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Department of Parasitology, Tulane Regional Primate Research Center, Tulane University Medical Center, Covington, LA 70433
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Borrelia burgdorferi, the spirochete that causes Lyme
disease, expresses a surface lipoprotein, VlsE (Vmp-like sequence,
expressed), that also undergoes antigenic variation (8).
In the B31 strain of B. burgdorferi, this lipoprotein has a
predicted molecular mass of 34 kDa and is expressed from a site
(vlsE) that is located near the right telomere of the linear
plasmid lp28-1 (8). The sequences of the 5'- and 3'-end
segments of the vlsE-coding region, each segment
encompassing less than one-third of this molecule’s length, are
preserved during the course of vlsE variation
(9). The central portion, namely the cassette segment,
which encodes the whole variable domain, recombines unidirectionally
with 15 similar cassette segments that are located upstream (8, 9) from the vlsE locus. The unidirectional
recombination events that result in VlsE antigenic variation only
affect six regions of the VlsE variable domain (8). These
six variable regions are 
16, 22, 6, 18, 13, and 6 aa in length,
respectively (9). The remainder of the VlsE variable
domain, which is composed of invariable regions of about 9, 16, 7, 25,
10, and 26 aa, remains essentially unchanged during the process of
antigenic variation (9).
The VSG, pilin, and Vmp each can be divided also into invariable and variable domains (10, 11, 12). As with VlsE, small invariable regions are scattered within the latter domains. Although the variable domains are highly immunogenic and serve as the major target of the host immune response (13), the small invariable regions within them as well as the larger invariable domains are not antigenic in natural infections and tend to be conserved among different isolates of the organism (10, 11). Conserved portions are probably responsible for the maintenance of a functional molecular conformation (10, 11). Abs directed to the variable domains are able to effectively kill the organisms that express the corresponding Ags. However, the organisms escape killing by successively expressing different variable domains. Pathogens thus use these variable Ags to divert the host immune response from other Ags that may be protective (13).
Nothing is known about the functional or antigenic properties of the invariable regions within the variable domain of the VlsE. As we analyzed the Ab response to a recombinant protein (P7-1), encoded by a cassette segment of the vls locus of the IP90 strain of Borrelia garinii, we made the following observation. In rhesus monkeys that had been infected with spirochetes of either the JD1 or the B31 strain of B. burgdorferi sensu stricto, Ab to P7-1 appeared within the first 3 wk postinfection (PI) and persisted thereafter, at least until wk 10 PI, the longest time point examined in this initial experiment. How could an Ag fragment so promiscuously and rapidly altered as the variable domain of VlsE (8, 14) elicit such a long-lasting Ab response to a cassette segment from another genospecies? A straightforward answer to this question was that one or more of the invariable regions within the variable domain are antigenic themselves and sufficiently conserved among Borrelia strains and genospecies to retain antigenic cross-reactivity.
To investigate this possibility, we analyzed the antigenicity of the invariable regions of the IP90 cassette segment using standard computer algorithms and determined the percent identity of their amino acid sequences with the corresponding invariable regions of published VlsE cassette segments of B. burgdorferi sensu stricto strains B31 (8) and 297 (15). One of the invariable regions (IRs), namely IR6, showed both the highest levels of antigenicity by the Hopp-Woods algorithm (16) and amino acid sequence identity among Borrelia genospecies and strains. We synthesized a peptide (C6) that encompassed the amino acid sequence of IR6 and assessed its antigenicity by ELISA in B. burgdorferi-infected mice, monkeys, and humans. We also performed antigenic competition experiments, using C6 as competitor, to determine what proportion of the mouse, monkey, and human Ab response to P7-1 was directed to IR6. Exposure of IR6 on the surface of the VlsE molecule was determined by immunoprecipitation of native VlsE of IP90 with a rabbit antiserum raised against the C6 peptide. Exposure of IR6 on the spirochetal surface was assessed by direct immunofluorescence with FITC-labeled anti-C6 Ab and by Ab-dependent, in vitro killing experiments using anti-C6 Ab and IP90 spirochetes. Here we report the results of these studies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys (2–4 years old, Macaca mulatta) were infected by the bite of Ixodes scapularis nymphal ticks that were themselves infected with B. burgdorferi sensu stricto strain JD1 or B31. Mice (6–8 wk old, C3H/HeN, The Jackson Laboratory, Bar Harbor, ME) were infected with B. burgdorferi sensu stricto strain Sh-2-82 (low passage, a gift from Denee Thomas, University of Texas Health Science Center, San Antonio, TX) by s.c. needle inoculation with 1 x 108 spirochetes administered in 1 ml of BSK-H medium (Sigma, St. Louis, MO) or by the bite of B31-infected Ixodes scapularis nymphal ticks. B. garinii strain IP90 (low passage) and B. burgdorferi sensu stricto strain HB19 (high passage, lacking the lp28-1 plasmid encoding VlsE) were originally obtained from the Centers for Disease Control and Prevention (CDC; Fort Collins, CO). When required, spirochetes were cultivated in BSK-H medium as described previously (17).
Cloning, sequencing, and expression of the 7-1 cassette segment of IP90
A library of randomly sheared total DNA from B.
garinii IP90 was constructed in the 
ZAP II bacteriophage vector
(Stratagene, La Jolla, CA) following a procedure described previously
(18). The library was screened with a pool of plasma
collected from rhesus monkeys within the first 10 wk after tick
inoculation with B. burgdorferi JD1. On immunoblots of whole
cell extracts of B. garinii IP90 this plasma pool reacted
strongly only with three components, namely flagellin, an unidentified
60-kDa protein, and an Ag that was the IP90 homologue of the 34-kDa
VlsE of B31 (8). After several rounds of screening, 11
clones were rescued into the pBluescript phagemid (Stratagene), and the
recombinant plasmids were purified and used to transform cells of the
SURE strain of E. coli (Stratagene). Several transformants
were selected from each original clone, the presence of the insert was
confirmed, and one such transformant from each clone was grown, induced
for expression, lysed, and analyzed by immunoblot analysis with the
original plasma pool. One of the 11 cloned fragments (named 7-1)
hybridized to all others by dot-blot hybridization. This fragment was
selected for overexpression and purification on the basis of the strong
reactivity of the expressed protein with the plasma Abs. Sequencing of
the 7-1 insert was performed using standard procedures
(19) after generating nested Bal31 deletions
and subcloning the partially deleted fragments. Ab from the original
plasma pool that was affinity purified using the recombinant protein
(P7-1) expressed by clone 7-1 as immunoabsorbant reacted with the
putative IP90 VlsE on immunoblot of B. garinii lysates. The
7-1 insert was subcloned into the pQE expression system for
overexpression and purification of the polypeptide (Qiagen, Chatsworth,
CA). The protein concentration was determined using the Bio-Rad protein
assay kit (Bio-Rad Laboratories, Richmond, CA) with BSA as a
standard.
P7-1 ELISA
This protocol was initially used to monitor monkey Ab responses
to P7-1. Ninety-six-well ELISA plates (Corning, Corning, NY) were
coated with 100 µl/well of a solution of P7-1 at 0.1 µg/ml in
coating buffer (0.1 M carbonate buffer, pH 9.2) at 4°C overnight.
Plates were blocked with 200 µl/well of 5% FCS in PBS/T (10 mM
sodium phosphate, 150 mM NaCl, and 0.1% Tween 20, pH 7.4) for 1 h
at room temperature. After washing three times with PBS/T, 50 µl/well
of serum samples from B. burgdorferi-infected monkeys
diluted 1/100 in 5% FCS in PBS/T were incubated for 1 h at
37°C. Plates were washed and incubated with 50 µl of 1) a mixture
of biotinylated goat anti-human IgG (
-chain-specific) and IgM
(µ-chain-specific) Abs at the dilution recommended by the
manufacturer (Vector Laboratories, Burlingame, CA) for 1 h at
37°C, 2) avidin-HRP complex also at the dilution recommended by the
manufacturer (Vector) for 30 min at 37°C, and 3) a solution
containing 2 g/l ortho-phenylenediamine and 0.03% hydrogen peroxide
(both from Sigma) in 0.1 M citrate-sodium-phosphate buffer, pH 5.0, for
10 min at room temperature. The reaction was stopped with 50 µl of 4
N H2SO4. The OD at 490 nm
was determined. Goat anti-human IgG and IgM cross-react with the
corresponding monkey Igs.
Identification of conserved sequences and prediction of antigenicity of invariable regions of the VlsE variable domain
The deduced amino acid sequence of P7-1 was compared with sequences available in the GenBank database (National Center for Biotechnology Information, Rockville, MD) using the BlastP algorithm (20). The antigenicity of the entire P7-1 polypeptide was analyzed using the Hopp-Woods scale (16), and identities of invariable regions of P7-1 with homologous cassette segments from strains B31 and 297 were calculated after aligning the cassette segments using the pam250 algorithm and MacVector 5.0 computer software (Eastman Kodak, New Haven, CT).
Peptide synthesis and conjugation to biotin
A 25-mer peptide (C6 = MKKDDQIAAAMVLRGMAKDGQFALK) and a 19-mer (C2 = DAASVNGIAKGIKGIVDAA) were prepared using the fluorenylmethoxycarbonyl synthesis protocol (21). N-terminal biotinylation was performed by the maleimide carboxylate method. The maleimide reagent was obtained from Molecular Probes (Eugene, OR), and the protocol suggested by the manufacturer was followed.
Human serum samples
A panel of 41 human serum samples was provided by the CDC. All samples were collected from Lyme disease patients who had signs and symptoms that satisfied the CDC clinical case definition (22). Four serum samples from chronic Lyme disease patients were obtained from the National Institutes of Health. Ninety-seven serum samples obtained from hospitalized patients in an area not endemic for Lyme disease were used as negative controls.
C6 peptide ELISA
Ninety-six-well ELISA plates were coated with 100 µl/well of 4
µg/ml streptavidin (Pierce, Rockford, IL) in coating buffer and
incubated at 4°C overnight. The remaining steps were conducted in a
rotatory shaker at room temperature. After two 3-min washes with 200
µl/well of PBS/T at 200 rpm, 200 µl of 5 µg/ml biotinylated
peptide dissolved in blocking solution (PBS/T supplemented with 5%
nonfat dry milk (Carnation, Nestle Food, Glendale, CA)) was applied to
each well. The plate was shaken at 150 rpm for 2 h. After three
washes with PBS/T as described above, 50 µl of serum (mouse, monkey,
or human) diluted 1/200 with blocking solution was added to each well.
The plate was incubated at 150 rpm for 1 h and then washed three
times with PBS/T. Each well then received 100 µl of 0.2 µg/ml goat
anti-monkey IgG (-chain specific; Kirkegaard & Perry
Laboratories, Gaithersburg, MD), 0.5 µg/ml anti-mouse IgG (heavy
and light chain specific; Sigma), or 0.1 µg/ml anti-human IgG
(heavy and light chain specific; Pierce), each conjugated to HRP and
dissolved in blocking solution. The plate was incubated for 1 h
while shaking. After four washes with PBS/T, each for 3–6 min, the
Ag-Ab reaction was probed using the TMB Microwell Peroxidase Substrate
System (Kirkegaard & Perry), and color was allowed to develop for 10
min. The enzyme reaction was stopped by addition of 100 µl of 1 M
H3PO4. OD was measured at
450 nm.
Competitive ELISA
Ninety-six-well ELISA plates were coated with 100 µl/well of 0.3 µg/ml of purified recombinant P7-1 dissolved in coating buffer at 4°C overnight. After blocking with blocking solution, 25 µl of this solution with 0, 1, 4, 16, 64, 256, or 1024 ng of the C6 or C2 peptides was added to each well. A 25-µl volume of a 1/100 dilution of the appropriate serum in blocking solution also was added to each well. The remaining steps were performed as related in the section describing the C6 peptide ELISA.
Preparation of rabbit anti-C6 peptide antiserum
The C6 peptide was covalently linked to keyhole limpet hemocyanin by the N-succinimidyl maleimide carboxylate method after adding a cysteine residue to the peptide’s N-terminus. The maleimide reagent was obtained from Molecular Probes (Eugene, OR), and the protocol suggested by the manufacturer was followed. Six-month-old New Zealand White rabbits were given three injections at biweekly intervals of 200 µg of conjugated Ag emulsified with CFA (first injection) or IFA (remaining injections). Ten days after the last injection the Ab titer was determined by the peptide ELISA and immunoblot analysis using IP90 spirochete whole cell lysates as Ag.
Immunoprecipitation and immunoblot
Immunoprecipitation was conducted at 4°C. About 1.5 x 1010 IP90 spirochetes harvested at stationary growth phase were extracted in 4.5 ml of solubilization buffer (50 mM Tris-HCl, 1% Triton X-100, and 1 mM EDTA, pH 7.6) for 30 min. The mixture was centrifuged at 13,000 x g for 30 min, and the supernatant was collected. Each 1.5 ml of this supernatant was mixed with 30 µl of preimmune or immune rabbit serum and incubated for 30 min. Fifty microliters of drained ImmunoPure Immobilized Protein G (Pierce) pre-equilibrated in solubilization buffer was then added and allowed to incubate for an additional 30 min. After washing the gel twice with excess volumes of this buffer by centrifugation at 3000 x g for 20 min, 150 µl of nonreducing SDS-PAGE sample buffer (125 mM Tris-HCl, 3% SDS, and 20% glycerol, pH 6.8) was added. The suspension was incubated at room temperature for 30 min and then centrifuged at 16,000 x g for 30 min. Ten microliters of supernatant was loaded onto each of 10 lanes of a SDS-12% polyacrylamide minigel. Separated proteins were electrotransferred to nitrocellulose in Towbin transfer buffer. After incubating in blocking solution for 2 h, the blot was incubated for 1 h in rabbit anti-C6 serum diluted 1/2,000 with blocking solution. After three washes with PBS/T, the blot was incubated in blocking solution with 0.5 µg/ml goat anti-rabbit IgG conjugated to HRP (Pierce) for 1 h. The Ag-Ab reaction was probed in PBS/T supplemented with 0.05% 4-chloro-naphthol (Sigma), 0.015% hydrogen peroxide, and 17% methanol.
Direct immunofluorescence
Rabbit IgG was purified from preimmune or immune serum using
conventional ammonium sulfate precipitation. The purity and
concentration of IgG preparations were assessed using SDS-PAGE and the
Bio-Rad protein assay kit, respectively. Purified IgG was conjugated to
FITC according to the manufacturer’s instruction (Pierce). For
labeling, spirochetes were either unfixed or acetone fixed. For the
fluorescent labeling of unfixed spirochetes,
108 spirochetes that were harvested from 1.0
ml of IP90 or HB19 culture grown to stationary phase by centrifugation
at 4,000 x g for 20 min were gently resuspended in 100
µl of PBS supplemented with 2 µg of rabbit
anti-C6 IgG-FITC conjugate, and incubated for
1 h. After two washes with excess volumes of PBS by centrifugation
at 16,000 x g for 5 min, spirochetes were resuspended
in 100 µl of PBS, applied to microscope slides, and counted under
both darkfield and fluorescent microscopes. The ratio of fluorescent to
total spirochetes was thus calculated. The same procedure was performed
on acetone-fixed spirochetes. For this purpose, organisms harvested
from 1.0 ml of culture fluid were suspended in 1.5 ml of acetone,
incubated for 20 min, and then centrifuged at 16,000 x
g for 5 min. After one wash with PBS, the fixed spirochetes
were stained with fluorescent anti-C6 Ab as
described for unfixed spirochetes. Goat anti-B.
burgdorferi Ab FITC conjugate (Kirkegaard & Perry Laboratories)
was used as a positive control.
In vitro Ab killing assay
As a source of complement, serum samples were collected from
normal rhesus macaques, pooled, and stored in small aliquots at
-70°C until used. Serum chosen for this purpose did not contain
cross-reactive anti-B. burgdorferi Abs as determined by
immunoblot analysis using whole cell lysates of B.
burgdorferi as Ag. To perform the killing assay, spirochetes were
cultured in BSK-H medium until they reached the mid-logarithmic phase
(2 x 107 cells/ml). A total of 5
x 105 spirochetes in 25 µl of BSK-H medium was
added to each well of a 96-well plate (Corning). A volume of 50 µl of
heat-inactivated (56°C, 30 min) serum sample appropriately diluted in
the same medium was already dispensed in each well. The plate was
incubated at 34°C for 30 min before the addition of 25 µl of
complement preparation (normal monkey serum). After 24 h of
incubation at 34°C in a humidified atmosphere of 3%
CO2, 5% O2, and the
balance of N2, 5 µl of each sample was removed,
and dead (nonmotile) and live (motile) spirochetes were counted under a
darkfield microscope. Monkey anti-outer surface protein A (OspA)
antiserum was used as a positive control (23).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As we assessed the Ab response to the recombinant P7-1 polypeptide
in monkeys that had been tick inoculated with either B31 or JD1
B. burgdorferi spirochetes, we noticed that the response was
detectable within the first 3 wk PI, and that it persisted at least
until wk 10 PI, the longest time point measured in this initial
experiment (Fig. 1
). We hypothesized that
one or more of the invariable regions within the P7-1 variable domain
were antigenic themselves and sufficiently conserved among
Borrelia strains and genospecies to exhibit antigenic
cross-reactivity. The deduced amino acid sequence of the B.
garinii P7-1 recombinant polypeptide is shown in Fig. 2. It is depicted aligned with sequences
from VlsE cassette segments of B. burgdorferi strains B31
(8) and 297 (15). In B31, the cassette
segment is comprised between the repeats EGAIKG (Fig. 2)
(8). The six variable regions of the B31 VlsE cassette
segment (8) are doubly underlined. The invariable regions
IR1–6 of the IP90 VlsE variable domain (Fig. 2)
are clearly conserved across both genospecies and strains of B.
burgdorferi sensu lato. With the exception of
IR1, which is 78% conserved in strain B31 and
only 11% conserved in 297, all other invariable regions are between
80–90% conserved with respect to the P7-1 VlsE cassette segment of
IP90 (Table I).
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). Experimental assessment of the antigenicity of IR6.
To evaluate experimentally the antigenicity of
IR6, we synthesized a peptide
(C6) whose primary structure encompassed that of
IR6. Serum samples from blood obtained from 10
rhesus macaques that were infected either with JD1 (six animals) or B31
spirochetes were used to examine the antigenicity of
IR6 in monkeys. Anti-C6
ELISA Ab levels shown in Fig. 3A were present in serum
samples at 4–6 wk PI and remained as high or higher for up to 3 yr PI
(data not shown).
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B). This result
further confirmed the antigenicity of IR6 and
reaffirmed this region’s antigenic conservation.
In humans, the antigenicity of IR6 was examined
with the aid of a panel of 41 serum samples provided by the CDC. Ab
directed to C6 was found in 35 of the 41 samples
(Table II). The result of this experiment
also underscores both the antigenicity and the antigenic conservation
of IR6, as the serum specimens in the panel
stemmed from several Lyme disease endemic areas in the United States
and must therefore contain Abs to multiple strains of B.
burgdorferi.
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IR6 may be the only immunodominant invariable region in both monkeys and humans
In addition to IR6, the VlsE variable domain
contains five other invariable regions (Fig. 2). To determine whether
any of these regions is antigenic, a competitive ELISA was performed,
using P7-1 as Ag attached to an ELISA plate, in the presence of
increasing concentrations of the C6 or
C2 peptides. The sequence of
C2, a 19 mer, was based on that of
IR2, plus three N-terminal amino acids
(DAASVNGIAKGIKGIVDAA). P7-1 includes invariable regions
IR1–6 (Fig. 2). Ten serum samples from infected
monkeys, four human serum samples randomly selected from the CDC panel,
and an additional four serum samples from chronic Lyme disease patients
provided by the National Institutes of Health were tested.
Representative results for four human and four monkey serum samples are
presented in Fig. 4. Addition of
C6 almost completely inhibited the binding of
monkey and human serum Abs to P7-1 (Fig. 4, A and
B). In contrast, addition of C2 in the
same range of concentrations caused no detectable inhibition (Fig. 4, C and D). Thus, inhibition of Ab binding to P7-1
by the C6 peptide is specific and, more
importantly, in both monkeys and humans IR6
appears to be an immunodominant invariable region within the VlsE
variable domain.
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40%, depending on the animal. Unlike
with monkeys and humans, however, addition of C2
competitively inhibited binding of mouse Abs to P7-1 (up to 40%; data
not shown). This indicates that other invariable regions may be
antigenic in mice. Exposure of IR6 on the surface of VlsE
We examined the exposure of IR6 on the
surface of the VlsE protein by immunoprecipitation with the rabbit
anti-C6 antiserum. VlsE from IP90 spirochetes
was extracted in solubilization buffer and precipitated with protein
G-agarose in the presence of either rabbit
anti-C6 antiserum or serum obtained from the
same rabbit before immunization. The presence of VlsE in the
immunoprecipitates was then assessed on immunoblots reacted with the
anti-C6 antiserum. The VlsE of IP90 was
immunoprecipitable with the anti-C6
antiserum, but not with normal preimmune serum (Fig. 5). This result indicates that
IR6 is exposed on the VlsE surface.
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Exposure of IR6 on the spirochetal surface
was assessed by immunofluorescence. While FITC-conjugated
anti-C6 Ab extensively labeled all the
acetone-fixed IP90 spirochetes (Fig. 6B), it labeled few (<5%) of
the unfixed spirochetes (Fig. 6A). The unfixed spirochetes
that were labeled exhibited a discontinuous fluorescent pattern,
whereas fixed spirochetes fluoresced uniformly (Fig. 6B). In
contrast, neither unfixed nor fixed spirochetes of the B.
burgdorferi sensu stricto strain HB19 high passage, which do not
express VlsE, fluoresced with the FITC-conjugated
anti-C6 Ab (Fig. 6, C and
D). Control anti-B. burgdorferi Ab-FITC
conjugate labeled both fixed and unfixed spirochetes (data not shown).
These results indicate that IR6 is not accessible
to Ab despite the fact that VlsE is itself surface exposed
(8). This is consistent with the result of the in vitro Ab
killing assay; anti-C6 antiserum had no
significant killing activity compared with preimmune serum, although
monkey anti-OspA antiserum killed all spirochetes in the same
experiment (data not shown). In vitro killing experiments were
performed at least twice with the same results.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table I). The sequence conservation of the six invariable regions across strain and genospecies barriers indicates that these regions are important in whichever role VlsE may play in the physiology of B. burgdorferi. One would therefore expect that such sequences are not antigenic in hosts with a chronic B. burgdorferi infection or would be otherwise inaccessible to Ab, either because they are conformationally buried within the VlsE molecule or are unavailable on the spirochetal surface. The Hopp-Woods algorithm indicated that, with the exception of IR6, all the other invariable regions were either not antigenic or had lower antigenicity.
The predicted antigenicity of IR6 was confirmed
in humans, monkeys, and mice (Fig. 3 and Table II). Sera from all these
hosts reacted with the C6 peptide early and
persistently in the course of infection, thus indicating that
IR6 contains one or more epitopes that may be
broadly antigenic regardless of host species. The antigenicity of
IR6 was not only manifest independently of host
species, but also regardless of whether the animals had been infected
with the JD1, B31, or Sh-2-82 strains of B. burgdorferi
sensu stricto. In addition, 35 of 41 human serum samples collected in
the Northeast and Midwest of the U.S. from patients with acute or
chronic Lyme disease also reacted with the C6
peptide. The five serum samples that had no detectable
anti-C6 Ab were obtained from patients who
were in the early stages of infection. Hence, the negative results may
reflect the presence of very low serum Ab titers rather than the
absence of cross-reactivity. The C6 peptide may
thus serve as a global diagnostic probe. We are currently investigating
this possibility.
No Ab reactivity was observed when the C6 peptide was not included in the assay, i.e., when plates were coated with streptavidin. Hence, the anti-C6 reactivity is specific and cannot be construed as originating from polyclonal B cell stimulation during B. burgdorferi infection (25, 26).
The absence of or relatively low antigenicity of
IR1–5 was underscored by the results of the
competition experiments (Fig. 4). The fact that the binding of
essentially all the human and monkey Ab that reacted with P7-1 could be
inhibited simply by adding an excess of the C6
peptide indicates that in these hosts IR6 is
possibly the only immunodominant invariable region within the VlsE
variable domain. In mice the results were different, as we were unable
to fully inhibit the P7-1 reactivity of mouse anti-Sh-2-82 or
anti-B31 Abs with an added excess of the C6
(or C6 and C2) peptide(s).
This result entails that some of the other IRs may be antigenic in
mice. Indeed, when synthetic peptides based on the five remaining IRs
listed in Fig. 2 were used as ELISA Ags, Ab responses to
IR2 and IR4 were evident in
infected mice, but not in monkeys or humans (34). The
immunodominance of IR6 was further underscored by
the long term persistence of anti-C6 Abs in
infected monkeys and in patients with chronic Lyme disease (not
shown).
The Hopp-Woods algorithm also predicted, as expected, that some variable regions of the cassette segment are strongly antigenic (data not shown). Thus, at least theoretically, the VlsE variable regions could satisfy the antigenic variation paradigm that variable domains are immunodominant. In the case of VlsE, however, variable region immunodominance of individual VlsE molecules may be difficult to achieve, for the recombination process that underlies the mechanism of VlsE variation occurs very rapidly (14). Despite strong antigenicity, no variable region might at any time be represented on a large enough number of bacterial cells or otherwise be expressed long enough to become immunodominant. Indeed, within the first 4 days PI, as many as 11 predicted amino acid changes per VlsE variant can occur during a B. burgdorferi B31 infection in C3H/HeN mice (14), and unlike with T. brucei, N. gonorrhoeae, and B. hermsii, VlsE serotypes of B. burgdorferi have never been found. In fact, it has been estimated that amino acid substitutions within the variable domain of VlsE could result in as many as 1030 different combinations (9), a number much larger than the diversity of the Ab repertoire of vertebrate host species (109–1011). Even though some of the combinations may share antigenic specificities, it seems unrealistic that VlsE serotypes may emerge during infection.
Our finding that IR6 is exposed on the VlsE
surface (Fig. 5) but not on the spirochetal surface (Fig. 6) is in
agreement with the antigenicity and immunodominance of this region.
Features of protein domains, such as surface accessibility,
hydrophilicity, flexibility, and proximity to a site recognized by Th
cells, are all important in positively determining domain antigenicity
(27). Except for the possibility that solubilization with
Triton X-100 may have partially denatured VlsE and thus artificially
exposed IR6, the result of the immune
precipitation experiment indicates that IR6 is
accessible on the surface of native VlsE. It is also hydrophilic, as
assessed by the Hopps-Wood algorithm. The molecular surface exposure of
IR6 was indicated also by the fact that a small
proportion of unfixed spirochetes fluoresced when incubated with
FITC-labeled anti-C6 Ab (Fig. 6A).
Most of the unfixed spirochetes failed to label with the
FITC-conjugated anti-C6 Ab (Fig. 6A). This indicates that IR6 is not
exposed on the spirochetal surface. The small proportion of unfixed
spirochetes that bound anti-C6 Ab probably
had some degree of membrane damage. Although our procedure for labeling
of unfixed spirochetes entailed very gentle manipulations, it is
possible that portions of the VlsE molecule not normally exposed on the
spirochete surface were nonetheless uncovered in a fraction of the
spirochetes. The fragility of the outer membrane of B.
burgdorferi has been noted by other investigators
(28). Loss of the lp28-1 plasmid, which encodes VlsE,
during in vitro cultivation (8) also could explain a
negative labeling result. However, the fact that nearly 100% of
acetone-fixed spirochetes from the same culture were labeled allows us
to rule out this possibility (Fig. 6B). Acetone fixation
probably exposed regions of the VlsE not accessible to Ab on an intact
spirochete. As expected, the HB19 spirochetes not expressing VlsE did
not bind anti-C6 Ab regardless of whether the
organisms were fixed or unfixed (Fig. 6, C and
D). Hence, the anti-C6 reactivity
is singularly specific.
The result of the in vitro Ab killing experiment was entirely consistent with our interpretation of the immunofluorescent observations, as absence of killing is most likely due to failure of the anti-C6 Ab to bind to the spirochetal surface. Complement-dependent killing of B. burgdorferi is facilitated by anti-surface Ab binding and does not depend on the complement-activating properties of the Ab. B. burgdorferi spirochetes are able to activate complement through an Ab-independent mechanism (29). Taken together, our immunofluorescence and in vitro killing results indicate that IR6 is inaccessible to Ab on the spirochetal surface. Because VlsE is surface exposed and is a lipoprotein (8), it is probably anchored to the spirochetal membrane only by its N-terminal lipid moiety and is not otherwise embedded in the membrane. It follows that VlsE is probably densely packed on the spirochetal surface, interacting with either neighboring VlsEs or other vicinal surface-exposed molecules.
In the variable Ags that have been thoroughly investigated to date, such as the VSG, Vmp, or pilin, the function attributed to variable portions, because of their immunodominance, has been to act both as target of the immune response and as decoy with respect to invariable portions of the same molecule or other molecules. On the other hand, the role ascribed to these Ags’ invariable portions, which are characteristically nonantigenic, has been to contribute to preserving a functional conformation (10, 11). What, then, is the role of the antigenicity and immunodominance of IR6? It has been hypothesized that chronic host exposure to immunodominant Ags or epitopes diverts the immune system from responding to less antigenic but functionally important Ags or epitopes, thus serving as a protective strategy for persistent pathogens (30). Recently, it was demonstrated that when the immunodominant V3 loop epitope of gp120 of HIV-1 is masked through site-directed targeting of N-linked glycosylation, the dominant, type-specific, neutralizing Ab response is shifted away from V3 to epitopes in the first variable domain (V1) of gp120 (31). Ab responses to conserved domains of gp120 also were observed (31).
We submit that IR6 may act as a decoy epitope and contribute to subvert the Ab response to B. burgdorferi. As mentioned before, it is conceivable that the promiscuity of VlsE antigenic variation is such that variable regions of VlsE do not, or do not always, become immunodominant. The variation may serve to inhibit the formation of high avidity Abs to the variable VlsE regions, but not to divert the response away from invariable VlsE regions and invariable domains or other less antigenic, but functionally neutralizing, B. burgdorferi Ag. It should be considered that only about half the length of the mature VlsE protein is variable, compared with more than two-thirds of proteins such as VSG, pilin, and Vmp. Moreover, more than half the variable domain of VlsE is encompassed by invariable regions, including one immunodominant IR6 identified in this study. Conserved regions of VlsE other than IR6 thus may be exposed, per force, on the spirochete surface. IR6 would serve as the decoy epitope for such domains by suppressing protective immune responses through mechanisms such as clonal restriction and/or idiotypic dysregulation, as has been invoked for HIV-1’s V3 (32, 33).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mario T. Philipp, Tulane Regional Primate Research Center, Tulane University Medical Center, 18703 Three Rivers Road, Covington, LA 70433. E-mail address:
3 Abbreviations used in this paper: VSG, variant surface glycoprotein; Vmp, variable major protein; VlsE, Vmp-like sequence, expressed; PI, postinfection; CDC, Centers for Disease Control and Prevention; Ospa, outer surface protein A.
Received for publication May 27, 1999. Accepted for publication September 8, 1999.
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