Download Use of human papiHomavirus type I I virions in an ELISA to detect

Journal of General Virology (1991), 71, 1343-1347.
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Use of human papiHomavirus type I I virions in an ELISA to detect
specific antibodies in humans with condylomata acuminata
William Bonnez,* Carrie Da Rin, Robert C. Rose and Richard C. Reichman
Infectious Diseases Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry,
601 Elmwood Avenue, Box 689, Rochester, New York 14642, U.S.A.
Human papillomavirus types 6 and 11 (HPV-6 and
HPV-11) are the major aetiological agents of condylomata acuminata. Serological studies of this disease
have been difficult to perform and interpret because
native, type-specific antigens have not been available.
In particular, since these viruses have not been
propagated in vitro and sufficient quantities of virions
are not present in lesions, virus particles have been
difficult to obtain. In the present study, we used HPV11 particles, obtained from human tumours produced
in athymic mice, as antigen in an ELISA to compare
antibody responses between 46 patients with biopsyproven condylomata acuminata and 44 controls. The
median [interquartile range] of the absorbance values
for the condylomata acuminata and the control groups
were respectively 0-324 [0-183, 1-029] and 0.118
[0-047, 0.286] (P= 0- 0001). Thirty-three per cent of
the absorbance values in the condylomata acuminata
group were higher than any of those of the control
group. Sera from patients whose biopsies contained the
papillomavirus common antigen were more reactive
than sera from patients whose biopsies did not contain
it (P= 0-0014).This study demonstrates the presence of
specific antibodies directed at native HPV-I 1 viral
particles in the sera of patients with condylomata
acuminata, and describes a test which can be used in
future serological studies of this common sexually
transmitted disease.
Introduction
variable. Over 60 different HPV types have now been
identified (Reichman & Bonnez, 1990), each with a
narrow epithelial tropism. There is generally no serological cross-reactivity between virions of different papillomavirus types (Gissmann et al., 1977; Orth et al., 1977,
1978; Orth & Favre, 1985). Indeed, in immunoassays
using intact viral particles of HPV-1, the causative agent
of plantar warts, plantar wart patients were more likely
to have specific antibodies than controls (Kienzler et al.,
1983; Anisimov~i et al., 1990; Steele & Gallimore, 1990;
Viac et al., 1990), whereas no difference in seroprevalence could be detected between controls and condylomata acuminata patients (Pfister & zur Hausen, 1978;
Pfister et al., 1979; Kienzler et al., 1983; Viac et al.,
1990). The paucity of virions in most warts, including
condylomata acuminata, and the inability to grow
papillomaviruses in vitro, have limited the availability of
suitable native antigens. The description by Kreider et
al. (1985, 1987) of the athymic mouse HPV-11-infected
human xenograft model has now provided investigators
with a method for the production of HPV-11 viral
particles. Using these particles in an ELISA, we detected
specific antibodies in sera of patients with documented
condylomata acuminata.
Human anogenital warts (condylomata acuminata) are
caused by distinct human papillomaviruses (HPV),
mostly types 6 and 11 (Oriel, 1990). These lesions are
usually diagnosed accurately by careful physical examination. However, because of their small size or location,
they may elude detection or proper identification (Oriel,
1990). Supplemental diagnostic methods such as histology or cytology, with or without HPV nucleic acid
detection, are useful but expensive and subject to
sampling error. None of these techniques is practical for
use in large surveys or in diagnosis of asymptomatic
infection. Thus, the availability of an accurate serological test would greatly augment our ability to study the
epidemiology, natural history, and response to treatment
of anogenital warts. Early serological studies of condylomata acuminata patients used extracts of unspecified
human warts as antigens in various immunoassays
(Ogilvie, 1970; Nel & Fourie, 1973; Viac et al., 1977,
1978; Pyrh6nen, 1978; Pyrh6nen et al., 1980). At that
time, the great diversity of HPV types was not
appreciated, and results of these studies were highly
0001-0053 © 1991 SGM
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I4I. Bonnez and others
Methods
HPV-11 purification. Fragments of human neonatal foreskins were
infected with an HPV-11 viral suspension and placed under the renal
capsule of nu/nu athymic mice according to the protocol described by
Kreider et al. (1985, 1987). The resulting tumours were collected several
months later, pooled, and HPV-11 virions were purified by a series of
high-speed and caesium chloride centrifugations, according to the
protocol described by Favre et al. (1975), but omitting trypsin
treatment and the final sucrose gradient centrifugation. Full viral
particles were identified by electron microscopy after staining with 2
neutral buffered phosphotungstic acid. By the same technique, viral
particles were counted after mixing with 109 nm polystyrene latex
beads (Ladd Research Industries) (Miller, 1974). The protein content of
the preparation was determined by comparative densitometry of
Coomassie blue-stained bands on polyacrylamide gels, using bovine
serum albumin as a standard and the major capsid protein of HPV-11
for the measurement.
Patient populations. Sera from three groups of patients were tested.
Group I consisted of 27 volunteer nuns and priests who upon answering
a detailed questionnaire denied any lifetime sexual activity or genital
warts; our aim was to have individuals with a low likelihood not only of
genital HPV-6 or -11 disease, but also of asymptomatic infection with
these viruses. The group's age (mean +__s.D., in years) was 49-6 _ 11-7.
Group 2 consisted of 17 additional volunteers with clinically
documented cutaneous warts, but no history, relating to themselves or
their partners, of HPV genital infection. This group was chosen to
assess potential serological cross-reactivity between HPV-11 on one
hand, and HPV-1 and -2, the aetiological agents of these most common
HPV diseases, on the other hand. The group's age was 22.8 + 4.4.
Subjects had hand (29~), plantar (59~) or both (12~) warts. Group 3
included 46 patients, age 24.6 + 5.2, with biopsy-proven external
condylomata acuminata (Table 1). In 41 biopsy specimens, HPV typing
was done by Southern blot and/or in situ hybridization, according to
techniques already described (Reichman et al., 1990). Thirty-two
specimens contained HPV-6, six contained HPV-II, and in three
biopsies, HPV nucleic acids were not detected. The common
papillomavirus antigen (Jenson et al., 1980) was present in 17 of 35
biopsy samples, when assayed by immunocytochemistry (Wilbur et al.,
1988).
E L I S A . The immunoassay was carried out in 96-well Maxisorp
polystyrene plates (Nunc-Vanguard International). One-hundred
microlitres of HPV-11 viral suspension (0.8 ng of protein/~tl, or
approximately 225 x 106 particles/~tl), diluted in carbonate buffer
(Voller et aL, 1976), was dispensed in alternate test wells; the remaining
wells (controls) were filled with buffer without antigen. After
incubation overnight at 4 °C in a humid chamber, plates were washed
three times with PBS-Tween (Voller et al., 1976) dispensed with a
manual washer (Immunowash 12, Nunc). Duplicate serum samples
were tested in a volume of 100 ~tl/well, at a 1 : 20 dilution in Blotto buffer
(Johnson et al., 1984). After a 90 min incubation at 37 °C in a humid
chamber, plates were washed, and 100 Ixl of a 10-3 dilution in Blotto
buffer of an affinity-purified goat anti-human IgG gamma chain
antibody conjugated to alkaline phosphatase (Tago) was added to each
well. Plates were incubated for 90 min at 37 °C in a humid chamber,
washed, and the substrate p-nitrophenol phosphate (Sigma) in
diethanolamine buffer (Voller et al., 1976) was added (200 Ixl/well) to all
wells plus a blank well. Incubation was carried out at room temperature
for 20 min before interruption by the addition of 50 p.1/well of 3 MNaOH. Plates were immediately read in an automated ELISA plate
reader at 405 nm. For each serum, the absorbance values of the control
wells were subtracted from the values of the test wells, and the mean
was calculated. The intra- and inter-assay coefficientsof variation were
calculated by testing a reactive serum sample 12 times in duplicate, in
plates prepared and used on separate days (Rodbard, 1974). The MannWhitney U test was used for all statistical comparisons, and a twotailed P value of less than 0.05 was considered statistically significant.
Results
Fig. 1 displays the absorbance values observed with the
sera from the three groups of patients. There was no
significant difference between groups 1 and 2 (P = 0.78);
thus, these patients were combined into a single control
group. The median [interquartile range] of the absorbance values for the control group and group 3 were
respectively 0.118 [0.048, 0-286] and 0-324 [0.183, 1.029],
a statistically significant difference (P = 0.0001). Taking
the highest absorbance value of the control group (i.e.
476) as a cut-off (that is, assigning to the test a specificity
of 100~), the sensitivity of the assay was 32.6~ (15/46).
Assay performance was also evaluated by determination
of the intra- a n d inter-assay coefficients of variation,
which were 8 ~ and 6.4~, respectively.
To analyse further the serological reactivity of patients
with condylomata acuminata, we examined the results
according to the presence or absence of the common
papillomavirus antigen in wart biopsies (Fig. 2). The
median [interquartile range] of the absorbance values in
the group without the antigen (n = 18) and with the
antigen (n = 17) were respectively 0.229 [0.065, 0.339]
and 0.463 [0-327, 1.296] (P=0.0014). There were no
detectable differences in antibody reactivity according to
HPV type (P = 0.46). Table 1 contains the typing, biopsy
antigen status and absorbance values of the condyloma
acuminatum group.
2000
= 1500
1000
U
x
e~
8
500
<
0
-500
I
A
i
B
i
C
Fig. 1. Reactivity of the sera in the three groups of patients: (A) nuns
and priests, (B) patients with cutaneous warts and (C) patients with
condylomata acuminata. For each group of values, a scatterplot is
superimposed on a boxplot. Each box includes the mid-50~ of the
values, and the horizontal bar within represents the median absorbance
measurement.
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Human HPV-11 antibodies and anogenital warts
Table 1. H P V typing, common papillomavirus antigen status
of the biopsy, and absorbanee values of the condyloma
acuminatum patient group
2000
=
1500
1000
Patient
no.
~t
I
t
500
e~
<
o
-500
1345
+
~
A
I
B
F i g . 2. R e a c t i v i t y o f t h e s e r a f r o m p a t i e n t s w i t h o u t (A) a n d w i t h (B) t h e
c o m m o n p a p i l l o m a v i r u s a n t i g e n p r e s e n t in t h e i r w a r t b i o p s y . E a c h b o x
i n c l u d e s the m i d - 5 0 ~ o f the values, a n d t h e h o r i z o n t a l b a r w i t h i n
represents the median absorbance measurement.
Discussion
Using HPV-11 viral particles as antigen in an ELISA we
demonstrated that the sera of condylomata acuminata
patients have greater reactivities than sera from controls
with or without non-genital warts. Since most of the
patients were infected with HPV-6, and because no
difference in seroreactivity according to type was
observed, we assume that there is antigenic crossreactivity between HPV-6 and HPV-11 virions, an
observation supported by the close, 85 ~o, DNA sequence
similarity between these two viruses (Chow et al., 1987).
Previous studies have documented cross-reactivity of
human antibodies for HPV-6b and HPV-11 fusion
proteins derived from the LI open reading frame, which
encodes the major capsid protein (Jenison et al., 1989,
1990). In addition to demonstrating seroreactivity
among infected patients, we also observed a strong
relationship between the presence of the common
papillomavirus antigen in wart biopsies and seroreactivity to HPV-I 1 particles. This observation supports the
specific nature of the serological response measured by
the ELISA, inasmuch as patients who are exposed to
detectable quantities of capsid protein antigens would be
expected to develop a more vigorous humoral response to
intact virions than patients not exposed to these
antigens. One should also note that most of the sera we
used in this assay had been previously tested against
various HPV-6 recombinant fusion proteins; no spurious
differences in seroreactivity were then observed between
control and patient sera (Strike et al., 1989; Bonnez et al.,
1990). The antigenic specificity of HPV-1 ! viral particles
that is apparent in our observation is not surprising:
HPV-I virions have the same specificity also (Kienzler et
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
HPV
type*
6
6
6
6
6
6
6
6
11
6
6
11
11
6
6
NA
NA
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
NA
11
-11
NA
6
6
Common
papillomavirus
antigent
A b s o r b a n c e ( x 10 3)
a t 405 n m
-
NA
--+
-NA
-NA
+
--NA
--NA
NA
--+
+
---+
+
+
+
+
-+
NA
-NA
+
-+
NA
+
+
6
+
11
NA
6
--
NA
NA
+
+
- 48.0
-23-0
-- 5-5
37"5
44.0
64'0
72.0
111'5
124"5
152"0
172"0
186"5
187"5
188"5
215"5
242'5
245"0
252"0
268"0
269'5
273"5
296"5
310"5
338"0
344-5
356"5
387'5
420'0
450"5
462"5
487"0
745'0
771"5
792"5
1021'0
1051'5
1078"5
1125"0
114l'0
1227'5
1363'5
1481"0
1485"5
1673'5
1925"0
1966-5
* - , N o H P V D N A d e t e c t e d ; NA, n o t a v a i l a b l e .
t + , P r e s e n t ; - , a b s e n t ; NA, n o t a v a i l a b l e .
al., 1983; Anisimov~t et al., 1990; Steele & Gallimore,
1990; Viac et al., 1990). However, the limits and
mechanisms of the antigenic specificity of viral particles
of different HPV types will have to be more precisely
defined.
This is the first report of an immunoassay capable of
detecting HPV type 6- and 11-specific antibodies in sera
of patients with biopsy-proven condylomata acuminata.
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1346
W. Bonnez and others
Other immunoassays, using recombinant or synthetic
HPV-6b-derived antigens have been reported, but have
not demonstrated a difference in specific seroreactivity
between control groups and patients with documented
HPV-6 or -11 infection. In a previous study, we used
Western blotting to assay sera from condylomata
acuminata patients for reactivity to each of seven flgalactosidase fusion proteins derived from contiguous
fragments of the L1 and L2 open reading frames (ORFs)
of HPV-6b (Strike et al., 1989). We were unable to find
specific antibodies. Jenison et al. (1989, 1990) used an
HPV-6b Ll-derived synthetic peptide in an ELISA, as
well as TrpE fusion proteins originating from the E2, E7,
L1 and L 2 0 R F s in a Western blot assay to detect
specific antibodies in a sexually transmitted diseases
clinic population. Surprisingly, the seroprevalence profile in that group was similar to the one in a control group
of children. These authors speculated that this observation may reflect frequent infection with HPV-6 early in
life. Using an HPV-6b L1 TrpE fusion protein encompassing the amino acid sequence of the synthetic peptide
of Jenison et al. (1989), we tested the sera of condylomata
acuminata patients and found a seroprevalence rate of
about 5 0 ~ (Bonnez et al., 1990). As in the observations
of the previous investigators, this rate did not differ
significantly from that of controls with no history of
sexual activity. A preliminary report of a Western blot
assay using an HPV-6b L1 clI fusion protein, indicated a
higher seroprevalence in colposcopy clinic patients than
in children (Li et al., 1987). However, these results were
not correlated with HPV type or disease status. Other
investigators utilized denatured bovine papillomavirus
(BPV) type 2 in ELISA and detected antibodies among
condylomata acuminata patients with an overall accuracy up to 9 9 ~ (Baird, 1983; Beiss et al., 1987). As
denatured BPV exhibits a broadly cross-reactive papillomavirus antigen (Jenson et al., 1980), the specificity of
these immunoassays for condylomata acuminata is
surprising, and we have not been able to replicate these
results (unpublished data).
The present work does not elucidate the nature of the
epitopes responsible for immunological reactivity. The
HPV-11 virions used in the assay appeared as full viral
particles under electron microscopy. Therefore,
conformational as well as non-conformational epitopes
could be involved. 'Generally, recombinant fusion proteins, when used in immunoassays, have been unable to
permit the distinction between condyloma acuminatum
patient and control sera; therefore, we are inclined to
believe that fragile conformational epitopes are required
for specific immunological reactivity. It should be noted
for instance, that an epitope overlapping two distinct
capsid proteins would not be duplicated by a single
recombinant viral protein, however faithful it might be
to the native conformation.
The relative insensitivity of our assay is possibly the
reflection of factors related to the assay methodology and
the immunopathogenesis of infection. The limited
availability of the antigen has precluded the optimization of the reaction conditions. However our limited
experience in this regard indeed suggests that higher
concentrations of antigen would result in greater
sensitivity, specificity being equal. Nonetheless, one has
to consider the possibility that not all patients infected by
HPV-6 or -11 mount a specific antibody response, or that
this response is not stable over the course of the infection.
These and other issues are the matter of future
investigations.
The difficulty and high cost associated with producing
HPV-11 particles in athymic mice, along with the
relatively low sensitivity of the test as currently used,
make widespread use of our ELISA impractical at the
present time. However the test can be used to conduct
small-scale seroprevalence studies, and also can be used
in other well defined settings. In addition, the assay
could serve as a reference for the development of cheaper
and more convenient immunoassays for the diagnosis of
HPV-11-induced disease. In addition to demonstrating
the feasibility of developing a specific serological test,
our observations provide the best currently available
evidence that patients with biopsy-proven condylomata
acuminata possess serum antibodies directed against
type-specific genital tract HPV antigens, and that
control subjects without anogenital warts do not.
We are indebted to John Kreider for instructing us in the use of the
nude mouse model, and providing us with the initial HPV-11 viral
suspension. We thank David Strike for providing us with some of the
HPV-11-infected human xenografts. Reid Mattison and Elizabeth
Woodward kindly gave us access to neonatal foreskins. We are also
grateful to Karen de Mesy Jensen for assistance with the electron
microscopy, and to Mark Stoler and David Wilbur for the in situ
hybridization and immunocytochemistryof the patients biopsies.
Finally, we are indebted to the cooperation of all the volunteers who
providedthe sera usedin this study.This work was supportedin part by
AI-82509 from the NIAID, NIH, Bethesda, Md., U.S.A., and was
presented in part at the 30thInterscienceConferenceon Antimicrobial
Agents and Chemotherapy, Atlanta, Ga., U.S.A., 21 to 24 October
1990.
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(Received 3 December 1990; Accepted 28 February 1991)
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