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Previous Article | Next Article 
Journal of Clinical Microbiology, December 1999, p. 3952-3956, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Acute-Phase-Specific Heptapeptide Epitope for
Diagnosis of Parvovirus B19 Infection
Leena
Kaikkonen,1
Hilkka
Lankinen,1
Irene
Harjunpää,1
Kati
Hokynar,1
Maria
Söderlund-Venermo,1
Christian
Oker-Blom,2
Lea
Hedman,1 and
Klaus
Hedman1,*
Department of Virology, Haartman Institute
and HUCH Diagnostic, University of Helsinki,
Helsinki,1 and VTT Biotechnology and
Food Research, VTT, Espoo,2 Finland
Received 20 April 1999/Returned for modification 5 July
1999/Accepted 16 August 1999
 |
ABSTRACT |
The major capsid protein VP2 of human parvovirus B19, when studied
in a denatured form exhibiting linear epitopes, is recognized exclusively by immunoglobulin G (IgG) antibodies of patients with acute
or recent B19 infection. By contrast, conformational epitopes of VP2
are recognized both by IgG of the acute phase and by IgG of past
immunity. In order to localize the VP2 linear epitope(s) specific for
acute-phase IgG, the entire B19 capsid protein sequence was mapped by
peptide scanning using well-characterized acute-phase and control sera.
A unique heptapeptide epitope showing strong and selective reactivity
with the acute-phase IgG was detected and characterized. By using this
linear epitope (VP2 amino acids 344 to 350) and virus-like particles
exhibiting conformational VP2 epitopes, an innovative approach,
second-generation epitope-typing enzyme immunoassay, was set up for
improved diagnosis of primary infections by human parvovirus B19.
| |
INTRODUCTION |
Human parvovirus B19 is a small
nonenveloped DNA virus, the icosahedral capsid of which consists of
structural proteins of two types, the minor protein VP1 (83 kDa) and
the major protein VP2 (58 kDa). VP2 is contained within VP1, which has
an additional unique portion of 227 amino acids (aa). Of the 60 capsid
protein molecules in the native virus, VP2 makes up more than 90%
(9).
Infection by the B19 virus may lead to a wide range of diseases. In the
early phase of infection, virus-induced cessation of erythropoiesis
among predisposed subjects can lead to abrupt anemia, aplastic or
hypoplastic crisis. In immunocompromised individuals persistent
parvovirus infection can cause prolonged bone marrow failure
(21). Fifth disease (erythema infectiosum) occurs frequently in children and young adults and is associated with varying forms of
autoimmune phenomena and transient, sometimes prolonged arthropathy, especially among older individuals. B19 infection during the first two
trimesters of pregnancy can lead to fetal hydrops and/or fetal death
(4, 19).
Serologic diagnosis of B19 infection is traditionally based on
measurement of virus-specific immunoglobulin G (IgG) and IgM and, in
particular cases, on measurement of other immunoglobulin classes or IgG
subclasses (6, 10, 12). The time of primary infection can be
determined by measurement of IgG avidity (14, 16, 28). An
even more novel approach is analysis of the epitope-type specificity
(ETS) of VP2-IgG (29). Virus-like VP2 particles exhibiting
conformational epitopes are recognized by IgG produced at all stages
after infection. By contrast, chemically denatured VP2 particles
exhibiting linear epitopes are recognized exclusively by acute-phase
IgG (29).
In order to understand the mechanism of the ETS phenomenon, we mapped
the linear epitopes that are recognized exclusively by antibodies of
the acute phase. The identification of an immunodominant acute-phase-specific IgG epitope led to the development of a
peptide-based second-generation ETS assay.
| |
MATERIALS AND METHODS |
Epitope mapping.
Epitope mapping was performed by using
20-aa peptides attached to cellulose membranes (SPOT peptides)
(11). The peptides covered the entire VP1-VP2 region with a
3-aa frameshift. One epitope was characterized further with another set
of SPOT peptides by alanine and glycine substitutions, by systematic
deletions, and by a 1-aa frameshift. The essential amino acids within
the epitope were further examined by replacement of each residue with alanine (alanine scanning). In all the SPOT peptide experiments, the
membranes were probed with sera pooled at different phases after B19 infection.
The SPOT peptides were synthesized by using an Abimed Auto-Spot Robot
ASP222 on cellulose membranes derivatized with polyethylene glycol
spacers (amino-PEG membranes; Abimed Analysentechnik GmbH, Langenfeld,
Germany). Peptide synthesis was performed by using 9-fluorenylmethoxy
carbonyl (Fmoc) amino acids and the synthesis protocols recommended by
the manufacturer. Stock solutions of amino acid derivatives were made
in 0.5 M N-hydroxybenzotriazole (HOBt) in
N-methylpyrrolidone (NMP) and stored in 180-µl aliquots at
20°C. Prior to use, the amino acid derivatives (final
concentration, 0.250 M) were activated in situ by addition of 0.275 M
N,N'-diisopropylcarbodiimide (DIC) in NMP. After
each twice-repeated amino acid addition, the unreacted amino groups
were capped with 2% acetic anhydride in N,N'-dimethylformamide (DMF). Fmoc deprotection
was carried out by washing the membranes in 20% piperidine in DMF for
10 min. After capping, the membranes were washed with DMF once for
30 s and twice for 2 min each time, and after Fmoc cleavage, they were washed once for 30 s and four times for 2 min each time, followed by methanol washes (once for 30 s and twice for 2 min each time) and drying. An additional capping was performed after the
last round of synthesis. The amino acid side chains were deprotected for 1 hour with a mixture of 5 ml of trifluoroacetic acid (TFA), 5 ml
of dichloromethane (DCM), 300 µl of tri-isobutylsilane, and 200 µl
of water, followed by rinses with DCM, DMF, and methanol. The membranes
were dried and stored at 20°C.
For the epitope-mapping assay, the SPOT peptide membranes were treated
in a shaker first with methanol followed by Tris-buffered saline, pH
8.0 (TBS), and then with blocking buffer (10% of casein-based blocking
buffer [10×] [Genosys, London, United Kingdom] and 0.05 g of
sucrose/ml in TBS containing 0.05% Tween 20 [TBS-Tween]) overnight
at 4°C. The membranes were washed once with TBS-Tween and incubated
at 37°C for 90 min with each serum pool diluted 1:5,000 in blocking
buffer. After three washes of 10 min each with TBS-Tween, the membranes
were treated with horseradish peroxidase-conjugated anti-human IgG
(DAKO, Glostrup, Denmark) diluted 1:5,000 in blocking buffer followed
by three washes and were incubated for 60 s in freshly prepared
enhanced chemiluminescence detection reagent (1.25 mM luminol and 0.2 mM p-coumaric acid in 0.1 M Tris-HCl buffer, pH 8.5, with
0.01% H2O2). Chemiluminescence was visualized on X-ray films exposed for various times (5 s to 10 min). The SPOT
peptide membranes were regenerated (twice for 15 min each time at
50°C) with a solution containing 8 M urea, 1% sodium dodecyl sulfate
(SDS), and 0.1% 
-mercaptoethanol followed by 50 mM glycine-HCl buffer, pH 2.2 (three times for 10 min each time at 20 to 23°C), rinsed with TBS-Tween and methanol, and dried. The conjugate alone was
tested between the analyses of serum pools.
Synthesis of peptide antigen for enzyme immunoassay (EIA).
A
24-aa peptide containing VP2 aa 335 to 359 was synthesized in the solid
phase (Applied Biosystems [Foster City, Calif.] 433A peptide
synthesizer) by using Fmoc chemistry. A portion of the peptide was
biotinylated with preactivated N-hydroxysuccinimide (NHS)
biotin overnight at 37°C, followed by washes with DMF and methanol,
and then dried. Final cleavage of the peptide from the solid matrix was
carried out under acidic conditions in the presence of scavengers (95%
TFA, 2.5% thioanisole, 1.25% ethanedithiol, and 1.25% water).
Peptide purity was assessed by reverse-phase high-performance liquid
chromatography and matrix-assisted laser desorption ionization-time of
flight mass spectrometry.
Synthesis of VP2 capsids.
By using viremic serum as a
template, the entire VP2 gene was amplified by PCR using a DNA
polymerase with strong 3'
5' proofreading exonuclease activity
(DeepVent; New England Biolabs, Beverly, Mass.). The PCR product was
cloned into a baculovirus transfer vector, p2Bac (Invitrogen, NV Leek,
The Netherlands), under the control of the polyhedrin promoter, by
standard methods. Bacteria (Escherichia coli DH5
)
containing the vector with the correct insert were identified by
restriction enzyme analysis.
Spodoptera frugiperda cells (Sf-9 cells) were cotransfected
with the recombinant vector DNA and linearized baculovirus DNA (BaculoGold; PharMingen, San Diego, Calif.). Transfection was done with
Lipofectin as recommended by the manufacturer (Bethesda Research
Laboratories, Life Technologies Inc., Gaithersburg, Md.). After 4 days
the cells were harvested and plaque purified twice (30).
SDS-polyacrylamide gel electrophoresis and immunoblotting showed VP2
production (3), and electron microscopy showed native-like B19 capsids (18), which were purified by ultracentrifugation in a 28% CsCl gradient (at 100,000 × g for 24 h)
followed by precipitation with 40% ammonium sulfate. The protein
pellet was resuspended in and dialyzed against phosphate-buffered
saline (PBS). For EIA, the VP2 capsids were biotinylated by using the
EZ-Link Sulfo-NHS-LC-biotinylation kit (Pierce, Rockford, Ill.)
according to the manufacturer's instructions.
Serum samples.
We had sera from 61 patients with acute
infection and, for most patients, one to four follow-up samples taken
during convalescence, up to 700 days after the onset of symptoms
(28). The total number of acute-phase and follow-up sera was
163. The diagnostic criteria were seroconversion or a 
4-fold rise in
B19-IgG and B19-IgM in each patient. All infections were primary, as
verified by significant VP1-IgG avidity maturation. Sera from 78 subjects with B19-IgG, but without IgM, were used as past-immunity
controls. Negative-control sera came from 46 nonimmune subjects. Serum
pools were made of 5 to 10 individuals' samples each. There were two
pools of sera from acute infection, two pools collected several years
after infection, and one pool devoid of B19 antibodies.
Antibody assays.
All the samples had been studied for
B19-IgG and -IgM with a reference radioimmunoassay (6, 7)
and/or a commercial EIA (IDEIA; DAKO). IgG avidity for VP1 was measured
by a protein-denaturing EIA (28). IgG binding to
conformational versus linear VP2 epitopes was measured with an ETS EIA.
Biotinylated VP2 capsids (DAKO) in native and denatured forms were used
in the first-generation ETS EIA as described previously
(29). In the second-generation ETS EIA, the biotinylated
antigens (VP2 capsid at 10 ng/well and VP2 peptide at 6 ng/well) in PBS
containing 0.05% Tween 20 (PBST) were immobilized on
streptavidin-coated plates (Labsystems, Helsinki, Finland) by
incubation for 60 min at room temperature in a rocking (400 rpm) EIA
incubator (iEMS; Labsystems). To minimize nonspecific background, the
antigen-sensitized plates were precoated three times, for 10 min each
time, with a sample diluent containing a protein and detergent additive
(Labsystems). Serum samples (1:200 in PBST) in 100-µl portions were
applied for 60 min at room temperature, followed by washes (three
times, for 5 min each time) with PBST and treatment for 60 min with
anti-human IgG-horseradish peroxidase conjugate (DAKO) diluted 1:2,000
in the sample diluent. After four washes with PBST, orthophenylene
diamine substrate and H2O2 were added, the
reaction was stopped after 10 min with 0.5 M
H2SO4, and the absorbances at 492 nm were
recorded. All tests were performed in duplicate.
In all the in-house EIAs, the cutoff values for IgG positivity were set
at the mean + 3 standard deviations (SD) of the absorbances of the
46 seronegative subjects.
| |
RESULTS |
Epitope scanning.
The entire B19 capsid sequence in SPOT
peptides was studied for IgG reactivity in two acute-phase serum pools,
one pool representing past immunity, and one pool devoid of B19 antibodies.
Antibody reactivity in 4 contiguous spots was regarded as indicative
of an antigenic region. As shown in Fig.
1 and Table 1, in the portion unique to VP1, four
such regions showed reactivity with the IgG of all the pools tested and
hence were not acute phase specific. In the portion common to VP1 and
VP2 (VP1 aa 228 to 780), IgG binding at eight antigenic regions was
observed. Of these eight regions, four were labeled with all the pools, and one of these four (VP1 aa 677 to 707) appeared to contain two or
more overlapping epitopes. One region (VP1 aa 316 to 326) was antigenic
only with the pool representing past immunity. Three regions were
specific for the acute-phase IgG: residues 292 to 302, 571 to 578, and
493 to 500 (weakly). By far the strongest IgG reactivity with the
acute-phase pools, and no reactivity with any other pool, came from
peptide spots containing VP1 aa 571 to 578.
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FIG. 1.
Epitope mapping with a pool of sera from individuals
with acute B19 infection (A) and a pool of sera from subjects
previously infected with B19 (B). Twenty-amino-acid synthetic peptides
covering the entire VP1-VP2 sequence were synthesized on cellulose
membranes, and IgG binding from the serum pools was visualized with
chemiluminescence. Panels A and B show the same membrane containing 255 partially overlapping (3-aa frameshift) B19 peptides. Each spot
corresponds to one peptide. After spot 1 (a technical control), the
VP1-VP2 sequence flows from left to right in each successive row. The
peptides containing VP1 aa 571 to 578 are marked with an arrow.
|
|
Epitope characterization.
The latter region was characterized
further with SPOT peptides by 1-aa frameshifting, systematic deletions,
and alanine and glycine scanning. These experiments were performed with
two acute-phase pools, two past-immunity pools, and one seronegative
pool, of which the latter three were nonreactive. Performed with 20-aa SPOT peptides covering the sequence 555 to 601, 1-aa frameshifting showed strong acute-phase IgG reactions with aa 571 to 577, whereas no
reactions were obtained with peptides lacking 1 or more of these 7 amino acids. Hence, the frameshifting experiments mapped the
acute-phase-specific epitope to a heptapeptide,
Lys-Tyr-Val-Thr-Gly-Ile-Asn (KYVTGIN), at VP1 aa 571 to 577.
In the alanine and glycine substitutions the KYVTGIN sequence and its
vicinity were replaced with increasing numbers of these amino acids,
from VP1 aa 568 downstream and from VP1 aa 581 upstream, without
changing the peptide length. The amino acid deletions followed a
similar design except that the peptide was shortened. All these
experiments gave the same result: substitutions or deletions extending
to the KYVTGIN sequence abolished its reactivity with the acute-phase
serum IgG, whereas perturbations in its surroundings had no such
effect. Interestingly, all the serum pools weakly recognized long
repeats of alanine or glycine; however, the intensity of these
reactions was only a fraction of that obtained with the acute-phase IgG
on the peptides containing the KYVTGIN sequence.
In alanine scanning, VP1 aa 562 to 581 in the SPOT peptides were
replaced one by one with alanine. As shown in Fig.
2, within KYVTGIN the essential amino
acids were K, T, G, I, and possibly N, whereas replacement of Y or V
did not markedly affect reactivity with the acute-phase IgG.
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FIG. 2.
Alanine scanning exploring the effect of point mutations
on the reactivity of IgG in a past-immunity serum pool and two
acute-phase serum pools. Each of the three rows contains the same set
of synthetic peptides, and all the spots contain VP1 aa 562 to 581 (VP2
aa 335 to 354). The first spot on the left contains peptides with the
native amino acid sequence, and the subsequent spots (from left to
right) contain peptides point mutated sequentially with alanine. The
boxed area marks the spots in which the KYVTGIN sequence has been
mutated, at the sites indicated by the letters.
|
|
KYVTGIN-antibody EIA.
The synthetic peptide and the native VP2
capsids, linked with biotin and streptavidin in the solid phase, were
studied by EIA for reactivity with IgG in individual sera (Fig.
3). In keeping with the definition of the
EIA cutoffs, among the 46 nonimmune controls, 45 (97.8%) showed IgG
absorbances below cutoff with the native VP2 antigen. Also with the
KYVTGIN antigen, 45 of 46 nonimmune controls gave negative results.
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FIG. 3.
Kinetics of IgG response for the native VP2 capsids and
the KYVTGIN peptide. Individual sera from 46 nonimmune subjects and 78 subjects with past immunity, and 163 acute-phase or followup sera from
61 patients, were used. The y axis shows
A492. Dashed lines mark cutoffs for IgG
positivity for the VP2 capsids (lower line; A492 = 0.198) and the KYVTGIN peptide (upper line;
A492 = 0.205).
|
|
During the acute phase and convalescence, all samples collected more
than 2 weeks after the onset of symptoms were IgG positive for the
native VP2 capsid. With the KYVTGIN peptide, most sera collected
between days 10 and 50 after onset were reactive, whereas most samples
collected >100 days after onset were devoid of KYVTGIN-IgG reactivity.
All the 78 control sera from subjects with preexisting immunity gave
positive IgG results with the native VP2 capsid antigen. Noticeably, of
the same past-immunity sera, none was reactive with the KYVTGIN peptide
(Fig. 3).
Second-generation epitope-typing EIA.
From the EIA absorbances
of the previous experiment, we calculated the ratios of ETS, i.e., the
ratios between IgG binding to the native VP2 and IgG binding to the
synthetic peptide epitope. In this assay the cutoff for separation of
acute infection from past immunity was set at the mean + 3 SD of
the ETS ratios of the sera collected within 3 months after onset. Among
105 B19-IgG-seropositive acute-phase samples, 103 (98.1%) yielded ETS
ratios below cutoff. From the donors of the other two samples, earlier
acute-phase sera showed low ETS ratios, compatible with acute B19
infection. By contrast, all the 78 past-immunity controls showed ETS
ratios exceeding the diagnostic cutoff (Fig.
4).
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FIG. 4.
Second-generation ETS EIA in diagnosis, showing ratios
of IgG absorbances for the native VP2 antigen and the KYVTGIN peptide.
The dashed line marks the diagnostic cutoff (ETS ratio, 1.3).
|
|
| |
DISCUSSION |
We mapped the acute-phase specificity of the primary structure of
the B19 major structural protein to an immunodominant epitope and
characterized its structure-function relation. Using this heptapeptide
(KYVTGIN) epitope and native VP2 capsids, we set up a new ETS assay for
diagnosis of B19 primary infections.
In terms of its acute-phase specificity, the KYVTGIN-IgG response is
strikingly different from the long-standing IgG response against the
unique part of VP1 (14, 20, 24, 27, 29, 31). On the other
hand, the kinetics of the KYVTGIN-IgG response was very similar to that
of the IgG response for the entire VP2 capsid protein in denatured form
(29). The epitope-scanning data suggested that, although it
was the most important, KYVTGIN was not the only acute-phase-specific
linear epitope in the VP2 molecule. Two relatively weaker acute-phase
IgG epitopes of our study (VP2 aa 65 to 75 and 266 to 273) have been
studied earlier for B19 antigenicity (13, 26).
As we have recently shown with chemically denatured protein antigens,
IgG binding to the primary structure of VP2 is specific for B19
infection, i.e., is not due to cross-reactive antibodies from other
acute viral infections (29). The IgG-KYVTGIN reactions presently observed by EIA could be completely abolished by the same
KYVTGIN peptide in solution (17a), adding proof for the specificity of the KYVTGIN-IgG response.
Recent studies on pathogens structurally and biologically unrelated to
B19, the lentiviruses human immunodeficiency virus (8) and
equine infectious anemia virus (15), have shown a transition
from a linear-epitope to a conformational-epitope specificity of IgG,
kinetically similar to our findings with VP2-IgG. Furthermore, a linear
epitope in the envelope glycoprotein of Sin Nombre virus appears to be
specific for acute-phase IgG (17). It therefore appears that
B-cell responses against certain entirely nonrelated viruses have a
feature in common, the molecular and cell biologic mechanism of which
is thus far incompletely understood. A simple explanation, cryptic
residence, appears unlikely for the B19 virus because of the external
localization of the KYVTGIN sequence on the surface protrusions between
the twofold and threefold axes of the VP2 capsid (5).
Within this immunodominant heptapeptide, VP1 aa 571 to 577, corresponding to VP2 aa 344 to 350, merely four or five residues (Lys344, Thr347, Gly348, Ile349, and Asn350) were the actual hot spots
for binding of acute-phase IgG. In this respect the KYVTGIN-specific human IgG resembles a well-characterized monoclonal antilysozyme antibody (1). By sequence mapping the KYVTGIN epitope is
part of a long region (VP2 aa 266 to 376) shown to be immunogenic in rabbits (23). Binding sites for neutralizing antibodies
(2, 22, 25) localize in the vicinity of KYVTGIN. However,
even if high-resolution data on the topology of this sequence in the B19 capsid are available (5), its function in the virus
warrants further study.
In conclusion, in the human parvovirus major structural protein we have
detected an immunodominant B-cell epitope eliciting antibodies
exclusively during the early phase of B19 infection. Using this linear
epitope specific for acute-phase IgG, and conformational VP2
antigenicity recognizing IgG at all stages after infection, we have set
up a sensitive and specific, technically straightforward new method. In
confirmatory use this second-generation ETS EIA should improve the
accuracy of diagnosis of B19 primary infections.
| |
ACKNOWLEDGMENTS |
This work was supported by the Helsinki University Central
Hospital Research and Education Fund, the Finnish Technology
Advancement Fund, the Finnish Science Academy, and the Päivikki
and Sakari Sohlberg Foundation.
We thank Antti Vaheri and Leena Kostamovaara for help with Sf-9 cell
culturing and Mavis Agbandje-McKenna, Matti Kaartinen, and Ilkka
Seppälä for stimulating discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Haartman
Institute and HUCH Diagnostic, Department of Virology, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. Phone:
358-50-5249 086. Fax: 358-9-1912 6491. E-mail:
klaus.hedman{at}helsinki.fi.
| |
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Journal of Clinical Microbiology, December 1999, p. 3952-3956, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
