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Journal of Clinical Microbiology, November 1999, p. 3718-3721, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effects of OspA Vaccination on Lyme Disease
Serologic Testing
Maria E.
Aguero-Rosenfeld,1,*
Janet
Roberge,2
Carol A.
Carbonaro,2
John
Nowakowski,3
Robert B.
Nadelman,3 and
Gary P.
Wormser3
Department of
Pathology,1 and Division of Infectious
Diseases, Department of Medicine,3 New York
Medical College, and Westchester Medical
Center,2 Valhalla, New York 10595
Received 7 May 1999/Returned for modification 15 July 1999/Accepted 11 August 1999
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ABSTRACT |
This study presents the effects of OspA vaccination on two-step
testing for Borrelia burgdorferi antibodies. Although
vaccinees developed enzyme-linked immunosorbent assay reactivity,
immunoblots did not fulfill Centers for Disease Control and Prevention
criteria for positivity. Furthermore, OspA reactivity did not interfere with interpretation of immunoblots with sera from patients who developed early Lyme disease despite vaccination.
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TEXT |
In December 1998, the Food and Drug
Administration approved a recombinant outer surface protein A (OspA)
vaccine preparation for prevention of Lyme disease (2). It
has been suggested that widespread use of this vaccine will affect the
performance of serologic testing for Lyme disease because the OspA
antigen is a component of the whole-cell sonicate of Borrelia
burgdorferi, which is used in most commercially available
enzyme-linked immunosorbent assays (ELISAs) (11). This study
evaluates the potential impact of OspA vaccination on two-stage
B. burgdorferi serologic testing.
Serum samples.
Sequential serum specimens were obtained from
17 healthy adults participating in a phase 2 safety and immunogenicity
study sponsored by Connaught Laboratories, Inc. (Swiftwater, Pa.).
Serum specimens were collected prior to receipt of the first 30-µg
intramuscular dose of the OspA vaccine (pre), at day 30, when a second
30-µg OspA vaccine dose was administered, and at days 60, 90, and 180 after the first vaccination. Serum samples were stored at 
70°C until time of testing. Linkage was removed before testing.
To illustrate vaccine effects further, serum samples from selected
individuals receiving the same OspA vaccine preparation in an efficacy
study (12), including patients who developed early Lyme
disease with erythema migrans despite vaccination, were studied. In the
efficacy study, three 30-µg doses of vaccine were given to the
volunteers at time 0, day 30, and 1 year.
ELISA.
Specimens were tested in an immunoglobulin M (IgM)-IgG
ELISA (Lyme Stat; Whittaker M. A. Bioproducts Inc., Walkersville,
Md.) in accordance with the manufacturer's instructions.
Immunoblots.
Separate IgM and IgG immunoblots (MarDx
Diagnostics, Inc., Carlsbad, Calif.) were used to test all the serum
specimens according to the manufacturer's instructions. The Centers
for Disease Control and Prevention-Association of State and Territorial
Public Health Laboratory Directors (CDC/ASTPHLD) criteria were used for
blot interpretation (3).
None of the 17 volunteers enrolled in the phase 2 trial had a positive
ELISA or immunoblot result prior to receiving vaccination. Nine (53%)
had a positive ELISA by 30 days after receipt of the first vaccine
dose, and all 17 (100%) were reactive by ELISA by 30 days following
the second dose (day 60) (Table 1). Six
(35%) still had a positive ELISA by day 180. The highest mean Lyme
index value (LIV) by ELISA was observed at day 60 (Fig.
1).
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TABLE 1.
Reactivity by ELISA and IgM and IgG immunoblot tests
after OspA vaccination in 17 individuals participating in phase 2 of
the Connaught Lyme vaccine trial
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FIG. 1.
LIVs determined by ELISA (± standard errors of the
means) are shown at different times postvaccination in 17 individuals
receiving two vaccine doses. The dashed line indicates the cut-off LIV
for a positive ELISA (LIV  1.09). The first vaccination dose was
given after collecting the pre serum specimen, and the second dose was
administered before the 30-day serum specimen was collected.
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Both IgM and IgG antibodies to OspA were observed by immunoblot tests
of serum samples from 65% of vaccine recipients by day
30. While IgM
reactivity to OspA at day 60 was nearly identical
to that at day 30, the IgG reactivity to OspA had risen to 93%.
By day 180, 88% still
showed IgG reactivity to OspA whereas only
18% had IgM reactivity. Of
interest, despite the high rate of
OspA reactivity by IgG immunoblot on
day 180, only 35% were still
reactive by ELISA. Importantly, none of
the IgM or IgG immunoblot
reactivities at any time point fulfilled the
CDC/ASTPHLD criteria
for a positive immunoblot result (Fig.
2).
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FIG. 2.
IgG (A) and IgM (B) immunoblot series of serum samples
obtained from a vaccinated individual participating in the phase 2 trial at different times postvaccination. Strip numbers are located at
the bottom of each strip. Results with pre (strips 1), day 30 (strips
2), day 90 (strips 3), and day 180 (strips 4) samples are shown.
Positive controls are shown in strips 5, with arrows indicating a few
relevant bands. Those specimens showing the most intense reactivity to
OspA in IgG blots (strips 3 and 4) also show reactivity to two other
bands below 23 kDa (OspC) (arrows positioned next to strip 4) and
diffuse darkening in the upper region of the blot. IgM reactivity to
OspA was less intense than IgG reactivity.
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Although none of the immunoblots was interpreted as positive, several
noteworthy effects on immunoblot reactivity were observed.
In addition
to the expected reactivity at the 31-kDa band (OspA),
those serum
samples with intense IgG OspA reactivity regularly
had several bands of
smaller molecular sizes, including bands
of approximately 22 and 16 kDa, particularly in individuals receiving
three vaccine doses (Fig.
3). The reason for the production of
these additional bands is not known, but they may represent reactivity
to degradation products of OspA or to other smaller-molecular-weight
proteins which have amino acid sequences in common with epitopes
of
OspA. The presence of these bands might lead to diagnostic
confusion if
they are interpreted as indicating reactivity to
the 18-kDa protein,
which is a band of diagnostic importance for
IgG immunoblot
interpretation. Sera with OspA antibodies were
also associated with
darkening of the immunoblot extending down
to the 31-kDa band and
somewhat beyond. Reactivity of high-titer
OspA antibodies to small
amounts of residual OspA protein that
failed to migrate to the 31-kDa
position is a possible explanation
for this. The darkening may make it
difficult to visualize bands
above 31 kDa or to judge their intensity
accurately, including
the six significant IgG bands with molecular
sizes greater than
31 kDa (
3). The darkening may be even
more pronounced with
serum samples from individuals after receipt of a
third dose of
recombinant OspA vaccine at the 12-month time point (Fig.
3,
4,
and
5B). OspA antibody titers after three
doses of vaccine are
substantially greater than those after two doses
(data not shown)
(
7). IgM reactivity to OspA, however, is of
lesser intensity
than the IgG response (Fig.
4).
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FIG. 3.
IgG immunoblot of serum samples from an individual who
received the third dose of the recombinant OspA vaccine preparation 2 weeks prior to collection of this serum specimen (strip 1). Besides the
band of strong reactivity to OspA (top left arrow), two other bands
below 23 kDa are visible (bottom two left arrows). These two bands are
located above and below the 18-kDa band, which is included in the
diagnostic criteria for IgG immunoblot interpretation (3).
In addition, there is a general darkening of the immunoblot extending
from the superior portion to beyond the 31-kDa band. Strip 2 shows a
positive control, with arrows indicating a few relevant bands.
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FIG. 4.
Representative IgG and IgM immunoblots of serum samples
from two individuals who received three doses of the OspA vaccine
preparation. Strip numbers are shown at the bottom of each strip. Strip
1 shows a positive control, and the arrows on the left indicate a few
relevant bands. Strips 2 and 3 show the IgG and IgM immunoblots,
respectively, of a serum sample from one individual collected 6 weeks
after the third dose of vaccine. Strips 4 and 5 show the IgG and IgM
reactivities of a serum sample collected from another individual 5 weeks following the third vaccine dose.
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FIG. 5.
IgM and IgG blots of serial serum specimens from two
patients who developed early Lyme disease associated with erythema
migrans despite OspA vaccination. Strip numbers are shown at the bottom
of each strip. (A) IgM (strips 1 to 4) and IgG immunoblots (strips 6 to
9) of serum samples from a patient who presented with erythema migrans
(confirmed by a positive culture for B. burgdorferi) 6 months after the second vaccine dose. Serum specimens were collected
prior to the first vaccine dose (strips 1 and 6) and 1 day (strips 2 and 7), 8 days (strips 3 and 8), and 3 weeks (strips 4 and 9) after
onset of erythema migrans. A positive control is shown in strip 5, with
arrows indicating a few relevant bands. The IgG immunoblots
postvaccination show weak reactivity to OspA, and new IgG bands were
observed in the acute- and convalescent-phase specimens. IgM
immunoblots showed the appearance of the 41- and 23-kDa bands in the
specimen collected 8 days postonset of illness, with expansion of
reactivity at 3 weeks. (B) IgM (strips 1 to 3) and IgG (strips 5 to 7)
immunoblot series of serum samples from a patient presenting with a
2-day history of multiple erythema migrans lesions when the serum
specimens shown in strips 3 and 7 were collected. Strips 1 and 5 show
the IgM and IgG immunoblots, respectively, of serum specimens collected
prior to the first vaccine dose, and strips 2 and 6 show the IgM and
IgG immunoblots of serum specimens collected prior to the third vaccine
dose. The serum specimens shown in strips 3 and 7 were obtained 12 days
after the third vaccine dose. Strip 4 shows a positive control, with
arrows indicating a few relevant bands. Note that the IgM bands of
diagnostic significance were not affected by the presence of IgM
reactivity to OspA (strip 3). Strip 7 shows intense IgG reactivity to
OspA and a general darkening of the immunoblot. Arrows located to the
right of strip 7 indicate the frequently observed bands after two to
three doses of vaccine, at 31 kDa and two bands below OspC.
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Figure
5 shows examples of immunoblots of serum samples from
participants in an efficacy study (
12) who despite OspA
vaccination
developed Lyme disease. Although such patients generally
had weak
OspA reactivity, the bands of diagnostic significance on IgM
immunoblots
were readily
visualized.
In our laboratory, similar findings to those presented in this study
were observed in serum samples from individuals receiving
the Food and
Drug Administration-approved Lyme disease vaccine
manufactured by
SmithKline Beecham (data not
shown).
If OspA vaccination becomes widely used, current ELISA testing using
B. burgdorferi that contains OspA will become superfluous.
Unfortunately, use of immunoblotting alone without a preceding
ELISA
will reduce specificity and increase costs. In studies to
date, the
reduction in specificity has ranged from 1.5 to 8% (mean,
4.1%),
depending on the particular group of control sera tested
and the
particular ELISA and immunoblot tests used (
4,
5,
9). Even a
small reduction in specificity would have a substantial
impact on the
predictive value of a positive test when testing
patients with a low
pretest probability of Lyme disease (
6).
Further, under the assumptions of an ELISA negativity rate of 75% in
clinical practice (unpublished data) and a cost of $52
for an ELISA and
$106 for an immunoblot test (
8), an additional
expenditure
of 77 million dollars per year would be incurred by
routinely
performing immunoblot tests instead of conditional two-stage
testing
for the estimated 2.8 million serum samples submitted
for Lyme disease
serologic testing annually in the United States
(
10).
Pending the introduction of assays without OspA (
13), we
recommend that serologic testing for Lyme disease be limited to
patients with at least a 20% pretest probability of Lyme disease
(
1) and that initial ELISA testing be omitted for
OspA-immunized
individuals.
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ACKNOWLEDGMENTS |
We thank Eleanor Bramesco, Donna McKenna, Diane Holmgren, Susan
Bittker, Denise Cooper, and Louis Rosenfeld for their assistance. We
also thank Daniel Benevento for his expert photographic assistance.
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FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Laboratories, Room 1J-11a, Westchester Medical Center, Valhalla, NY
10595. Phone: (914) 493-7389. Fax: (914) 493-5742. E-mail:
maria_aguero-rosenfeld{at}nymc.edu.
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Journal of Clinical Microbiology, November 1999, p. 3718-3721, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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