ONCHOCERCIASIS
2017 © The Authors,
some rights reserved;
exclusive licensee
American Association
for the Advancement
of Science.
Nodding syndrome may be an autoimmune reaction to
the parasitic worm Onchocerca volvulus
Tory P. Johnson,
1
Richa Tyagi,
1
Paul R. Lee,
1
Myoung-Hwa Lee,
1
Kory R. Johnson,
2
Jeffrey Kowalak,
3
Abdel Elkahloun,
4
Marie Medynets,
5
Alina Hategan,
1
Joseph Kubofcik,
6
James Sejvar,
7
Jeffrey Ratto,
8
Sudhir Bunga,
8
Issa Makumbi,
9
Jane R. Aceng,
9
Thomas B. Nutman,
6
Scott F. Dowell,
10
Avindra Nath
1
*
Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is
an epidemiological association with Onchocerca volvulus, the parasitic worm that causes onchocerciasis (river
blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding
syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoanti-
bodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls
from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients
with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro
and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cor-
tical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting
leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome
were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that
nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and
suggests that patients may benefit from immunomodulatory therapies.
INTRODUCTION
Nodding syndrome is an epileptic disorder typified by atonic seizures
that affects children between 5 and 15 years of age in geographically
localized regions of Tanzania, Uganda, and the Republic of South Su-
dan (1). The atonic seizures are characterized by a head-dropping mo-
tion, but patients may also develop clonic-tonic seizures, atypical
absence seizures, mild to severe cognitive impairment, and cerebellar,
cerebral, and hippocampal atrophy (1–5). Nodding syndrome is a
disabling disease, resulting in neurological deterioration and, in some
cases, death (1, 2). The clinical characteristics of nodding syndrome are
distinct from other epileptic disorders in children.
Because of an increase in reports of nodding syndrome (6), rigorous
efforts to understand this disease have been undertaken (2). These studies
have resulted in a consensus case definition and clinical characterization
of nodding syndrome (1–4). However, the pathophysiology and etiology
of nodding syndrome remain unknown. Extensive investigation of
environmental neurotoxins, nutritional deficiencies, genetic disorders, or
infectious organisms has been unrevealing (2). An increased rate of
nodding syndrome in areas where the parasite Onchocerca volvulus is
endemic led to the hypothesis that the infection may play a role in
nodding syndrome pathogenesis (6). Case-control studies have
consistently documented an association between nodding syndrome
and O. volvulus infection but have failed to find evidence of invasion
of the brain or cerebrospinal fluid (CSF) by the mature parasite (2, 5, 7),
although prelarval worms (microfilariae) have been detected in the CSF
(8). It has thus been hypothesized that an immune-mediated mecha-
nism may be involved. Previous investigations of autoantibodies known
to be associated with neurological illness have been unrevealing in
nodding syndrome [as described in (2, 9)]. The aim of the current study
was to further investigate whether autoantibodies could be a contributing
factor to the pathogenesis of nodding syndrome.
RESULTS
Autoantibodies in patients with nodding syndrome
An unbiased approach for profiling autoantibodies using a protein
array detected a >2-fold increase in reactivity to 167 probes represent-
ing 137 individual proteins and a >100-fold increase in four proteins
in pooled sera from patients with nodding syndrome compared to
pooled sera from unaffected control villagers (Fig. 1A and table S1).
The top two signals were from autoantibodies to leiomodin-1 (increased
33,000-fold) and autoantibodies to DJ-1 (increased 750-fold). Further
examination of the top four enriched autoantibodies in patients with
nodding syndrome (table S2) demonstrated differential immuno-
reactivity by immunoblot analyses between pooled serum samples
from patients with nodding syndrome and controls for only two
of the proteins, leiomodin-1 and DJ-1 (Fig. 1B). However, only anti-
bodies to leiomodin-1 (and not to DJ-1) were detected in the CSF of
patients with nodding syndrome (Fig. 1C). Serum samples from each
of the patients with nodding syndrome and unaffected village controls
were analyzed for reactivity to leiomodin-1 by enzyme-linked immu-
nosorbent assay (ELISA) (Fig. 1D and Table 1); a subset of samples was
1
Section of Infections of the Nervous System, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
2
Bioinformatics Section, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, MD 20892, USA.
3
Clinical Proteomics Unit,
National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, MD 20892, USA.
4
Microarray Core Facility, National Human Genome Re-
search Institute, National Institutes of Health, Bethesda, MD 20892, USA.
5
Neural
Differentiation Unit, National Institute of Neurological Disorders and Stroke, National
Institutes of Health, Bethesda, MD 20892, USA.
6
Helminth Immunology Section, Lab-
oratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Na-
tional Institutes of Health, Bethesda, MD 20892, USA.
7
Division of High-Consequence
Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Dis-
eases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
8
Division
of Global Health Protection, Center for Global Health, Centers for Disease Control and
Prevention, Atlanta, GA 30333, USA.
9
Ministry of Health, Kampala, Uganda.
10
Bill &
Melinda Gates Foundation, Seattle, WA 98109, USA.
*Corresponding author. Email: natha@ninds.nih.gov
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confirmed by immunoprecipitation (fig. S1). Leiomodin-1 antibodies
were more frequently detected in patients with nodding syndrome com-
pared to unaffected village controls: 29 of 55 (52.7%) patients with
nodding syndrome versus 17 of 55 (30.9%) unaffected village controls
[P = 0.024, mOR, 2.7; 95% confidence interval (CI), 1.1 to 6.5]. In
patients with nodding syndrome with determined O. volvulus status
(n = 54), 44 patients were O. volvulus–positive.Ofthese,24patients
(54.5%) were positive for both O. volvulus and leiomodin-1 antibodies.
Twenty patients (45.5%) were O. volvulus–positive and leiomodin-1 antibody–
negative. In unaffected village controls, there were 54 individuals with
determined O. volvulus status. Of these controls, 29 were O. volvulus–positive.
Twelve of these 29 controls (41.4%) were leiomodin-1 antibody–positive
and O. volvulus–positive, and 58.6% were leiomodin-1 antibody–
negative and O. volvulus–positive. Thus, in patients with nodding
syndrome, there was a higher percentage of O. volvulus–positive status
in leiomodin-1 antibody–positive cases than in leiomodin-1 antibody–
negative cases. The same did not hold
true for unaffected village controls.
Additionally, patients with nodding
syndrome had increased titers of anti-
bodies to leiomodin-1 compared to un-
affected village controls (P =0.04,
ANOVA with Holm-Sidak correction
for multiple comparisons). Both immu-
noglobulin G (IgG) and IgM antibodies
directed against leiomodin-1 were pres-
ent in the sera of patients with nodding
syndrome (fig. S2). Fifty per cent (8 of
16) of patients with nodding syndrome
showed antibodies to leiomodin-1 in
the CSF, whereas none (0 of 8) of the
North American patients with epilepsy,
as a control, demonstrated antibodies to
leiomodin-1 in their CSF (P =0.022,
Fisher’sexacttest).
To confirm that antibodies in the sera
from patients with nodding syndrome
recognized leiomodin-1, human embry-
onic kidney (HEK) cells transfected
with leiomodin-1 DNA were co-stained
with rabbit anti–leiomodin-1 antibody
and sera from a patient with leiomodin-1
antibodies or sera from an unaffected
village control without antibodies to
leiomodin-1. Colocalization of nodding
syndrome patient sera, but not the sera
from unaffected village controls, with
leiomodin-1 was observed (Fig. 1E and
fig. S3). To confirm that the antibodies
in the CSF from patients recognized
neuronal leiomodin-1, we stained hu-
man neurons with CSF from two pa-
tients with leiomodin-1 antibodies in
conunction with rabbit anti–leiomodin-1.
Colocalization of the human anti-
bodies from CSF and the rabbit anti–
leiomodin-1 antibodies was observed
on neurons (fig. S4).
Expression of leiomodin-1 in the CNS
Because leiomodin-1 has been reported to be expressed primarily in
smooth muscle tissue and in the thyroid (10, 11), we next examined
the expression of leiomodin-1 in the central nervous system (CNS).
Using multiple approaches, we confirmed that leiomodin-1 is ex-
pressed in the CNS and in neurons. Immunostaining of human neurons
with rabbit anti–leiomodin-1 antibody demonstrated leiomodin-1 expres-
sion throughout the cytoplasm of neurons (Fig. 2A). Immunoblots
demonstrated the presence of leiomodin-1 in adult human brain homo-
genates (Fig. 2B). Localization of leiomodin-1 in mouse brain by im-
munohistochemistry using rabbit anti–leiomodin-1 antibody showed
that it was focally expressed in cortical neurons in the cerebral cortex,
Purkinje cells in the cerebellum, and pyramidal cells in the CA3 region
of the hippocampus. Other regions of the hippocampus incl ud in g CA1
and the dentate gyrus did not express leiomodin-1 (Fig. 2C). Smooth
muscle from blood vessels and skeletal muscle (Fig. 2D) were used as
A BC
185
115
80
50
30
15
10
Leiomodin-1
UVC NS
1
10
100
1000
10,000
100,000
Leiomodin-1
DJ1
COX4I2
C7orf10
Log
10
signal NS/HC
185
115
80
50
30
15
Pooled CSF
DJ-1
Leiomodin-1
Brain
Leiomodin-1 + tag
123
Leiomodin-1 DAPI
Patient sera
Merge
DE
Leiomodin-1
NS
UVC
HC
0.0
0.5
1.0
1.5
OD
P = 0.04
P = 0.0001
185
115
80
50
30
15
10
DJ-1
UVC NS
Fig. 1. Leiomodin-1 autoantibodies in patients with nodding syndrome. (A) Log
10
-fold distribution plot depict-
ing autoantibody reactivity differences between patients with nodding syndrome (NS) and unaffected village controls
(UVC). Annotated on the graph are four proteins observed to have a >100-fold difference between nodding syndrome and
unaffected village controls. (B) Immunoblot analyses of leiomodin-1 and DJ-1 immunoreactivity in sera from unaffected
village controls or nodding syndrome patients. (C) Immunoblot analysis of recombinant DJ-1, recombinant leiomodin-1,
and human brain homogenate probed with CSF pooled from 16 patients with nodding syndrome. In lane 1, there is no
immunoreactivity to recombinant DJ-1. In lane 2, there is immunoreactivity to histidine-tagged leiomodin-1 (arrow;
~80 kDa). Lane 3 shows immunoreactivity to a single protein in brain homogenate at ~60 kDa, the molecular mass of
leiomodin-1 (arrow). (D) Scatterplot depicting optical density (OD) of individual patient serum’s immunoreactivity to
leiomodin-1 as determined by ELISA. The cutoff for determining a positive sample was set at 3 SD above the mean for
U.S. healthy control sera (HC; n = 20). Data were analyzed by analysis of variance (ANOVA) that showed an overall
significant difference (P = 0.0001), and a Sidak-Holms correction was applied to correct for multiple comparisons:
nodding syndrome (n = 55) versus unaffected village controls (n = 55), P = 0.04. Data were also log-transformed
and reanalyzed with consistent findings. (E) Coimmunostaining with patient sera (green) and rabbit anti–leiomodin-1 anti-
body (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 20 mm.
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positive controls. Mining publicly available RNA sequencing data, we con-
firmed that leiomodin-1 is expressed in the human brain (fig. S5). This was
confirmed by detecting leiomodin-1 messenger RNA (mRNA) in human
brain tissue extracts and human neuronal stem cells and cultured neurons
(Fig.2EandtableS3).Therewasahigher expression of leiomodin-
1 mRNA in brain extracts compared to the expression in human neural
stem cells and differentiated neurons.
Leiomodin-1 antibodies are neurotoxic
To determine whether antibodies to leiomodin-1 could cause neuronal
dysfunction, we treated cultured human neurons with leiomodin-1 anti-
bodies or control serum and assayed them for viability. Neurons treated
with anti–leiomodin-1 antibody showed decreased viability (mean vi-
ability ± SD, 40.9 ± 9.3%) compared to treatment with normal rabbit
sera (mean viability ± SD, 75.5 ± 12.8%; P =3.4×10
−9
,two-wayStudent’s
paired t test) (Fig. 3A and table S4). Neurons treated with sera from
a patient with nodding syndrome (1:200) with detectable leiomodin-
1 antibodies demonstrated neurotoxicity (mean viability ± SD, 14.7 ±
1.0%) that was abrogated by antibody depletion from the patient sera
(mean viability ± SD, 78 ± 12.8%; P = 0.0003, two-way Student’s
paired t test) (Fig. 3B and table S5). Neurons exposed to sera from
patient s with detectable leiomodin-1 antibodies (n = 4) showed
increased neurotoxicity (mean toxicity ± SD, 52.71 ± 24.9%) com-
pared to sera selectively depleted of le iomo din- 1 an ti bodi e s by af fi nity
purification (mean toxicity ± SD, 33.16 ± 19.8%; P = 0.0048, two-way
Student’spairedt test) (Fig. 3C and table S6). The neurotoxicity induced
by autoantibodies to leiomodin-1 from patients with nodding syndrome
in conjunction with the expression of leiomodin-1 in the CNS suggests
that antibodies directed against leiomodin-1 may play a role in nodding
syndrome disease pathogenesis.
Leiomodin-1 antibodies cross-react with
O. volvulus proteins
Epidemiological studies have indicated that nodding syndrome is
associated with O. volvulus (2, 4–6, 12–14). Therefore, we investigated
whether autoantibodies to leiomodin-1 could be related to infection
with O. volvulus. Screening of antigen prepared from adult male
and female O. volvulus with pooled sera from patients with nodding
syndrome and from unaffected village controls by immunoblotting
showed limited differential immunoreactivity profiles (Fig. 4A). Bands
to which nodding syndrome patient sera demonstrated increased im-
munoreactivity were excised from the gel and identified by mass spec-
trometry (table S7). O. volvulus tropomyosin was one of the proteins
identified. O. volvulus tropomyosin protein has 25.5% sequence identity
and 37% sequence similarity to leiomodin-1 (Fig. 4B). Although the
overall homology is low, regional homology analyses indicated that
the conserved N-terminal DAIKK sequence of O. volvulus tropomyosin
(15), amino acids 2 to 14, showed 57.1% identity and 71.4% similarity to
amino acids 107 to 117 of human leiomodin-1. The conserved signature
sequence LKEAExRAE (15), amino acids 230 to 240 of O. volvulus
tropomyosin, showed 66.7% identity and 83.3% similarity to amino
acids 362 to 372 of human leiomodin-1. Although many of the proteins
from O. volvulus remain uncharacterized, we hypothesized that there
may be homology between human leiomodin-1, a member of the tro-
pomodulin gene family (10), and tropomodulin from O. volvulus.
Modeling of human leiomodin-1 and O. volvulus tropomodulin demon-
strated regions of structural overlap (Fig. 4C, left). Further modeling
showed that the second hit in our screen, human D J - 1 , also h a d st r ong
homology to the O. volvulus homolog (Fig. 4C, right). This in silico analysis
su ggested that infectio n with
O. volvulus may generate antibodies cross-
reactive to host proteins. We therefore aimed to determine whether
autoantibodies to leiomodin-1 cross-reacted with O. volvulus proteins.
Affinity-purified leiomodin-1 antibodies from four patients with
nodding syndrome showed a single band after addition of lysates fr o m
HEK cells expressing leiomodin-1, indicating that these antibodies were
specific for human leiomodin-1 (Fig. 4D). However, these antibodies
also robustly recognized several proteins in an O. volvulus whole-organism
lysate, suggesting that several O. volvulus proteins may share homology with
human leiomodin-1 (Fig. 4D). Further confirmation of the cross-reactive
nature of these antibodies was demonstrated by competing for the
binding of leiomodin-1 by nodding syndrome patient sera (n =4)after
preincubati on of patient sera with either O. volvulus whole-organism lysate
or a nonspecific protein, BSA. Sera that were preincubated with O. volvulus
lysate showed decreased immunoreactivity to leiomodin-1 (mean OD, 78.3 ±
89.3) compared to sera preincubated with BSA (mean OD, 236.7 ± 124; P =
0.042, one-way Student’sunpairedt test) (Fig. 4E and table S8).
DISCUSSION
Nodding syndrome is a devastating disease for children, families, and
communities. A 2012–2013 survey estimated that there were about 2000
cases in Northern Uganda alone (2, 16). Desp it e inte rn at io nal efforts to
identify the etiological factors, the pathophysiology of nodding syn-
drome remains elusive, and no cure is available (12). Here, we hypothe-
sized that an autoimmune process contributes to nodding syndrome.
To address this hypothesis, we compared affected patients and un-
affected village controls by conducting detailed immunotyping for auto-
reactive antibodies. Our initial attempt to discover autoantibodies by
Table 1. Patient demographics, Onchocerca infection status, and presence of leiomodin-1 antibodies. mOR, matched odds ratio; NS, not significant.
Patients with nodding syndrome (n = 55) Unaffected village controls (n = 55) P; mOR (range)
Age (years), mean (range) 11.8 (5–16) 10.8 (6–17) NS
Gender (% male) 52.7% 45.5% NS
Onchocerca status (% positive)
80% (44 positive,
10 negative,
1 indeterminate)
52.7% (29 positive,
25 negative,
1 indeterminate)
0.008; 3.8 (1.4–10.2)
Serum leiomodin-1 antibody status
(% positive)
52.7% 30.9% 0.024; 2.7 (1.1–6.5)
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using a protein chip showed the presence of antibodies to multiple anti-
gens. Four of these had levels greater than 100-fold higher in patients
compared to the unaffected village controls, demarcating them from
the other antibodies that showed less than a 10-fold increase. Of the four
proteins, autoantibodies to only two, DJ-1 and leiomodin-1, were con-
firmed biochemically to have increased reactivity i n pa t i ents co mpare d
to controls. Although we were able to con-
firm the presence of antibodies to DJ-1 in
sera from patients with nodding syn-
drome, it was not detectable in CSF. In
contrast, antibodies to leiomodin-1 were
present in the sera and CSF of about half
the patients with nodding syndrome.
Heretofore, leiomodin-1 was known to
be present primarily in muscle (10, 11),
but its location in the brain had not been
characterized. Further analyses in our
study showed that leiomodin-1 transcripts
and protein were detectable in the CNS.
The immune response to leiomodin-1 is
of particular interest because we demon-
strated that leiomodin-1 is expressed in
neurons in distinct regions of the mouse
brain, the cerebral cortex, the CA3 region
of the hippocampus, and Purkinje cells in
the cerebellum, areas that correspond with
those hypothesized to be associated with
the clinical manifestations of nodding syn-
drome. For example, patients with nodding
syndrome have cerebral atrophy and asso-
ciated cognitive abnormalities (1, 3, 4, 17).
In animal models of epilepsy, retraction
of dendrites and loss of neurons in the
CA3 region are associated with epilepto-
genesis (18). This is also consistent with
hippocampal and cerebellar atrophy
seen upon magnetic resonance imaging
of some patients with nodding syndrome
(1, 3–5). Leiomodin-1, previously described
as human 64-kDa autoantigen D1 (19),
ac t s as an actin nucleator in muscle cells
(20 ). Except for the description of col d
extremi ti es (3), patients with nodding syn-
drome are not known to develop vascu-
lar symptoms. The specificity of CNS
involvement may indicate that leiomodin-1
is differentially expressed, modified, or regu-
lated in the CNS compared to other tissue
compartments.
In contrast to other autoimmune epi-
lepsies, where the antibodies are targeted
against surface receptors such as AMPAR
(AMPA-selective glutamate receptor),
NMDAR (N-methyl-
D-aspartate receptor),
AMPAR (AMPA-selective glutamate recep-
tors), GABAR (g-aminobutyric acid recep-
tor), VGKC (voltage-gate d potassium
channel), and the glycine receptor (21),
leiomodin-1 is an intracellular antigen.
Other intracellular antigens that have been associa te d with ence ph al it is
and epilepsies include Hu, CRMP-5 (collapsin response mediator pro-
tein 5), GAD-65 (glutamic acid decarboxylase 65), and Ma (21). It may
be that autoreactive T cells are the main mediators of the immuno-
pathology of nodding syndrome with the production of leiomodin-
1 autoantibodies as a consequence of this T cell activation. Alternatively,
A
DAPI
Leiomodin-1
Vybrant Dil
Vybrant Dil
2nd only
185
115
80
50
30
15
10
12
Leiomodin-1
BC
Leiomodin-1
Brain
E
HeLa
NSC
Neuron
Brain 1
Brain 2
Myoblast
0.0
5.0 × 10
–7
1.0 × 10
–6
1.5 × 10
–6
2.0 × 10
–6
0.0002
0.0004
0.0006
D
Leiomodin-1 mRNA (normalized to GAPDH)
Cerebellar vermis
CA1
CA3
CC
Cortex
Fig. 2. Leiomodin-1 is expressed in human brain. (A) Cultured human neurons immunostained with rabbit anti–
leiomodin-1 antibody (green channel). Vybrant Dil labels the cell membrane (red), and DAPI (blue) indicates the
nucleus. Vybrant Dil and secondary antibody (2nd) only (rightmost) . Scale bars, 20 mm. (B) Immunoblot analysis
of human brain homogenates with rabbit anti–leiomodin-1 antibody and recombinant leiomodin-1 plus histidine-
tag as a positive control. (C) Immunohistochemistry demonstrates leiomodin-1 staining in specific areas of the murine
brain. Scale bar, 50 mm. (D) Murine skeletal muscle (top; scale bar, 100 mm) and murine smooth muscle in the wall of a
blood vessel (bottom; scale bar, 50 mm) as positive controls. (E) Quantitative polymerase chain reaction (qPCR) measuring
leiomodin-1 transcripts in HeLa cells (as a low-expressing positive control), human neuronal stem cells (NSCs), human
neurons, brain homogenates from two individuals (brain 1 and brain 2), and H9C2 myoblast cells (as a high-expressing
positive control). Data are expressed as the mean leiomodin-1 transcript levels [normalized to glyceraldehyde-3-
phosphate dehydrogenase (GAPDH)] ± SD from three independent replicates. (Tabulated data are available in the
Supplementary Materials.)
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antibodies to leiomodin-1 may be directly contributing to disease. Al-
though antibodies to intracellular antigens are not classicall y thought
to be pathogenic, antibodies to intracellular neuronal targets such as am-
phiphysin have been shown to cause stiff person syndrome in an animal
model (22). While the molecular mechanisms for antibodies that target
intracellular antigens are not completely elucidated, some studies suggest
cell-penetrating antibodies (22), alterations in antigen localization during
apoptosis (23, 24), and changes in antigen expression within the target
tissue during repair of injury (25). Additional studies of leiomodin-1 ex-
pression in the CNS, especially confirming subcellular localization during
damage and repair processes, will be important.
Epidemiological studies have consistently shown an association
between nodding syndrome and infection with O. volvulus (2, 4–6).
We found that leiomodin-1 antibodies cross-reacted robustly with
O. volvulus antigens and found sequence and structural homology
between leiomodin-1 and O. volvulus tropomyosin and tropomodulin.
Not all O. volvulus proteins have been fully characterized, and other
parasite proteins could share homology.Further,oursecondhit,DJ-1,
had structural homology to its O. volvulus homolog. We speculate that
the typical immune response to O. volvulus infection may be generating
multiple cross-reactive antibodies and that, in some patients, autoanti-
bodies develop, enter the CNS, and cause distinct pathology recognized
as nodding syndrome. However, not all patients with nodding syndrome
had detectable antibodies to leiomodin-1. We speculate that nodding
syndrome may not be a single antibody syndrome, and other investi-
gations have suggested that patients with nodding syndrome may have
antibodies to other neuronal proteins (26).Thepatientsweinvesti-
gated had multiple autoantibodies, as demonstrated by proteomics.
Here, we have characterized the autoantibody with the highest titer
that best distinguished the patients with nodding syndrome from
unaffected village con trols. This sy ndrome is like ly not a diseas e
mediated by a single immune specificity.
We postulate that the age of onset of nodding syndrome may be
related to the initial burden of exposure to O. volvulus in early childhood.
However, it is not clear why the syndrome is not seen in adults. It is
possible that the developing immune system, or ongoing brain devel-
opment, may make children more vulnerable to nodding syndrome. Ad-
ditionally, some patients also have delayed sexual development, suggesting
pituitary-hypothalamic dysfunction (27). In the current study, we have
not investigated whether leiomodin-1 is expressed in these regions of
the brain. The geographical restriction of nodding syndrome relative to
the presence of O. volvulus may be due to interruption of parasite con-
trol in these regions (28) or may suggest that other as yet unidentified
cofactors may also contribute to the pathobiology of nodding syndrome.
This study has several limitations that future studies need to ad-
dress. We examined only a limited number of well-defined patients
and unaffected village controls collected in the public health responses
in Uganda and South Sudan in this cross-sectional discovery study.
Although we found a statistically significant association between
leiomodin-1 antibodies and nodding syndrome, one-third of unaffected
village controls also had detectable antibodies. It may be that these in-
dividuals had early, asymptomatic disease that would progress to recog-
nizable nodding syndrome. However, in this cross-sectional study, it
was not possible to determine whether leiomodin-1 serum antibodies
were an early marker of disease. Future studies, especially longitudinal
studies examining leiomodin-1 antibody titers, are needed, but because
ofthenatureoftheepidemicandtheremote location, such studies will
be challenging. It is possible that other pathophysiological changes in
patients with nodding syndrome allow for penetration of leiomodin-1
antibodies into the CNS. However, at the time of sample collection,
it was not appropriate to co llect CSF from village children not dis-
playing neurological disease. Future studies may be able to determine
whether differences in antibody status in the CNS correlate with dis-
ease. Additionally, using a limited number of patients with antibodies to
leiomodin-1, we demonstrated that these antibodies were neurotoxic.
Although we did not investigate the toxicity of serum samples from un-
affected village controls or patients with nodding syndrome who lacked
detectable leiomodin-1 antibodies and hence could not exclude the pos-
sibility of other neurotoxic substances in these patients, we think that
this is unlikely because the depletion of leiomodin-1 antibodies de-
creased the neurotoxic ity. Another limitation of this study was that only
sera and CSF were investigated, and therefore, potentially important
immune cells such as leiomodin-1–specific T cells could not be charac-
terized. Autoreactive T cells may be the mediators of nodding syndrome
with the production of leiomodin-1 autoantibodies as a result of this
immune response. Further studies investigating the T cell repertoire
of patients in both the periphery and CNS would be informative. De-
spite these limitations, the work reported here provides insight into the
pathophysiological mechanisms that contribute to the development
of nodding syndrome. This syndrome can now be added to a grow-
ing list of autoimmune epilepsies (29, 30).
One mechanism through which autoreactive immune components
may be generated is molecular mimicry. Four criteria have been
established to define a molecular mimicry process, including (i) evi-
dence of an epidemiological association between the pathogen and the
immune-mediated disease, (ii) demonstration of immune cells or anti-
bodies directed against patient tissue containing the antigen, (iii) dem-
onstration of cross-reactivity of immune cells or antibodies specific
to the host antigen with the pathogen, and (iv) immune response to
and reproduction of disease in an animal model by infection or im-
munization (31). Here, we have demonstrated (i) the presence of
AB C
0
20
40
60
80
100
0
20
40
60
80
100
P = 0.0003
0
20
40
60
80
100
P = 3.4 × 10
−9
P = 0.0048
Viability (%)
relative to control
Viability (%)
relative to control
Toxicity
(% of saponin)
Patient sera
Patient sera
anti−leiomodin-1−
depleted
PS
PS–
depleted
Patient 1
Patient 1–
depleted
Normal sera
Anti–leiomodin-1
Saponin
Fig. 3. Leiomodin-1 antibodies are neurotoxic. (A) Viability of primary human neu-
rons treated with rabbit polyclonal anti–leiomodin-1 antibody compared to normal
rabbit sera. Saponin was used as a positive neurotoxic control. Data shown are percent
viability relative to treatment with vehicle only (P =3.4×10
−9
;analyzedbyrepeated-
measures ANOVA with Scheffe’s procedure), with the horizontal bars indicating the
mean viability ± SD for n = 10 replicates. (B) Viability of neurons treated with sera from
patients with nodding syndrome with detectable anti–leiomodin-1 antibodies com-
pared to antibody-depleted sera from the same patient (P = 0.0003, two-way Student’s
paired t test). Horizontal bars are the mean percent viability ± SD relative to cells in culture
medium only of n =5replicates.(C) Neurotoxicity induced by each patient’ssera(PS)was
compared to sera specifically depleted of leiomodin-1 antibodies (PS-depleted). Data
shown are percent toxicity relative to saponin (n =4;P = 0.0048, two-way Student’s
paired t test). [Tabulated data are available for (A) to (C) in the Supplementary Materials.]
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RESEARCH ARTICLE
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