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Molecular Immunologic Strategies to Identify Antigens and B-Cell Responses Unique to Multiple Sclerosis
Donald H. Gilden, MD;
Mark P. Burgoon, PhD;
B. K. Kleinschmidt-DeMasters, MD;
R. Anthony Williamson, PhD;
Omar Ghausi, BS;
Dennis R. Burton, PhD;
Gregory P. Owens, PhD
Arch Neurol. 2001;58:43-48.
ABSTRACT
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Identification of the causative agent of multiple sclerosis (MS) has
long eluded investigators and has become the "Holy Grail" of researchers in
the field. The immune response in cerebrospinal fluid of patients with MS,
indicated by an increased IgG level and the presence of specific oligoclonal
bands after electrophoresis, strongly parallels that found in various infectious
diseases of the central nervous system. To understand the nature of B-lymphocyte
activation in MS, 4 laboratories studied the antigen-binding regions of antibodies
found in MS brain demyelinative plaques and cerebrospinal fluid. Each analysis
revealed (1) limited germline expression, results not expected for a random
bystander response; (2) features consistent with a specific antigen–targeted
process; and (3) the clonal expansion of populations of B lymphocytes in MS.
The screening of libraries expressing protein products derived from chronic
MS plaque messenger RNA with antibodies purified from plaques, cerebrospinal
fluid, or serum of patients with MS has thus far not revealed the antigenic
target(s) of the MS antibody response. Because putative MS antigens could
be in low abundance, the screening of large libraries of random peptides expressed
on phage surfaces might offer an alternative approach to identify peptide
sequences recognized by MS antibodies. New sophisticated molecular immunologic
techniques described herein should enhance our ability to identify putative
antigen(s) targets in MS.
INTRODUCTION
Normal cerebrospinal fluid (CSF) has an IgG content that is less than
13% of total protein. In multiple sclerosis (MS), there is an unexplained
elevation of IgG in the CSF to 15% to 30%, visualized as oligoclonal bands
(OGBs) after electrophoresis. Besides MS, OGBs in CSF are typically found
in patients with infectious disorders of the central nervous system (CNS),
such as neurosyphilis, tuberculous and fungal meningitis, and subacute sclerosing
panencephalitis (SSPE) caused by measles virus. In these diseases, OGBs have
been shown to be antibodies directed against the disease-causing organism.
The specificity of OGBs in MS CSF and brain is unknown, but their presence
suggests the possibility of an underlying infection. This review focuses on
analyses of intrathecally synthesized oligoclonal IgG to identify specific
antigens in MS brain against which the humoral immune response might be directed.
Exactly which tissue to target to identify the putative MS antigen is
problematic. Frozen tissue samples of acute MS brain plaques would be the
best choice for analysis, but they are usually unavailable. Even the definition
of what are the earliest actively demyelinating plaques is uncertain. Attempts
to identify a specific and uniform temporal stage–dependent cascade
of specific inflammatory cells and immune mediators in demyelinative lesions
have been problematic1 because immunohistochemical
analyses of MS plaques have revealed great heterogeneity.2
The presence of macrophages expressing the MRP-14 protein (a marker of presumed
early macrophage activity) seems to identify the earliest phases of myelin
breakdown in some patients (John Prineas, MD, oral communication, December
6, 1999). Targeting plaque tissues for T cells is also useful because T cells
appear early in lesions, followed by B-cell influx, but both may be transient
and hence are less reliable markers of acute-hyperacute lesions.
In the late 1990s, 4 laboratories independently used molecular immunologic
and biologic strategies and techniques to study the humoral immune response
in MS. Two groups studied MS brain and 2 studied MS CSF. Each laboratory used
equivalent strategies that extracted RNA from brain or CSF, reverse transcribed
the RNA into complementary DNA (cDNA), amplified the cDNA with primers specific
for immunoglobulin heavy-chain genes, cloned the amplified IgG-specific DNA,
and sequenced and analyzed the expressed genes. We describe their findings,
which were remarkably similar, and their significance, with implications for
further study of the humoral immune response in MS.
ANTIBODY STRUCTURE AND FUNCTION
A brief review of antibody structure and function serves as a starting
point for understanding recent studies that examined the humoral immune response
in MS. Antibody molecules are proteins, known collectively as immunoglobulins,
and are produced by activated B cells. Immunoglobulin antibodies bind specifically
to particular molecules known as antigens. Immunoglobulin binding can neutralize
pathogens or mark them for uptake and destruction by phagocytes. There are
5 main classes of immunoglobulins (IgM, IgD, IgA, IgG, and IgE) that are distinguished
by the immunoglobulin heavy-chain constant region. IgG is the most abundant
class of antibody in human serum, human CSF, and the brain of patients with
MS.
Although all antibodies have the same overall structure, each antibody
molecule has unique sequence differences that confer specificity for a particular
antigen. Figure 1A shows the 2 identical
heavy- and 2 identical light-chain regions of IgG. Each heavy- and light-chain
region contains V (variable) and C (constant) domains. One pair of heavy-
and light-chain V regions at the "arms" of the IgG molecule binds to a specific
antigen. Thus, each intact IgG molecule has 2 binding sites. Within the V
regions of the heavy and light chains, there are 3 hypervariable regions (1,
2, and 3), the most diverse of which is the third region. The rest of the
V domains between the hypervariable regions are termed the framework regions
(FR1, FR2, FR3, and FR4).
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Figure 1. A, The IgG molecule comprises
2 identical pairs of heavy (H) and light (L) chains, covalently linked by
disulfide bonds. The H chains contain a variable (VH) and 3 constant
(CH) domains, whereas each L chain contains only 1 variable (VL) and 1 constant (CL) domain. The variable domains (VH and VL, in blue) combine to bind a specific antigen. B,
Polymerase chain reaction (PCR) strategy to generate the IgG antibody repertoire.
Nested pairs of primers (Rev L and Zap L in the adjoining vector sequence,
CH-1 in the constant region, and CH-J in the junction between the constant
and variable regions) were used to PCR amplify the variable regions (VH) of complementary DNAs representing IgG H-chain messenger RNA. Within
each VH domain are 4 framework regions (in green) that provide
the scaffolding for the complementarity-determining regions (CDRs) that bind
to antigen in the intact antibody.
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The framework regions provide the structural backbone of the antigen-binding
domain. When the VH and VL domains pair in the antibody
molecule, the hypervariable loops from each domain are brought together to
form the antigen-binding site. Because the 3 hypervariable loops constitute
the binding site for antigen and determine specificity by forming a surface
complementary to the antigen, these hypervariable loops are termed the complementarity-determining
regions (CDRs) and are denoted CDR1, CDR2, and CDR3. The combination of heavy-
and light-chain CDRs determines the final antigen specificity. Thus, one way
in which the immune system is able to generate antibodies of different specificities
is by generating different combinations of heavy- and light-chain variable
regions.
The complete collection of antibody specificities available within an
individual is known as the antibody repertoire and in humans consists of as
many as 1011 or more different antibody molecules. The entire antibody
repertoire is generated by recombination events involving limited numbers
of heavy- and light-chain genetic elements. The heavy-chain V region (VH) is more diverse and is generated by recombination of a heavy-chain
variable segment, a D segment, and a J segment during B-cell development.
In humans, there are approximately 51 functional VH segments, 30
functional D segments, and 6 functional J segments. Recombination of these
segments provides an enormous number of antibody combinations. Whereas the
VH segment determines CDR1 and CDR2, the entire D segment and the
amino terminal end of the J segment (Figure
1B) encodes the heavy-chain (H) CDR3. Because the recombination
mechanism that joins these segments is not precise and additional nucleotides
can be added or deleted at the V-D and D-J junctions during recombination,
the HCDR3 sequence is the most diverse. In fact, the chances of 2 different
B cells forming identical CDR3 sequences during development is so remote that
the HCDR3 sequence can be used as a clonal marker. Thus, in the VH
repertoire analyses we review herein, any VH sequences that share
the same or almost identical CDR3 sequence, but contain other discrete sequence
differences, are considered clonally related variants. These variants have
originated from a common B-cell ancestor that was activated and underwent
clonal expansion and somatic mutation (detailed in the following section).
Any substance can elicit an antibody response, and the response even
to a simple antigen is diverse, comprising many different antibody molecules,
each with a unique affinity and fine specificity. The diversity of the antibody
repertoire reflects not only the fact that there are separately inherited
genes for each different antibody chain, but, more important, that somatic
mutation occurs. Somatic mutation refers to the alterations that occur in
the V region sequences of activated B cells after antigenic stimulation.
EVIDENCE FOR AN ANTIGEN-DRIVEN IMMUNE RESPONSE IN MS
The target of the humoral response in MS is unknown, but several possibilities
exist. First, B-cell activation may result from antigenic stimulation (foreign
or self) targeted to a specific molecule(s). Second, activation could result
from a nonconventional mechanism such as B-cell superantigen stimulation.
B-cell superantigens are proteins, usually viral or bacterial, that activate
B cells polyclonally irrespective of antigen specificity but dependent on
some feature of variable domain framework architecture. Third, activation
could be a random bystander effect of the inflammatory response seen in MS
plaques. The first studies to distinguish between random and targeted humoral
immune responses were based on the premise that OGBs are synthesized within
the brain because plasma cells and large quantities of IgG messenger RNA (mRNA)
are present in MS plaques but not in normal human brain white matter. A strategy
using the polymerase chain reaction yielded a representative sampling of the
IgG VH repertoire expressed in MS plaques that was sequenced and
analyzed (Figure 1).3
The VH repertoire shared many hallmarks associated with antigen-driven
responses, including a skewed distribution of VH germline segments
in multiple brain plaques compared with peripheral blood, extensive somatic
mutation, preferential accumulation of amino acid replacement mutations in
the antigen-binding CDRs, and the presence of distinct sequence differences
in some overexpressed populations (clonal variants), indicative of clonal
expansion.
A second, larger study4 compared the
VH repertoire in MS plaques with that found in SSPE in which CSF
and brain OGBs result from a continuous antigen-driven response directed against
measles virus. First, when the IgG sequences in SSPE brain were expressed
as antibody, they were shown to be directed against measles virus antigen.5 Second, when the VH sequences in MS and
SSPE brain were further analyzed, sequence variation was found. Instead of
the 98% to 100% homology normally found among VH genes in different
individuals (human polymorphisms account for 1%-2% variation), the average
homology with germlines was only 92%, indicative of extensive somatic mutation
in MS and SSPE brains. Third, specific VH sequences in SSPE and
MS brains were overrepresented, and many sequences preferentially accumulated
replacement mutations in CDRs relative to the framework regions. Comparison
of VH family and germline usage showed that patients with SSPE
or MS had a response characteristic of a targeted, antigen-driven process.
Similar conclusions regarding the humoral response in MS were also reached
in studies6 that analyzed the length and amino
acid structure in the CDR3 regions of IgG in 10 additional MS brains. Compared
with the peripheral VH repertoire in normal blood, most MS brains
displayed skewed VH gene usage, arguing against a random B-cell
response. Furthermore, 2 independent studies7, 8
of the VH repertoire in CSF of different patients with MS also
revealed the same features. Overall, these molecular immunologic findings
in MS, where the antigen is unknown, are similar to the humoral response in
SSPE, where the antibodies are known to be directed against measles virus.
If the antibody responses in MS were due to superantigen stimulation, consistent
restricted use of the same germline segments would be expected. While VH4 germline segments predominated in the first MS brain analyzed,3 analyses of additional MS brains revealed a unique
response profile in each brain, again inconsistent with superantigen stimulation
but consistent with an antigen-driven, targeted response.
cDNA EXPRESSION LIBRARY SCREENING
The IgG antibody in MS brain and CSF may be directed against an antigen
(protein) crucially involved in the disease process. Furthermore, this putative
MS antigen has probably been translated from mRNA expressed in MS brain. Thus,
it would be valuable to analyze all the mRNAs expressed in MS brain to survey
their encoded proteins for antibody specificity. To do so, all the RNA extracted
from MS brain is first reverse transcribed into cDNA. The cDNA is then cloned
into a suitable cloning vector to create a cDNA library. This approach uses
special cloning vectors, called  expression vectors, in which the
cloned DNA is transcribed into the complementary mRNA, which in turn is translated
into the encoded protein.
When replicate nitrocellulose filters are prepared from a cDNA library
constructed in a expression vector, fusion proteins expressed from
each individual clone are bound to the nitrocellulose filter. Thousands of
individual clones can be screened on 1 filter. The replicate filter can be
screened by procedures capable of detecting specific fusion proteins. For
example, an antibody specific for a protein of interest, eg, IgG from MS brain
or CSF, can be incubated with replicate filters of a cDNA expression
library. If one of the clones expresses a fusion protein that includes
the region of the protein bound by the antibody, antibody molecules will bind
to the filter at the position of that specific clone. After washing the filter
to remove unbound antibody, the position of the specific clone is detected
by incubation with a second radioactively labeled antibody that recognizes
the first antibody, followed by autoradiography of the filter.
We screened cDNA expression libraries prepared from acute and chronic
plaques in MS brain. Two cDNA libraries prepared from pathologically verified
chronic plaques were screened with pooled MS CSF containing OGBs and with
IgG extracted from MS CSF or MS serum, but they did not reveal MS-specific
antigens.9 Furthermore, immunoblotting with
pooled MS CSF IgG or IgG eluted from MS plaque-periplaque white matter did
not reveal myelin proteins or any MS-specific antigens in MS or normal brain
white matter.
Another group searched for possible CNS-specific autoantigens in MS
by screening cDNA expression libraries generated from an oligodendrocyte precursor
cell line.10 A library screened with pooled
CSF from 54 patients with MS detected 6 positive clones, of which 5 contained
a common 7–amino acid sequence highly homologous to the translation
product of an alu repeat sequence. Subsequent screening
with serum and CSF samples from patients with MS showed that nearly half reacted
with these so-called alu peptides. Although alu sequences compose 5% of the human genome, the expression
of these encoded peptides in the brain or their relevance to the pathogenesis
of MS is not known.
PHAGE DISPLAY LIBRARIES TO IDENTIFY RELEVANT ANTIBODIES AND ANTIGENS
IN MS
Identification of the antibodies in the CNS and CSF of patients with
MS that correlate with disease is a logical first step in discerning their
corresponding antigens. Recombinant antibody technology combined with phage
display offers a novel strategy to identify disease-relevant IgG and their
corresponding antigens in MS. Phage display technology involves the fusion
of foreign DNA fragments to genes encoding filamentous phage coat proteins.
A phage (abbreviated from bacteriophage) is a virus that infects bacteria.
Antibody-phage libraries rapidly generate specific high-affinity antibodies
from immune donors.11 Recombinant antibody
libraries have been selected against purified or crude antigenic preparations,
yielding antibodies that specifically recognize a variety of viral pathogens
and autoantigens.12, 13 In antibody
libraries, sequences encoding IgG Fab (antibody binding) fragments from target
tissue (eg, MS brain) are amplified by polymerase chain reaction and cloned
sequentially into a phage display vector, yielding random combinations of
IgG heavy- and light-chain binding fragments. Each Fab fragment is fused to
the gene encoding phage coat protein III, which generally affords monovalent
display of the recombinant antibody on the phage surface. This approach has
been used to create phage libraries containing diverse repertoires of more
than 107 antibody clones. Repeated testing of the library against
a chosen antigenic preparation ("panning") leads to the recovery of monoclonal
antibodies that react specifically with the selecting antigen. Successive
panning rounds also reduce the diversity of phage-Fabs binding to the antigen
to yield antibody clones with the highest affinity for antigen. Figure 2 outlines the production and application of phage-antibody
libraries in chronic CNS infections. This technology can be applied to MS
by cloning the antibody response localized in and around acute MS plaques
and in CSF of patients with MS. Antigenic preparations of MS brain tissues
can then be used to select specific disease-associated antibodies within the
phage library.
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Figure 2. Strategy for cloning human monoclonal
antibodies elicited by disease-relevant antigens in central nervous system
inflammatory diseases. Recombinant antibody–phage display libraries
are constructed using RNA prepared from active-acute plaque regions of flash-frozen
multiple sclerosis (MS) brains or from B cells present in cerebrospinal fluid
(CSF). Monoclonal antibodies of interest are then selected from the phage
libraries through successive rounds of panning against antigenic presentations
of active-acute tissues. HC and LC IgG indicate heavy and light chains, respectively.
PCR indicates polymerase chain reaction.
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The feasibility of this approach was demonstrated in a test system using
an antibody Fab-phage library prepared from gray and white matter of SSPE
brain. Panning against lysates of measles virus–infected cells14 or sections of SSPE brain15
selected Fabs that immunostained SSPE brain tissue and reacted with measles
virus–specific antigens. Thus, IgG mRNA expressed in brain during a
chronic CNS infection can be used to generate high-affinity antibodies that
recognize antigens of the disease-causing pathogen.15
Applying recombinant antibody technology combined with phage display
in a CNS inflammatory disease of unknown cause such as MS requires that panning
be performed on diseased tissue. The presence of endogenous antibody in brain
tissues may reduce the efficiency of the panning, particularly when the target
antigen is present in low abundance and possibly masked by being bound to
endogenous antibody. In such cases, endogenous antibody can be eluted before
panning. However, when panning on SSPE brain, elution was not necessary to
recover measles-specific recombinant antibodies.15
This might reflect a high level of unbound measles virus antigens in SSPE
brain, in addition to antigen that is complexed by endogenous antibody to
measles virus.16
These findings demonstrate a potential strategy to identify antigens
of potentially infectious agents in the brains of individuals with inflammatory
disease of unknown cause. The recent technical success in identifying disease-relevant
antibodies in SSPE awaits application to MS, once specific reactivity of intrathecal
IgG extracted from MS brain has been demonstrated.
PEPTIDE LIBRARIES FOR SELECTION OF MS-SPECIFIC TARGET EPITOPES
Random peptide libraries displayed on the surface of phage represent
another potentially valuable technique to characterize the humoral immune
response in human disease. Antibodies from the brain or CSF of patients with
MS can be used to identify their corresponding protein epitopes, however rare,
from a peptide library. Epitope libraries contain exhaustive arrays of short
nucleotide sequences that encode random amino acids at each of 6 or more positions.
These sequences are displayed on the surfaces of phage.17
Libraries containing all possible hexapeptides, ie, the functional size limit
of most antibody epitopes, would contain 1 billion combinations and challenge
the upper size limit for a workable library. But libraries of several hundred
million independent clones with longer peptide insertions of about 15 amino
acids also contain enough potential hexapeptide sequences and are easier to
construct.
Using panning protocols similar to the Fab display libraries described
previously, approximately 1012 phage-displayed peptides can be
selected over immobilized antibody preparations. The peptide regions in bound
phage are readily sequenced to reveal consensus peptides that correspond to
the antibody's target. Comparison of consensus peptide epitopes to databases
can then reveal clues to the in vivo antigen against which the antibody was
generated, particularly as databases expand. The peptide library technique
is most useful to identify linear epitopes of antibodies, and even perhaps
competing epitopes of antibodies against nonpeptide targets. One potential
pitfall of this technique is the detection of mimotopes, peptides that mimic
the original antigen binding but do not share sequence homology with the native
antigen and therefore are not useful in database searches. Because this technique
can be confounded by polyclonal antibody responses, it is best suited for
panning on single antibodies. So far, one group has reported18
that antibody from MS CSF identified a 5–amino acid consensus sequence
in 5 of 14 patients. The same peptide sequence is present in the Epstein-Barr
virus nuclear antigen and in  ß-crystallin, a heat-shock protein
active in some MS myelin extracts. The relevance of these findings to MS is
still being investigated.
FUTURE DIRECTIONS
In addition to the strategies and techniques described herein that have
been applied to MS, another immunologic strategy for pathogen detection and
identification is based on the use of new gene microchip array techniques.
Screening which genes are activated in response to a specific infectious agent
may result in characteristic host molecular signatures generated in response
to infection or toxins. Specific microbial stimuli may elicit a characteristic
gene expression response profile that can serve as a diagnostic signature
of infection. The collection and analysis of expression response profiles
from cells exposed to infectious agents and toxins in vitro, and expression
response profiles from peripheral blood mononuclear cells of healthy individuals,
uses custom-designed human cDNA microarrays with 18 000 elements, representing
more than 15 000 expressed genes. Early results suggest that expression
profiles can discriminate between different members of the same bacterial
genus and between specific virulence factors, eg, toxins.19
Multiple studies have elucidated the nature of the humoral immune response
in MS and have revealed the hallmarks of an antigen-driven response. Now,
the presence of a novel antigen can be investigated with the most sophisticated
and sensitive techniques of molecular immunology.
AUTHOR INFORMATION
Accepted for publication June 29, 2000.
This work was supported in part by grants NS 32623 (Dr Gilden) and A1
39162 (Dr Burton) from the Public Health Service, Bethesda, Md.
We thank Marina Hoffman for editorial review and Cathy Allen for preparing
the manuscript.
From the Departments of Neurology (Drs Gilden, Burgoon, Kleinschmidt-DeMasters,
and Owens and Mr Ghausi), Microbiology (Dr Gilden), and Pathology (Dr Kleinschmidt-DeMasters),
University of Colorado Health Sciences Center, Denver; and the Department
of Immunology, Scripps Research Institute, La Jolla, Calif (Drs Williamson
and Burton).
Corresponding author and reprints: Donald H. Gilden, MD, Department
of Neurology, University of Colorado Health Sciences Center, 4200 E Ninth
Ave, Mail Stop B182, Denver, CO 80262 (e-mail: don.gilden{at}uchsc.edu).
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