Anti-neural antibody reactivity in patients with a history of Lyme borreliosis
and persistent symptoms
Abhishek Chandraa, Gary P. Wormserb, Mark S. Klempnerc, Richard P. Trevinoc, Mary K.
Crowd, Norman Latova, Armin Alaedinia*
aDepartment of Neurology and Neuroscience, Cornell University, New York, NY, USA
bDivision of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla,
cDepartment of Microbiology, Boston University, Boston, MA, USA
dDivision of Rheumatology, Hospital for Special Surgery, New York, NY, USA
*Corresponding author: Armin Alaedini, Department of Neurology and Neuroscience, Weill
Medical College of Cornell University, 1300 York Ave., LC-819, New York, NY 10065; Phone:
212-746-7841; Email: firstname.lastname@example.org.
Running title: Anti-neural antibodies in PLS
Word count: 3,808
Number of figures: 4
Some Lyme disease patients report debilitating chronic symptoms of pain, fatigue, and
cognitive deficits despite recommended courses of antibiotic treatment. The mechanisms
responsible for these symptoms, collectively referred to as post-Lyme disease syndrome (PLS) or
chronic Lyme disease, remain unclear. We investigated the presence of immune system
abnormalities in PLS by assessing the levels of antibodies to neural proteins in patients and
controls. Serum samples from PLS patients, post-Lyme disease healthy individuals, patients
with systemic lupus erythematosus, and normal healthy individuals were analyzed for anti-neural
antibodies by immunoblotting and immunohistochemistry. Anti-neural antibody reactivity was
found to be significantly higher in the PLS group than in the post-Lyme healthy (p<0.01) and
normal healthy (p<0.01) groups. The observed heightened antibody reactivity in PLS patients
could not be attributed solely to the presence of cross-reactive anti-borrelia antibodies, as the
borrelial seronegative patients also exhibited elevated anti-neural antibody levels.
Immunohistochemical analysis of PLS serum antibody activity demonstrated binding to cells in
the central and peripheral nervous systems. The results provide evidence for the existence of a
differential immune system response in PLS, offering new clues about the etiopathogenesis of
the disease that may prove useful in devising more effective treatment strategies.
Keywords: post-Lyme disease syndrome, chronic Lyme disease, immune dysregulation,
Lyme disease is a multisystem infection, caused by bacteria of the Borrelia burgdorferi
species complex and transmitted by Ixodes ticks (Stanek and Strle, 2003). It is the most
commonly reported tick-borne disease in the northern hemisphere, widespread in Europe and
endemic in more than 15 states in the United States (Stanek and Strle, 2008; Steere, 2001). The
initial skin rash (erythema migrans) may be followed by complications affecting joints, heart,
and the nervous system (Stanek and Strle, 2003; Wormser et al., 2006). The neurologic
complications involve both the central and peripheral nervous systems. These include
lymphocytic meningitis, encephalitis, cranial neuropathy, radiculopathy, and alterations of
mental status, all of which usually respond well to antibiotic treatment (Halperin, 2008).
However, some patients with Lyme disease continue to have persistent complaints despite
treatment and in the absence of objective evidence of infection, as determined by currently
available methods (Feder et al., 2007; Marques, 2008). The symptoms in these patients are
generally accepted to include mild to severe musculokeletal pain, fatigue, and/or difficulties with
concentration and memory (Feder et al., 2007; Marques, 2008). The condition, variably referred
to as chronic Lyme disease, post-treatment Lyme disease syndrome (PTLDS), and post-Lyme
disease syndrome (PLDS or PLS), is associated with considerable impairment in the health-
related quality of life in some patients (Klempner et al., 2001).
Considering the lack of evidence for the presence of live spirochetes in PLS patients who
have received recommended antibiotics, persistent infection is currently not thought to account
for the symptoms of PLS by most investigators (Baker, 2008; Feder et al., 2007). However,
despite several years of debate and a number of treatment clinical trials (Fallon et al., 2008;
Klempner et al., 2001; Krupp et al., 2003), few clues to the causes of the symptoms have
emerged. Lack of any biomarkers to aid in the diagnosis and follow up, or to help in
differentiating between PLS patients and post-Lyme healthy individuals, has also compounded
the problem of understanding the disease. Mechanisms other than active infection, including the
possibility of involvement of adaptive or innate immune system abnormalities, have been
suggested, but experimental evidence has been scarce (Marques, 2008; Sigal, 1997). The aim of
this study was to characterize the level and specificity of antibody reactivity to neural antigens in
PLS patients. Here, we show evidence of heightened anti-neural antibody levels in PLS,
indicating the presence of objective immunologic abnormalities in affected patients that may be
relevant to the pathogenic mechanism of the disease.
Serum samples from 83 individuals with a history of Lyme borreliosis and persistent
symptoms, recruited as part of a previous clinical trial (Klempner et al., 2001), were used in this
study (37 female, 46 male; mean age 55.6 ± 12.0 y (SD); mean elapsed time since the original
diagnosis of Lyme disease 5.0 ± 2.9 y (SD)). Selection of these specific specimens from the
original cohort was based on limiting the elapsed time between diagnosis of acute Lyme disease
and serum specimen collection to between 1 and 12 years. Patients had at least one of the
following: a history of erythema migrans (EM) skin lesion, early neurologic or cardiac symptoms
attributed to Lyme disease, radiculoneuropathy, or Lyme arthritis. Documentation by a physician
of previous treatment of acute Lyme disease with a recommended antibiotic regimen was also
required. Patients had one or more of the following symptoms at the time of enrollment:
widespread musculoskeletal pain, cognitive impairment, radicular pain, paresthesias, or
dysesthesias. Fatigue often accompanied one or more of these symptoms. The chronic symptoms
had to have begun within 6 months after the infection with B. burgdorferi. Control subjects
included 27 individuals who had been treated for early localized or disseminated Lyme disease
associated with single (n=18) or multiple (n=9) EM, but had no post-Lyme symptoms after at
least 2 years of follow-up (12 female, 15 male; mean age 54.4 ± 14.7 y (SD); mean elapsed time
since the original diagnosis of Lyme disease 5.4 ± 3.8 y (SD)). The diagnosis of acute Lyme
disease in control subjects was confirmed by recovery of B. burgdorferi in cultures of skin and/or
blood sample. The elapsed time between diagnosis of acute Lyme disease and serum specimen
collection was limited to between 1 and 12 years for post-Lyme healthy subjects. In addition to
the above, serum samples from 15 patients with systemic lupus erythematosus (SLE) and 20
healthy individuals were analyzed in the study. All SLE patients met four or more of the
American College of Rheumatology classification criteria for diagnosis (Tan et al., 1982).
Serum specimens were stored at -80 oC prior to use. This study was approved by the
Institutional Review Board of the Weill Medical College of Cornell University.
2.2. Total IgG
Total IgG concentration of serum specimens was measured using an ELISA kit (ICL),
according to the manufacturer’s instructions.
2.3. Anti-borrelia antibodies
IgG anti-borrelia antibody levels were determined by ELISA. 96-well polystyrene plates
(BD Biosciences) were incubated overnight with 0.5 µg/well of B. burgdorferi B31 antigen
(Meridian) in 0.1 M carbonate buffer (pH 9.6). Blocking of wells was done with 1% BSA in
phosphate-buffered saline containing 0.05% Tween-20 (PBST) for 1.5 h. Incubation with
diluted serum samples (50 µL/well at 1:800 in blocking buffer) was done for 1 h. Each plate
contained 1 negative and 2 positive controls. Incubation with HRP-conjugated sheep anti-human
IgG (Amersham) secondary antibody was for 1 h. Incubation with developing solution,
comprising 27 mM citric acid, 50 mM Na2HPO4, 5.5 mM o-phenylenediamine, and 0.01% H2O2
(pH 5), was for 20 min. Absorbance was measured at 450 nm and corrected for non-specific
binding by subtraction of the mean absorbance of corresponding wells not coated with the
borrelia antigen. Absorbance values were normalized based on the mean for the positive
controls on each plate. Cutoff for positivity was assigned as two standard deviations above the
mean for the healthy control group results.
2.4. Anti-neural antibodies
2.4.1. Immunoblotting. Antibodies to brain proteins were detected by immunoblotting for all
specimens as follows. Mouse brain was utilized in order to avoid artifactual bands that result
from the binding of secondary anti-human antibodies to endogenous immunoglobulins when
using the sensitive chemiluminescence method of detection. Mouse tissue was specifically
chosen among non-primate sources due to the high level of known homology and orthology
between human and mouse proteomes (Southan, 2004), a strategy that has been used in other
studies as well (Maruyama et al., 2004; Shoenfeld et al., 2003; Tin et al., 2005). Mouse
(C57BL/6J strain) brain lysate was prepared as previously described (Alaedini et al., 2007).
SDS-PAGE (4-15% pre-cast 2D-prep gel from Bio-Rad) was carried out on 400 μg protein
aliquots of lysate at 200 V in tris-glycine-SDS buffer for 35 min, followed by transfer to
nitrocellulose membrane at 33 V in tris-glycine buffer containing 20% methanol for 16 h. Each
gel contained the Precision Plus molecular weight marker mix (Bio-Rad) in one lane. The
membrane was incubated in blocking buffer, containing 5% milk and 0.5% BSA in Tris-buffered
saline containing 0.05% Tween-20 (TBST) for 2 h. Incubation with patient serum (1:2000 in
dilution buffer containing 10% blocking buffer and 10% fetal bovine serum in TBST) was
carried out for 1 h in a Mini-PROTEAN II Multiscreen apparatus (Bio-Rad). A positive control
sample was included on every membrane. HRP-conjugated sheep anti-human IgG (Amersham)
was used as the secondary antibody. Detection of bound antibodies was by the ECL system
(Millipore) and BioMax MR film (Kodak) after 10s exposure. Each membrane was treated with
stripping buffer (Pierce) at 58 oC for 30 min, and reblotted with HRP-conjugated rabbit anti-β
tubulin antibody (Novus). Detection of bound antibodies was as before. Conversion of
immunoblots to line graph, density analysis, and subtraction of background were performed by
the Unscan-It program (Silk Scientific). Measurement of total antibody reactivity towards neural
proteins in each sample was done by calculating the sum of gray-level intensities for all
software-assigned and background-subtracted reactive bands. Total gray-level intensity for each
specimen was corrected for 1) inconsistencies within each membrane (e.g., for variation in
sample loading and efficiency of protein transfer) according to the gray-level intensity of the
tubulin band for each lane, and 2) inconsistencies in experimental conditions between
membranes (e.g., for variation in sample loading, efficiency of protein transfer, and
autoradiography exposure time) according to the total gray-level intensity for the positive control
on each membrane.
2.4.2. Immunohistochemistry. Immunohistochemical analysis was similar to previously
described procedure (Alaedini et al., 2008). Formaldehyde-fixed and paraffin-embedded
sections of human cerebral cortex and dorsal root ganglia (DRG), obtained at post mortem, were
cut (10 μm thickness) and placed on slides. Sections were deparaffinized and rehydrated by
sequential incubation in xylene, ethanol (100%, 90%, 80%, and 70%), and PBS. Antigen
retrieval was done by incubation in 0.05% citraconic anhydride buffer (pH 6.0) for 20 min at 95
oC. Endogenous peroxidase was quenched with 1% H2O2. Tissue sections were blocked for 30
min with 15% goat serum (Sigma-Aldrich) in PBS. Sections were then incubated for 1.5 h with
1:100 dilutions of representative serum specimens from each group in duplicate. HRP-
conjugated goat anti-human IgG was used as secondary antibody. Tissues were washed and
colorimetric detection was carried out using the metal-enhanced DAB (3,3’-diaminobenzidine)
2.5. Cross-reactivity of anti-borrelia antibodies towards neural antigens
2.5.1. Affiinity-purification of antibodies. Anti-borrelia antibodies were obtained from the
pooled serum IgG fraction of rabbits immunized with B. burgdorferi B31 strain (Virostat). The
antibodies were affinity purified with a column coupled with B. burgdorferi proteins as follows.
The affinity column was prepared using the AminoLink activated agarose gel bead support
(Pierce). After packing the column with slurry, it was equilibrated with PBS, followed by the
addition of 2 mL of a 0.9 mg/mL solution of desalted proteins from a B. burgdorferi B31 lysate
(Meridian) and 200 μL of 1 M NaCNBH3 in 10 mM NaOH. The coupling reaction was allowed
to continue while gently rotating the column (6 h, room temperature). Remaining reactive sites
were blocked by incubation with 1 M Tris (pH 7.4). Affinity purification was initiated by the
introduction of antibody solution into the column and continuous flow for 1 h. The column was
washed and bound antibodies were eluted with 100 mM glycine buffer (pH 3.0). The eluted
antibody fraction was neutralized with 1 M Tris (pH 7.5) and concentrated by centrifugal
2.5.2. Binding of anti-borrelia antibodies to neural antigens. The interaction of the anti-
borrelia antibodies with neural proteins was characterized by Western blotting and
immunohistochemistry. One- and two-dimensional electrophoresis was carried out on 40-80 μg
aliquots of mouse brain lysate protein. The two-dimensional electrophoresis was based on
previously described procedure (O'Farrell, 1975) in which isoelectric focusing was carried out in
glass tube using pH 3.5-10 ampholines (GE Healthcare) and SDS slab gel electrophoresis was
done for 4 h at 15 mA/gel. The proteins were transferred to nitrocellulose membrane. The
membrane was blocked as before and then incubated with the prepared affinity-purified anti-
borrelia antibody or with non-immunized rabbit serum IgG (Sigma-Aldrich) (0.5 μg/mL) for 1 h.
The HRP-conjugated secondary antibody used was anti-rabbit IgG (Amersham). Detection of
bound antibodies was as before. Immunohistochemical analysis was as was described above for
human samples, but instead using the affinity-purified anti-borrelia antibodies or rabbit IgG at
0.01 mg/mL as primary antibody, and HRP-conjugated donkey anti-rabbit IgG (Amersham) as
2.6. Data analysis
Group differences were analyzed by two-tailed Welch’s t-test (continuous data with unequal
variances), and Chi-square test or Fisher’s exact test (nominal data). Calculated gray-level
intensity data were normalized by square root transformation prior to statistical analysis.
Adjustment for covariate effect was carried out by analysis of covariance (ANCOVA), using the
general linear model. Differences with p<0.05 were considered to be significant.
3.1. Total IgG
The mean total IgG concentration (± standard error of mean) for the PLS group (14.1 ± 0.35
mg/mL) was not significantly different from that of the post-Lyme healthy (13.0 ± 0.50 mg/mL),
SLE (14.7 ± 0.67 mg/mL), and normal healthy (12.8 ± 0.68 mg/mL) groups.
3.2. Anti-borrelia antibodies
Serum samples from 54 of 83 PLS and 14 of 27 post-Lyme healthy individuals were found to
be positive for IgG anti-borrelia antibodies by ELISA. None of the SLE and healthy control
samples were positive for anti-borrelia antibodies.
3.3. Anti-neural antibodies
At the dilution and exposure used in this study, anti-neural antibody reactivity (as represented
by the presence of one or more reactive protein bands on Western blots) was seen in serum
specimens from 41 of 83 (49.4%) PLS patients, 5 of 27 (18.5%) post-Lyme healthy individuals,
11 of 15 (73.3%) patients with SLE, and 3 of 20 (15.0%) normal healthy subjects. A
significantly higher number of PLS patients exhibited anti-neural antibody reactivity to one or
more protein bands than post-Lyme healthy (p<0.01) and normal healthy (p <0.01) individuals.
The anti-neural antibody reactivities in PLS and SLE patients were directed at multiple protein
bands (Fig. 1). The mean number of reactive protein bands per specimen (± standard error of
mean) for the PLS group (1.2 ± 0.16) was similar to that for the SLE group (1.6 ± 0.34), but
significantly higher than the post-Lyme healthy (0.22 ± 0.10) (p<0.005) and normal healthy
(0.10 ± 0.10) (p<0.005) groups. The differences in antibody reactivity were even more
significant when taking into account both the number and intensity of bands (total antibody
reactivity), measured as described in the methods section. The total antibody reactivity was
significantly higher in the PLS group in comparison to the post-Lyme healthy (p<0.001) and
normal healthy (p<0.001) groups (Fig. 2A). The difference between PLS and post-Lyme healthy
groups remained significant, even after adjusting for differences in age, gender, and elapsed time
since exposure to pathogen (p<0.001). The differences in the frequency and level of total
antibody reactivity between PLS and SLE groups did not reach statistical significance.
When considering only the borrelial seropositive subjects in the study, total anti-neural
antibody reactivity was significantly higher in the PLS group than the post-Lyme healthy group
(p<0.005) (Fig. 2B). Similarly, total anti-neural antibody reactivity was higher in the PLS
seronegative group than the post-Lyme healthy seronegative group (p<0.005) (Fig. 2B). On the
other hand, the difference in the anti-neural antibody reactivity between borrelial seropositive
and seronegative patients in either the PLS group or the post-Lyme healthy group did not reach
the level of significance (Fig. 2B).
One of 9 post-Lyme healthy subjects with multiple EM was positive for anti-neural
antibodies (11.1%), a rate that was even lower (though not statistically significant) than that for
those with single EM (4 of 18; 22.2%), indicating a lack of correlation between dissemination of
B. burgdorferi infection and anti-neural antibodies in the post-Lyme healthy group.
In order to ascertain the presence of antibodies against human central and peripheral nervous
system tissue and assess target cell specificity, reactivity of serum antibodies from representative
patients in each group was also analyzed by immunohistochemistry. Serum antibodies from PLS
patients found to be positive for anti-neural antibody reactivity by immunoblotting (both
borrelial seropositive and seronegative specimens) stained cortical pyramidal neurons, as well as
neurons of the DRG (Fig. 3). Antibody binding to some glial cells of the brain and DRG was
also observed. Patterns of staining varied for different PLS patients, with preferential binding to
cell membrane seen in some cases. Serum specimens from borrelial seropositive and
seronegative post-Lyme healthy individuals with anti-neural antibody reactivity showed faint or
no binding of antibodies to neural tissues. Serum antibodies from control SLE patients with anti-
neural antibody reactivity bound strongly to neurons and glial cells in the cerebral cortex and the
DRG, with preferential staining of the nuclei in some cases. Sera from normal healthy subjects,
however, did not stain tissues specifically.
3.4. Cross-reactivity of anti-borrelia antibodies toward neural proteins
In order to assess the extent of cross-reactivity of the anti-borrelia antibodies towards brain
proteins using our system of anti-neural antibody detection, we examined the binding of affinity-
purified anti-borrelia antibodies to brain proteins by one- and two-dimensional immunoblotting.
The purified antibodies bound to approximately 20 different protein bands (Fig. 4A),
demonstrating the potential for substantial cross-reactivity of the anti-borrelia antibody response
towards neural proteins. The cross-reactivity was confirmed by immunohistochemical analysis,
which showed anti-borrelia antibody binding to neurons and glial cells of the cerebral cortex and
the DRG (Fig. 4B).
Much of the controversy surrounding PLS arises from the lack of sufficient knowledge about
the etiology and pathology of the disease. This is compounded by the fact that there are few or
no objective methods available for diagnosis and follow-up of affected individuals. In light of
the results from the aforementioned clinical trials of antibiotic treatment and the lack of
convincing evidence for active infection in PLS, other hypotheses, including a role for
involvement of the immune system, have been suggested (Bolz and Weis, 2004; Marques, 2008).
If present, immune abnormalities—possibly triggered by the original infection—may offer clues
about the disease (Jarefors et al., 2007; Segal and Logigian, 2005). Considering the neurologic
and psychiatric nature of post-Lyme symptoms, we sought to assess the presence of nervous
system-specific antibodies in patients and control subjects. Approximately half of the examined
PLS patients had heightened levels of antibodies to neural proteins, compared with 18.5% of
post-Lyme healthy subjects and 15% of normal healthy controls. In fact, the heightened
antibody response level in PLS was statistically similar to that in SLE, a multisystem
autoimmune disease. Immunohistochemical analysis with representative PLS patient sera
demonstrated binding of the antibodies to pyramidal neurons in the cerebral cortex and neurons
of the DRG, highlighting their relevance in the context of central and peripheral nervous system
It is important to note that our method of analysis only detected antibodies against
prominently expressed proteins. Elevated antibodies to minor proteins or non-protein antigens
might also exist in some cases that were reported to be negative. Therefore, examination of
antibody binding to antigens in specific regions of the nervous system might reveal reactivity in
more individuals. In addition, although this work focused on antibodies against neural proteins,
antibodies to specific antigens in other tissues (e.g. muscle, thyroid, etc.) may also be found in
some patients and could be relevant to PLS. At the same time, the absence of anti-neural
antibodies in many patients might provide evidence for the heterogeneous nature of the
population under study.
We can make some conjectures about the possible reasons for the observed increased
antibody reactivity to self antigens in PLS. First, our experiments with affinity-purified
antibodies generated in rabbits against B. burgdorferi antigens clearly show that anti-borrelia
antibodies can cross-react with several neural proteins. A number of earlier studies have also
demonstrated the potential for cross-reactivity of the anti-borrelia immune response towards
neural antigens (Alaedini and Latov, 2005; Dai et al., 1993; Garcia-Monco et al., 1995; Maier et
al., 2000; Sigal and Tatum, 1988). A portion of the observed anti-neural antibody reactivity in
PLS patients is, therefore, likely to be the result of such cross-reactivity. However, the observed
anti-neural antibody reactivity cannot be attributed solely to positive anti-borrelia serology, as
increased anti-neural antibody reactivity was also seen in the borrelial seronegative PLS group.
Second, considering the non-specific pattern of immunologic reactivity, the presence of these
antibodies might signify an activated immunologic response to neural injury caused by the
original borrelial infection or another disease. Tissue injury can, in fact, result in the release of
autoantigens and lead to an increase in post-translational modification of proteins and production
of novel self-epitopes that elicit a strong immune response (Doyle and Mamula, 2005). Third,
borrelial infection has been shown to be a potent polyclonal B cell activator, capable of inducing
the non-specific proliferation and differentiation of antibody-secreting cells (Ma and Weis, 1993;
Yang et al., 1992). The ability of borrelia to act as a B cell activator is likely to be enhanced the
longer the infection is left untreated (Soulas et al., 2005). Therefore, the observed non-specific
increase in autoreactive antibodies in PLS may be due to the mitogenic effect of the borrelial
antigens, including OspA and OspB, and point to a possible association between post-Lyme
disease symptoms and the duration of the course of active infection prior to treatment. Finally,
immune abnormalities stemming from genetic predisposition might also play a significant role in
the form of B cell and effector cell dysregulation that leads to elevated levels of released
autoantibodies (Hostmann et al., 2008).
At this point, it is difficult to know what role, if any, the anti-neural antibodies might play in
the pathogenesis of PLS. Several immune-mediated diseases of the nervous system, including
multiple sclerosis, paraneoplastic nervous system disorders, autoimmune neuropathies,
myasthenia gravis, and stiff-person syndrome, are associated with elevated levels of antibodies to
neural antigens. A disease-causing role for such antibodies has been demonstrated in some of
these disorders (Dalakas, 2008). In general, antibodies might have a pathogenic effect in the
body through direct binding to a molecule and interference with its function, by activation of
complement and initiation of an inflammatory response, or by inducing tissue injury through
binding to Fc receptors on macrophages, neutrophils, and NK cells (Diamond et al., 2009).
Considering the non-specific antibody response seen in the examined PLS cohort, however, a
direct pathogenic role for the antibodies is doubtful. Nevertheless, even without a direct role,
antibodies have the potential to be involved in disease mechanism through the activation of toll-
like receptor pathways and secretion of various inflammatory molecules, which can affect the
function of other cells responsible for neuropsychiatric defects (Crow, 2007; Halperin, 2008;
Nawa and Takei, 2006).
The aim of this study was to begin a process of examining potential immune abnormalities in
PLS that would be relevant to the reported neurologic and cognitive symptoms of affected
patients. Results of the antibody analysis demonstrate the presence of a heightened, but
apparently non-specific, production of antibodies to neural antigens in PLS. We speculate that
these antibodies may either 1) be indicative of past injury to the nervous system during the active
phase of the Lyme disease infection, resulting in the immune system being exposed to and
activated by novel self antigens, or 2) point to the enhanced B cell mitogenic effect of the
borrelia pathogen in cases of delayed treatment and prolonged infection in genetically
predisposed individuals. As such, this study points to the presence of a differential immune
response in PLS in comparison to healthy individuals. Obviously, these findings are preliminary
and must be extended in future studies using a larger number of subjects and additional cohorts,
including healthy individuals with past Lyme arthritis and neurologic Lyme, as well as patients
with similar complaints and no history of Lyme disease. At this juncture, it is logical to assume
that further study of immune system response in PLS is likely to yield more clues about the
etiopathogenesis of the disease and provide insights that may pave the way for developing safe
and effective treatments.
This work was supported by the National Institutes of Health (NIH) [grant number
AI071180-02 to A. Alaedini] and involved the use of specimens derived from an NIH-supported
repository [contract number N01-AI-65308]. We are indebted to Dr. Phillip J. Baker at the
National Institutes of Health for his invaluable support and guidance. We thank the New York
Brain Bank for the human neural tissues [5 P50 AG08702-15 and NS 16367-24] and the HSS
Core Center for Musculoskeletal Repair and Regeneration for access to microscopy facilities
[NIH AR 46121]. We would also like to thank Ms. Diane Holmgren, Ms. Donna McKenna, and
Ms. Susan Bittker for their assistance with specimen collection and organization. We are
grateful to all of the research participants involved in this project.
Conflict of interest statement
All authors declare that there are no conflicts of interest.
Alaedini, A., Latov, N., 2005. Antibodies against OspA epitopes of Borrelia burgdorferi cross-
react with neural tissue. J Neuroimmunol 159, 192-195.
Alaedini, A., Okamoto, H., Briani, C. et al., 2007. Immune cross-reactivity in celiac disease:
anti-gliadin antibodies bind to neuronal synapsin I. J Immunol 178, 6590-6595.
Alaedini, A., Xiang, Z., Kim, H., Sung, Y.J., Latov, N., 2008. Up-regulation of apoptosis and
regeneration genes in the dorsal root ganglia during cisplatin treatment. Exp Neurol 210,
Baker, P.J., 2008. Perspectives on "chronic Lyme disease". Am J Med 121, 562-564.
Bolz, D.D., Weis, J.J., 2004. Molecular mimicry to Borrelia burgdorferi: pathway to
autoimmunity? Autoimmunity 37, 387-392.
Crow, M.K., 2007. Type I interferon in systemic lupus erythematosus. In: Pitha, P.M. (Ed.),
CTMI Vol. 316, Springer Berlin Heidelberg, pp. 359-386.
Dai, Z., Lackland, H., Stein, S. et al., 1993. Molecular mimicry in Lyme disease: monoclonal
antibody H9724 to B. burgdorferi flagellin specifically detects chaperonin-HSP60.
Biochim Biophys Acta 1181, 97-100.
Dalakas, M.C., 2008. B cells as therapeutic targets in autoimmune neurological disorders. Nat
Clin Pract Neurol 4, 557-567.
Diamond, B., Huerta, P.T., Mina-Osorio, P., Kowal, C., Volpe, B.T., 2009. Losing your nerves?
Maybe it's the antibodies. Nat Rev Immunol.
Doyle, H.A., Mamula, M.J., 2005. Posttranslational modifications of self-antigens. Ann N Y
Acad Sci 1050, 1-9.
Fallon, B.A., Keilp, J.G., Corbera, K.M. et al., 2008. A randomized, placebo-controlled trial of
repeated IV antibiotic therapy for Lyme encephalopathy. Neurology 70, 992-1003.
Feder, H.M., Jr., Johnson, B.J., O'Connell, S. et al., 2007. A critical appraisal of "chronic Lyme
disease". N Engl J Med 357, 1422-1430.
Garcia-Monco, J.C., Seidman, R.J., Benach, J.L., 1995. Experimental immunization with
Borrelia burgdorferi induces development of antibodies to gangliosides. Infect Immun
Halperin, J.J., 2008. Nervous system Lyme disease. Infect Dis Clin North Am 22, 261-274.
Hostmann, A., Jacobi, A.M., Mei, H., Hiepe, F., Dorner, T., 2008. Peripheral B cell
abnormalities and disease activity in systemic lupus erythematosus. Lupus 17, 1064-
Jarefors, S., Janefjord, C.K., Forsberg, P., Jenmalm, M.C., Ekerfelt, C., 2007. Decreased up-
regulation of the interleukin-12Rbeta2-chain and interferon-gamma secretion and
increased number of forkhead box P3-expressing cells in patients with a history of
chronic Lyme borreliosis compared with asymptomatic Borrelia-exposed individuals.
Clin Exp Immunol 147, 18-27.
Klempner, M.S., Hu, L.T., Evans, J. et al., 2001. Two controlled trials of antibiotic treatment in
patients with persistent symptoms and a history of Lyme disease. N Engl J Med 345, 85-
Krupp, L.B., Hyman, L.G., Grimson, R. et al., 2003. Study and treatment of post Lyme disease
(STOP-LD): a randomized double masked clinical trial. Neurology 60, 1923-1930.
Ma, Y., Weis, J.J., 1993. Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess
B-cell mitogenic and cytokine-stimulatory properties. Infect Immun 61, 3843-3853.
Maier, B., Molinger, M., Cope, A.P. et al., 2000. Multiple cross-reactive self-ligands for Borrelia
burgdorferi-specific HLA-DR4-restricted T cells. Eur J Immunol 30, 448-457.
Marques, A., 2008. Chronic Lyme disease: a review. Infect Dis Clin North Am 22, 341-360.
Maruyama, T., Saito, I., Hayashi, Y. et al., 2004. Molecular analysis of the human autoantibody
response to alpha-fodrin in Sjogren's syndrome reveals novel apoptosis-induced
specificity. Am J Pathol 165, 53-61.
Nawa, H., Takei, N., 2006. Recent progress in animal modeling of immune inflammatory
processes in schizophrenia: implication of specific cytokines. Neurosci Res 56, 2-13.
O'Farrell, P.H., 1975. High resolution two-dimensional electrophoresis of proteins. J Biol Chem
Segal, B.M., Logigian, E.L., 2005. Sublime diagnosis of Lyme neuroborreliosis. Neurology 65,
Shoenfeld, Y., Nahum, A., Korczyn, A.D. et al., 2003. Neuronal-binding antibodies from
patients with antiphospholipid syndrome induce cognitive deficits following intrathecal
passive transfer. Lupus 12, 436-442.
Sigal, L.H., 1997. Lyme disease: a review of aspects of its immunology and
immunopathogenesis. Annu Rev Immunol 15, 63-92.
Sigal, L.H., Tatum, A.H., 1988. Lyme disease patients' serum contains IgM antibodies to
Borrelia burgdorferi that cross-react with neuronal antigens. Neurology 38, 1439-1442.
Soulas, P., Woods, A., Jaulhac, B. et al., 2005. Autoantigen, innate immunity, and T cells
cooperate to break B cell tolerance during bacterial infection. J Clin Invest 115, 2257-
Southan, C., 2004. Has the yo-yo stopped? An assessment of human protein-coding gene
number. Proteomics 4, 1712-1726.
Stanek, G., Strle, F., 2003. Lyme borreliosis. Lancet 362, 1639-1647. 498
Stanek, G., Strle, F., 2008. Lyme disease: European perspective. Infect Dis Clin North Am 22,
Steere, A.C., 2001. Lyme disease. N Engl J Med 345, 115-125.
Tan, E.M., Cohen, A.S., Fries, J.F. et al., 1982. The 1982 revised criteria for the classification of
systemic lupus erythematosus. Arthritis Rheum 25, 1271-1277.
Tin, S.K., Xu, Q., Thumboo, J., Lee, L.Y., Tse, C., Fong, K.Y., 2005. Novel brain reactive
autoantibodies: prevalence in systemic lupus erythematosus and association with
psychoses and seizures. J Neuroimmunol 169, 153-160.
Wormser, G.P., Dattwyler, R.J., Shapiro, E.D. et al., 2006. 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
Yang, L., Ma, Y., Schoenfeld, R. et al., 1992. Evidence for B-lymphocyte mitogen activity in
Borrelia burgdorferi-infected mice. Infect Immun 60, 3033-3041.
Figure 1. Pattern of antibody reactivity in the serum of representative PLS patients and
control subjects towards electrophoresis-separated and transferred brain proteins. A) PLS
patients P1-P6; B) post-Lyme healthy individuals H1-H6 (H1-H4 had presented with single EM,
while H5 and H6 had presented with multiple EM); C) systemic lupus erythematosus patients
S1-S6; D) normal healthy individuals N1-N6. Lane C in each panel is the positive control.
Molecular weight markers are indicated to the left of each panel (kDa).
Figure 2. Mean total anti-brain antibody reactivity in patient and control groups. A)
Comparison between PLS patients, post-Lyme healthy subjects, normal healthy subjects without
serologic evidence of prior Lyme disease, and patients with systemic lupus erythematosus.
Reactivity was significantly higher in the PLS group than in post-Lyme healthy (p<0.001) and
normal healthy (p<0.001) groups. B) Comparison between seropositive and seronegative
patients in PLS and post-Lyme healthy groups. PLS seropositive and seronegative subgroups
had significantly higher anti-brain antibody reactivity than their counterparts in the post-Lyme
healthy group (p<0.005). The difference between PLS seropositive and seronegative patients did
not reach statistical significance. Error bars represent the standard error of the mean. Groups
indicated by different superscripts are significantly different from one another.
Figure 3. Immunohistochemical analysis of serum antibody reactivity towards cells in the
brain cerebral cortex (left panel) and DRG (right panel). A) Staining of sections with serum
from borrelial seropositive (A1) and borrelial seronegative (A2) patients with anti-neural
antibody reactivity (as determined by immunoblotting) showed specific binding to neurons of the
cerebral cortex and the DRG. B) Staining of sections with serum from borrelial seropositive
(B1) and borrelial seronegative (B2) post-Lyme healthy individuals with anti-neural antibody
reactivity showed faint or no specific binding of antibodies to cerebral cortex and DRG tissues.
C) Serum antibodies from two representative SLE patients (C1 and C2) with anti-neural antibody
reactivity bound strongly to neurons and glial cells in the cerebral cortex and the DRG. D) Sera
from two normal healthy subjects (D1 and D2) did not stain tissues specifically. Bars = 50 μm.
Figure 4. Cross-reactivity of the anti-borrelia immune response in immunized rabbits
towards neural proteins. A, B) One- and two-dimensional immunoblots of mouse brain lysate
with rabbit affinity-purified anti-borrelia antibody indicated cross-reactivity towards several
neural proteins. Numbers to the left of each panel indicate molecular weight markers (kDa). C,
D) Immunohistochemical analysis of the interaction of affinity-purified anti-borrelia antibodies
with human cerebral cortex (C) and DRG (D) showed binding to neurons and glial cells. Bars =
C S6S5 S4S3S2 S1C N6N5 N4N3 N2N1
PLS Post-Lyme NormalSLE
SLEPLS Post-Lyme healthy
Total antibody reactivity (AU)
Total antibody reactivity (AU)
Dorsal root ganglia
A Download full-text