Journal of General Virology (1992), 73, 429-432.
429
Printed in Great Britain
Epitope mapping of the human papillomavirus type 16 E4 protein by
means of synthetic peptides
A. Such~nkovfi, 1. V. Krchfi~ik, 2 J. V~igner, 2 E. Ham[ikovfi, 1 M. Kr~.mfi~,1 L. Ritterov~d
and V. Vonka 1
1Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Korunni 108, 101 03 Prague 10
and 2Department of Synthetic Peptides, Institute of Sera and Vaccines, Prague, Czechoslovakia
Eight overlapping icosapeptides covering the entire
sequence of the E4 protein of human papillomavirus
type 16 (HPV-16), were prepared and tested for their
reactivity with human sera in IgG-specific ELISA. The
strongest reactivity of sera from HPV-16 DNApositive invasive cervical carcinoma (INCA) patients
was detected with the peptide denoted 16/E4-6,
covering amino acids 51 to 70. Subsequently nearly 200
sera were tested for the presence of the 16/E4-6-
specific antibody. Reactivity was more frequent in
cervical intraepithelial neoplasia patients and INCA
patients than in matched control subjects. Sera from
INCA patients were also tested for antibody reactive
with peptide 16/E7-2 covering the major type-specific
reactive region of the HPV-16 E7 protein. Only four of
13 sera possessing the 16/E4-6-specific antibody were
reactive with the 16/E7-2 peptide.
The development and evaluation of type-specific serological tests for diagnosis of human papillomavirus (HPV)related cervical cancer has recently received much
attention (Dillner et al., 1989; Dillner, 1990; JochmusKudielka et al., 1989; Jenison et al., 1989, 1990, 1991;
Krchfi~k et al., 1990; Mann et al., 1990; Suchfinkovfi et
al., 1990, 1991; K6chel et al., 1991). Although the
majority of the research has been focused on the E7
protein, the product of the E4 open reading frame
(ORF), which is poorly conserved among different HPV
types (Doorbar et al., 1989), has also emerged as a
possible candidate antigen in such tests. The E4 protein
of HPV-1 is expressed abundantly in HPV-l-associated
warts (Croissant et al., 1985; Doorbar et al., 1986) and is
believed to play a role in virus maturation. Although an
abundant E4-specific m R N A spliced to the 5' end of the
E 1 0 R F was found in genital warts (Chow et al., 1987)
and in a human cell line immortalized by HPV-16 D N A
(Diirst et al., 1987), little is known about the extent of E4
ORF expression in genital precancerous and cancerous
lesions associated with HPV-16. Distribution of anti-E4
antibodies in human sera was first reported by JochmusKudielka et al. (1989) who used the HPV-16 E4 fusion
protein as the antigen in Western blot (WB) assays. They
have detected IgG anti-E4 antibody in 16% of invasive
cervical carcinoma (INCA) patients and in 8% of
matched control women; however, up to 40% anti-E4positive sera were found among healthy teenagers and
among women suffering from cervical intraepithelial
neoplasia (CIN). On the basis of these data the authors
concluded that the presence of E4-specific antibody
correlates more closely with virus replication than with
the development of cancer. K6chel et al. (1991) who also
employed HPV-16 fusion proteins as antigens in WB
detected anti-E4 antibody in three out of four genital
carcinoma patients but not in any of the 63 healthy
women tested. Using an ELISA with synthetic peptides
derived from the ORF of HPV-16 E4, Dillner (1990)
found the major IgG- and IgA-reactive epitope(s) in a
peptide covering amino acids 47 to 66. The IgG antibody
reactive with this peptide was detected in 37% of INCA
patients and in 28 % of control subjects; the prevalence of
the IgA antibody was 67 and 33%, respectively,
suggesting that the positions of the immunoreactive
epitopes can differ for different Ig classes. To identify
seroreactive regions of the HPV-16 E4 protein, Miiller et
al. (1990) used HPV-16 E4 antigens expressed as part of
the capsid protein of genetically modified phage fd and
synthetic octapeptides derived from the HPV E4 ORF.
Employing rabbit sera prepared by immunization with
the HPV-16 E4 fusion protein they located the major
immunoreactive region between amino acids 34 and 47.
Recently we have reported the mapping of HPV-16 E7
epitopes reactive with human sera using a series of
overlapping peptides covering the entire sequence of this
protein (Krchfifik et al., 1990; Such~nkov/t et al., 1991).
In the present experiments we adopted a similar
approach to delineate regions of the HPV-16 E 4 0 R F encoded protein containing immunoreactive epitopes.
Sera used in the present study originated from both
0001-0507 © 1992 SGM
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430
Short communication
2.5
t
t
i
i
i
i
i
i
2
1.5
Group
Healthy subjects*
CIN patients
CIN controls
I N C A patientsl"
INCA controls
1
0.5
0
Table 1. Presence of 16/E4-6 antibody in sera of healthy
women and patients with C I N or INCA and their matched
control subjects
16/E4-1
16/E4-3
16/E4-5
16/E4-7
16/E4-2
16/E4-4
16/E4-6
16/E4-8
Fig. 1. Reactivity (A6o0) in ELISA of pooled sera with individual
peptides derived from HPV-16 E 4 0 R F . ~ ,
HPV-18 DNA-positive;
IININ, HPV-16 DNA-positive; ~ ,
HPV-16 E4 WB-positive; gT~,
HPV 16 E4 WB-negative; [ ~ ] , children, 1 to 3 years.
healthy women and those suffering from either CIN or
INCA. The group of healthy women consisted of 87
subjects aged 19 to 49 years who had been investigated at
the Center of Cervical Cancer Prevention, Third
Medical Faculty, Charles University, Prague, and found
to be free of any pathological colposcopical and
cytological lesions. Fifteen sera originated from women
with CIN (I to III) aged 24 to 50 years and 30 sera from
women suffering from INCA aged 33 to 79 years. Biopsy
specimens for hybridization tests (Krchfi~ik et al., 1990)
were available from 12 INCA patients. HPV-16 DNA
was demonstrated in six and HPV-18 DNA in three
specimens. Control groups of healthy women matched
by age and the area in which they lived were constituted
for each group of patients; these subjects were not
investigated gynaecologically. In addition, sera from 30
children aged 1 to 11 years were also included. Eight
icosapeptides consecutively overlapping each other by 10
amino acids and covering the entire sequence of the E4
protein were prepared by continuous-flow solid-phase
multiple peptide synthesis and were purified as described
previously (Krchfi/tk et al., 1990). An ELISA for
detecting IgG antibodies was performed as described
(KrchlSfik et al., 1990). The HPV-16 E4 fusion protein
and WB technique were the same as those used by
Jochmus-Kudielka et al. (1989).
For screening tests we used 2 lag of each peptide per
well and five serum pools each containing at least three
sera. Pool no. 1 originated from INCA patients in whose
biopsies HPV-18 DNA was detected. Pool no. 2 was
formed by sera from HPV-16 DNA-positive INCA
patients which possessed antibody reactive with HPV-16
E4 in WB. Another pool (no. 3) was formed by sera from
INCA patients whose sera were HPV-16 E4 antibodypositive in WB but were not tested for the presence of
No. tested
No. reactive
Percentage
reactive
87
25
25
30
30
19
8
4
13
4
21.8
32.0
16-0
43.3
13.3
* Prevalence of antibody in age groups < 25, 25 to 35, > 35 years was
29-1, 17.1 and 21.4~, respectively,
t Hybridization tests were performed with materials from 12 I N C A
patients; HPV-16 D N A was detected in six and HPV-18 D N A in three
patients.
HPV DNA. Pool no. 4 consisted of INCA patients' sera
which were free of HPV-16 E4 antibody in WB and were
not tested for the presence of HPV DNA. Finally, pool
no. 5 was created by mixing sera from healthy children.
The ELISA results with the eight peptides are shown in
Fig. 1. The strongest reactivity occurred for peptide
16/E4-6 (amino acids 51 to 70) with the two serum pools
(nos. 2 and 3) originating from INCA patients, positive
in WB for the HPV-16 E4 antibody. Sera which were free
of the E4 antibody in WB assay, originating from HPV18 DNA-positive patients and from other INCA
patients, were non-reactive with this peptide. Some
reactivity, although of lower intensity, was also observed
with peptides 1 to 5. However, due to the relatively high
reactivity of control children's sera, the specificity of the
reactions with peptides 16/E4-1, 16/E4-2 and 16/E4-5 is
questionable. None of the serum pools was reactive with
peptides corresponding to the C-terminal portion of the
E4 protein.
For subsequent tests only the 16/E4-6 peptide was
used. In all the tests 4 antigen units of the 16/E4-6
peptide were used, 1 antigen unit being defined as the
highest antigen dilution reactive with a 1:20 dilution of
serum pool no. 3. All sera were tested in a 1 : 20 dilution.
To assign a cutoff value we tested sera from the 30
control children which we assumed had not experienced
HPV-16 infection. The cutoff value selected was the
mean ELISA absorbance value of these sera plus 3 S.D.
(~ = 0.395; S.D. 0"231; cutoff point 1.08). The icosapeptide derived from the HPV-1 E7 ORF (amino acids 11 to
30) served as control antigen; only one of nearly 200 sera
tested was found reactive with this antigen.
Results of 16/E4-6 antibody determinations are summarized in Table 1. Nineteen out of 87 (i.e. 21.8~0)
women with normal colposcopical and cytological
findings possessed the antibody. As indicated in the
footnote to Table 1 within this group the prevalence of
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Table 2. Presence of 16/E4-6 and 16/E7-2 antibodies in
sera from I N C A patients
16/E4-6
+
16/E7-2 +
-
Total
-
4
4
9
13
13
17
Total
8
22
30
the 16/E4-6 antibody was not clearly age-dependent. The
antibody was detected in eight of 25 CIN patients (32~)
but only in four of 25 (16~) matched control women.
However this difference was not statistically significant
(P > 0.05). The prevalence of 16/E4-6 antibody among
the INCA patients was even higher; it was detected in 13
of 30 patients (i.e. 43.3~o); among the matched control
subjects it was found only in four instances (13-3 ~). This
difference was statistically significant (P < 0-01). Of the
six INCA patients in whose biopsies HPV-16 D N A had
been demonstrated, three possessed the 16/E4-6 antibody, which was also detected in sera of all three HPV-18
DNA-positive patients; it may be of interest, however,
that the absorbance values of these three sera were the
lowest among the sera scored as positive. These three
sera were also weakly positive in the WB assay using the
HPV-16 E4 protein as antigen (not shown). Sera from the
three HPV DNA-negative patients were free of the
16/E4-6 antibody.
Sera from INCA patients were also tested for the
presence of antibody to the 16/E7-2 peptide (corresponding to amino acids 11 to 30 of the E7 protein); this
peptide has been found to cover the major type-specific
reactive region of the HPV-16 E7 protein (Krchfifik et
al., 1990; Such~mkovfi et al., 1991 ; Jenison et al., 1991).
As indicated in Table 2, of the 13 16/E4-6-positive sera
only four possessed the 16/E7-2 antibody. Thirteen sera
were negative in both tests. Thus the overall concordance
was 56.7~, showing a rather low degree of correlation.
The results presented here indicate that the major
immunoreactive region of the HPV-16 E4 protein is
located between amino acids 51 and 70. This finding is
consistent with the previous observation by Dillner et al.
(1989). The immunoreactive region recognized is different from that reported by Miiller et al. (1990), which
indicates that the antibody raised by immunizing with
bacterial fusion protein was reactive with epitopes
distinct from those recognized by human antibody. The
prevalence of the 16/E4-6 antibody was higher than that
of the 16/E7-2 antibody which had been quite rare
among healthy subjects and women with CIN (Such~nkov~t et al., 1991). However, there seems to be an
association between cervical neoplasia and the presence
431
of the 16/E4-6 antibody. Prevalence of this antibody was
higher among the CIN and INCA patients than among
matched control women; this does not necessarily imply
that the occurrence of the 16/E4-6 antibody was
associated with the development of cancer. It is possible
that the difference in antibody prevalence simply
reflected a higher proportion of HPV-16-infected subjects among I N C A patients compared to healthy women.
It is noteworthy that the antibody response to 16/E4-6
did not predict the reactivity with 16/E7-2, and vice
versa. It should be recalled that Mann et al. (1990)
also reported poor correlation between the presence of
HPV-16 E7 antibody and antibody to peptide 245, a
nonadecapeptide derived from a region close to the
carboxyl terminus of the E2 protein of HPV-16 (Dillner
et al., 1989). It is not clear whether the present
observation was due to the variation in the response of
HPV-16-infected subjects to different epitopes. Such
variation may be dependent on individual sensitivities to
different viral antigens as reported recently by Jenison et
al. (1991) for HPV-16 and HPV-18 L2 proteins, or be a
consequence of specific features of the virus-host
interactions, such as the rate of virus replication or the
extent of the viral expression in tumour tissue. Provided
that both reactions are HPV-16-specific, the simultaneous use of the two antigens could markedly increase
the capability of monitoring HPV-16 infections and/or
HPV-16-associated cervical cancer by serological means.
It is also possible, however, that the discrepancy reported
here and that observed by Mann et al. (1990) were
associated with different strain specificies of the respective antigens and that a considerable proportion of the
positive reactions was due to fortuitous cross-reactions.
The present observation of the positive albeit weak
reactivity of three sera from HPV-18 DNA-positive
patients may favour this possibility. More information
and more rigorous analyses of the reactivities monitored
are needed to clarify the significance of the association of
the various antibodies with HPV-16 infection and,
especially, with HPV-16-1inked neoplasia.
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(Received 23 July 1991; Accepted 31 October 1991)
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