombinant antigen and an appropriate strongly positive patient serum
sample. After incubation overnight at 4°C, the plates were washed
with 0.05% phosphate-buffered saline-Tween 20 and incubated with 5%
nonfat dried milk in phosphate-buffered saline-Tween 20 (milk buffer)
for 45 min at 37°C. After washing, 200 µl of patient serum samples
(1:50 dilution, except 1:200 for P35 and P37) were plated in duplicate
and incubated for 45 min at 37°C. After washing again, the plates
were incubated with alkaline phosphatase-conjugated, goat anti-human
IgG (1:750) or IgM (1:500) in milk buffer (Tago). The substrate was
freshly prepared p-nitrophenyl phosphate. The plates were
read at 405 nm when the lowest dilution of the positive control sample,
which was included on each plate, reached 1.0. The cutoff for a
positive value was defined as 3 standard deviations above the mean
optic density of seven negative control samples which were also
included on the same plate. The negative control samples were obtained
from healthy individuals with no prior history of Lyme disease. Samples
from the same patient were always tested together on the same plate.
Statistics.
For each of the three time points, the IgM or
IgG absorbance value for each recombinant protein was correlated with
the severity and duration of maximal arthritis, using the Spearman rank
correlation test. The P values are two tailed. Because
testing was done with 10 borrelial proteins, a P value of
0.005 rather than
0.05 (a Bonferroni correction) was considered
statistically significant.
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RESULTS |
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Natural history of the antibody responses to B. burgdorferi during the course of Lyme disease. When erythema migrans was present (2 weeks to 3 months after disease onset), 60 to 80% of the 25 patients had IgM or IgG antibody responses to OspC, P35, P37, and P41; the most common early IgG response was to P35 (Table 1). The percentage of patients with reactivity with each of these proteins remained high during early, brief attacks of arthritis or arthralgia (2 to 12 months after disease onset) and during the most prolonged period of arthritis (7 months to 4.5 years after disease onset). Although many patients had IgM responses with OspE early in the illness, IgM reactivity with OspF, P39, or P93 was less common at that time. The number of patients with IgG responses to OspE, OspF, P39, and P93 increased at each subsequent time point. Only three to five patients (12 to 20%) had weak IgM or IgG reactivity with OspA or OspB early in the illness, and no additional patients developed responses to these proteins during early periods of arthritis or arthralgia. Instead, 16 of the 25 patients (60%), including all 5 with chronic arthritis, had IgG responses to OspA and OspB during periods of maximal arthritis. Thus, the only new responses that sometimes developed during the period of maximal arthritis were to OspA and OspB; reactivity with the other spirochetal proteins developed prior to that time.
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Correlation of antibody responses with duration and severity of
arthritis.
Early in the illness, the levels of IgG antibody to
P35, the most common early response, correlated inversely with the
subsequent duration of arthritis (r =
0.54, P = 0.005) (Table 2). Similarly, patients who had higher levels of antibody to OspC or P41 early in the
illness tended to have milder and briefer arthritis. However, these
trends were not significant at the 0.005 level, a Bonferroni correction
for the 10 borrelial antigens tested in this analysis. Several weeks to
months later during early, brief attacks of arthritis or arthralgia,
IgG reactivity with P35 again correlated inversely with the amount of
joint swelling during subsequent, prolonged attacks of arthritis
(r =
0.67, P = 0.0004). In addition, during early arthritis, higher levels of antibody to OspE tended to correlate with less subsequent joint swelling. Thus, the greater the early responses to P35, and to a lesser degree to OspC, OspE, and P41, the
milder and briefer the subsequent arthritis.
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Antibody responses in representative patients. To further illustrate these correlations, Fig. 2 shows the antibody responses to OspA, OspB, and P35 for four representative patients. At the mild end of the spectrum, patient A had only one short attack of arthritis, and patient B had only short episodes of arthralgia. During erythema migrans or during early periods of arthritis or arthralgia, both of these patients had high levels of IgG antibody to P35. Neither patient had responses to OspA or OspB at any time in the illness. In contrast, patients C and D had chronic Lyme arthritis with severe and prolonged episodes of knee swelling lasting more than 1 year. Both had the HLA-DR4 specificity. Both patients had only moderate or low levels of IgG antibody to P35 throughout the illness, but both developed marked IgG responses to OspA and OspB more than 1 year after disease onset during periods of maximal arthritis with marked knee swelling. In both, the response was stronger to OspA than OspB.
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DISCUSSION |
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In this study of 25 untreated patients monitored longitudinally throughout the course of Lyme disease, common IgM and IgG responses early in the infection were to OspC, P35, P37, and P41. Within weeks, many patients also had responses to P39, P93, OspE, and, to a lesser extent, OspF. Reactivity with most of these proteins increased at each time point, suggesting that these proteins are often expressed by the spirochete throughout the infection. Although many patients had IgM reactivity with OspC early in the infection, the levels of IgG antibody to this protein remained low until periods of prolonged arthritis. Individual differences in the strength and timing of antibody responses may be due to differential expression of these proteins by the spirochete or to variations in the host immune response. Although strain variation among spirochetes may be a factor, the 25 study patients came from the Lyme, Conn., area, where this infection is thought to be caused by a single borrelial species, B. burgdorferi sensu stricto.
When patients were stratified according to the severity and duration of arthritis, higher levels of IgG antibody to P35 earlier in the infection correlated with milder and briefer arthritis. Based on cloning studies, P35 was reported to be a newly recognized spirochetal protein that is expressed only in vivo, not in culture (14). However, with the recent publication of the complete sequence of the B. burgdorferi genome (18), it became apparent that P35 contains the C-terminal 261 aa of a 354-aa protein encoded by a gene called bbk32. Most recently, Probert and Johnson reported that bbk32 encodes a differentially expressed, fibronectin binding protein (32). Thus, we would postulate that a marked antibody response to this fibronectin binding protein early in the illness reduces the number of spirochetes that reach or remain in the joints, resulting in milder arthritis of shorter duration.
In contrast with the responses to all other antigens, only a small percentage of patients had reactivity with OspA and OspB early in the infection or during early attacks of arthritis or arthralgia. Although the spirochete down-regulates expression of these two related proteins in the tick prior to transmission to the vertebrate host (17), we and other investigators have noted that patients with early infection or acute neuroborreliosis may have an ephemeral immune response to OspA, primarily of the IgM isotype (24, 35, 36), suggesting that some spirochetes may still express this protein early in the illness. However, in this study, this early response did not correlate with the severity or duration of subsequent arthritis. Instead, as we have shown previously (23, 24), IgG reactivity with these proteins developed in the majority of patients more than 1 year after disease onset in association with periods of maximal arthritis. Thus, in most patients, B. burgdorferi may not express OspA and OspB until late in the illness in joints.
This study confirms that the stronger the responses to OspA and OspB, particularly to the C-terminal epitope of OspA, the longer and more severe the arthritis. To explain this association, one must postulate either that the expression of OspA and OspB has survival value for the spirochete in the joint or that host immunity to one or both of these closely related proteins enhances the severity and duration of joint inflammation. It is surprising that expression of OspA and OspB would have survival value in joints since high levels of antibody to a C-terminal epitope of OspA kill spirochetes in the midgut of the tick (17), thereby protecting mice as well as human subjects from reinfection with B. burgdorferi (13, 34, 45). Perhaps spirochetes in joints express OspA only intermittently; OspA might be masked by other antigens, or it may undergo antigenic variation in the joint (16). In one patient, an OspA frameshift, identified from DNA in Lyme arthritis synovial fluid, resulted in an OspA that did not bind protective antibodies (15).
Alternately, autoreactive immune phenomena may be a factor in explaining severe and prolonged Lyme arthritis. The first clue to this possibility was the observation that a small percentage of patients have persistent Lyme arthritis for months or even several years after prolonged courses of antibiotic therapy (43). Although B. burgdorferi DNA can frequently be detected in the joint fluid of such patients prior to antibiotic therapy, it cannot usually be demonstrated there after treatment (29). Moreover, this outcome is associated with HLA-DR4 alleles (23, 42, 43) and with cellular as well as humoral immunity to OspA (23, 24, 26). We recently showed that in HLA-DRB1*0401-positive individuals, there is molecular mimicry between the dominant T-cell epitope of OspA and human leukocyte function-associated antigen (hLFA-1) (20). Both hLFA-1 and OspA induced T-cell reactivity in 9 of 11 patients tested with treatment-resistant Lyme arthritis but not in those with other forms of chronic inflammatory arthritis. A cross-reactive T-cell response to OspA and hLFA-1 would provide an amplification mechanism to explain more severe and prolonged arthritis in the natural infection, and it would also explain the persistence of joint inflammation after the apparent eradication of the spirochete from the joint in antibiotic-treated patients. We do not think that anti-OspA antibody is pathogenic itself; rather, we suspect that it is simply a marker for this critical T-cell response. We observed in this study a statistically significant association with the C-terminal fragment of OspA presumably because this is usually the dominant antibody epitope of OspA (24, 38). The similar but less significant association with OspB, which has 56% sequence homology with OspA (5), may result from shared antibody epitopes with OspA.
In summary, the higher the IgG antibody response to P35 earlier in the infection, the shorter the severity and duration of subsequent arthritis, whereas during the period of maximal arthritis, the higher the IgG response to OspA and OspB, especially to a C-terminal epitope of OspA, the more severe and prolonged the arthritis. These clinical correlations presumably result from both the differential expression of these proteins by the spirochete and genetically determined differences in the host immune response.
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ACKNOWLEDGMENTS |
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We thank John M. Robinson for the recombinant P39, P41, and P93 proteins used in this study; Manchuan Chen for preparing P35, P37, OspE, and OspF, and Robin Ruthazer for assistance with the statistical analysis.
This study was supported by grant AR-20358 from the National Institutes of Health (A.C.S.), grant U5-CCU-106581 from the Centers for Disease Control and Prevention (R.A.F. and E.F.), and the Eshe Fund (A.C.S.). E.A. received support from the Lincoln National Foundation of Fort Wayne, Ind. E.F. is the recipient of the Wellcome Clinical Scientist Award in translational research.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Rheumatology/Immunology, New England Medical Center, NEMC 406, 750 Washington St., Boston, MA 02111. Phone: (617) 636-5789. Fax: (617) 636-4252.
Editor: J. R. McGhee
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