VIROLOGY

190,724-732

(1992)

Independent Association of Antibodies against Human Papillomavirus Type 16 El /E4 and E7 Proteins with Cervical Cancer TADAHITO KANDA,*r’ TAKASHI ONDA,” SOICHI ZANMA,t TOSHIHARU YASUGI,* AKEMI FURUNO,” SUMIE WATANABE,* TAKASHI KAWANA,+ MOTOYASU SUGASE,§ KUNIAKI UEDAJ TAKAHIKO SONODAJ SABURO SUZUKI,B TAKENOBU YAMASHIRO,# HIROYUKI YOSHIKAWA,“” AND KUNITO YOSHIIKE* *Division of Molecular Genetics, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 14 1, Japan; tDiagnostics Laboratory, Chugai Pharmaceutical Company, Ltd., Tokyo 171; *Department of Obstetrics and Gynecology, Tokyo University Branch Hospital, Tokyo 112; SDepartment of Obstetrics and Gynecology, Nagano Red Cross Hospital, Nagano 380; IjDepartment of Gynecology, Tokyo Metropolitan Komagome Hospital, Tokyo 113; J-Department of Gynecology, National Cancer Center Hospital, Tokyo 104; lIDepartment of Obstetrics and Gynecology, Narashino National Hospital, Chiba 275; #Department of Obstetrics and Gynecology, University of the Ryukyus, Okinawa 903-O 1; **Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113 Received

March

11, 1992;

accepted

July 1, 1992

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an El /E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac-El/E4 and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-El/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-El/E4 and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma. o 1992 Academic press, IIIC.

INTRODUCTION

bridization techniques, because of the lack of the cell culture producing appropriate viral antigens. Jochmus-Kudielka et a/. (1989) first reported on the seroepidemiologic studies to detect human antibodies against HPV 16 E4 and E7 proteins in the healthy (or presumably unrelated to HPV infection) control population and the patients with cervical cancer. A protein (Western) blot method using bacterially expressed fusion proteins as antigens has shown that the anti-E4 is detectable in 30 to 40% of the age group 0 to 20 years and in 18% of the overall control population and that the anti-E7 is found 14 times as frequently in the patients with cervical cancer (20% positive) as in the agematched control (1.4% positive), whereas the difference in anti-E4 positivity between the two groups is not significant. From their data, they have concluded that the anti-E7 antibody may represent a marker for cervical cancer development and that the anti-E4 antibody may be correlated with viral replication. Whereas HPV 16 oncoprotein E7 has been shown to be the most abundant viral protein in some of the cell lines derived from cervical cancer (Smotkin and Wettstein, 1987; Oltersdot-f et a/., 1987) little is known about the HPV 16 E4 protein. The HPV 1 E4 protein is an abundant (30%) cytoplasmic protein in the HPV linduced warts (Doorbar et al., 1986). The mRNA-en-

Human papillomavirus type 16 (HPV 16) (Diirst et al., 1983) is considered to be one of the agents that cause cervical cancer, from its close association with the cancer and premalignant cells (zur Hausen, 1989) and from its oncogenic potential as shown by the capacity to encode two viral oncogenes E6 and E7 for rodent (Kanda et al., 1988a,b; Phelps et al., 1988; Yutsudo et al,, 1988) and human (Watanabe et al., 1989; Mijnger et al., 1989; Hawley-Nelson et al., 1989) cells. The two oncogenes appear to be involved in development of cancer in humans, because their expression can immortalize primary human keratinocytes in vitro (MOnger et a/., 1989; Hawley-Nelson et a/., 1989) and is detectable in the human cell lines derived from cervical cancer (Androphy et al., 1987; Seedorf et a/., 1987), but the sequence of events during the malignant change and the possible involvement of the other HPV 16 genes and unidentified factors are virtually unknown at present. Epidemiologic studies would be important and useful to define the role of HPV 16 in cancer development, but serologic studies have been scarce, as compared with studies by nucleic acid hy-

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be addressed. 724

ANTIBODIES

AGAINST

coding E4 protein constitutes the most abundant viral mRNA species in the HPV 1 warts and in the HPV 6-or HPV 1 l-induced condylomas, and analyses of the E4 mRNA has shown that splicing fuses the El and E4 open reading frames (ORF’s) (Chow et al., 1987a,b; Nasseri et al., 1987; Palermo-Dilts et al., 1990). A major transcript of HPV 16 in the transformed rodent cells is polycistronic mRNA encoded by the E7, E5, and E4 ORFs (Tanaka et al., 1989; Taniguchi and Yasumoto, 1990). In this mRNA, splicing fuses a short segment of the El ORF (encoding 5 amino acids (AAs)) to the E4 ORF, and thus the El/E4 fusion gene encodes a protein 92 AAs long. Expression of the E4 gene is not required for bovine papillomavirus type 1 (BPV 1) mediated transformation of mouse Cl 27 cells (Hermonat and Howley, 1987; Neat-y et a/., 1987), but the functions of BPV and HPV E4s are not known. In this study we constructed the HPV 16 El/E4 fusion gene and expressed it in bacteria and monkey cells. We examined 421 human sera for anti-E4 and anti-E7 antibodies by enzyme-linked immunosorbent assay (ELISA) using highly purified, bacterial lac-El/E4 fusion protein and nonfusion E7 protein as antigens. The data will show that the anti-E4 is not prevalent among young healthy females and that both anti-E4 and anti-E7 are specifically but independently associated with cervical cancer. These findings agree with those recently reported by Kochel et al, (1991 a,b), who showed that antibodies to E4 and/or E7 of HPVl6/18 are associated with cervical cancer. MATERIALS Human

AND

METHODS

sera

Sera were collected from 421 Japanese women: from 140 healthy women (20 from each of the age groups; 0 to 10, 11 to 20, 21 to 30, 31 to 40,41 to 50, 51 to 60, and over 61 years), 30 prostitutes, 53 pregnant women, 108 patients (25 to 93 years old, average 55.3 years) with cervical cancers (80 squamous cell carcinomas, 12 adenocarcinomas, and 5 adeno/squamous cell carcinomas, 11 unknown cases), 28 patients with ovarian cancer (24 to 80 years old, average 54.9 years), 6 patients with endometrial cancer (36 to 72 years old, average 56.5 years), 3 patients with choriocarcinoma (24, 31, and 52 years old), a patient with vulvar cancer (59 years old), 12 patients with cervical intraepithelial neoplasia (30 to 61 years old, average 45.5 years), 3 patients with vaginal intraepithelial neoplasias (43, 60, and 64 years old), a patient with vuluvar intraepithelial neoplasia (35 years old), 33 patients with condyloma acuminata (17 to 60 years old, average 33.5 years), 2 patients with wart, and a patient with bowenoid papulosis. Sera were obtained from patients

HPV16

El/E4

AND

E7

725

before treatment. For antibody assays, serum samples were preabsorbed twice with YA21 -PlO solution in which PlO fraction (see “Purification of antigens,” below) of YA2 1 transformed with pUC 18 plasmid was suspended in PBS containing 0.2% gelatin (Difco Laboratories, Detroit, Ml) at the concentration of $ of original bacterial culture volume. Thirty microliters of each serum sample was mixed with 135 ~1 of the YA21 -PlO solution and centrifuged at 10,000 g for 10 min after incubation at 0” overnight. The supernatant was again mixed with 135 ~1 of the YA2 1-PlO solution, incubated at 0” for 6 hr, and centrifuged at 10,000 g for 10 min. The second supernatants were used for assays. Expression

plasmids

Plasmid pUCl8-/ac-El/E4 was used to express in Escherichia co/i strain YA21 (Mizushima and Yamada, 1975) fusion protein lac-El/E4, in which the first 24 AAs of lac-Za-peptide are fused to the El/E4 protein at its N-terminal. Plasmid pSRcu-El/E4 was used to express nonfusion El/E4 protein in monkey cells. Plasmids pUCE7 (Kanda et al., 1991 a) and pUCE6 (Kanda et al., 1991 b) were used to express nonfusion E7 and E6 proteins, respectively, in E. co/i. Construction

of expression

plasmids

Plasmids pUC18-lac-El/E4 and ~SRLY-El/E4 were made by insertion of the constructed fusion gene El/ E4 into expression vectors pUCl8 and pSRcu-0 (Kanda et a/., 1991 a), respectively. From the structure of the reported splice junction for El/E4 (Tanaka et a/., 1989; Taniguchi and Yasumoto, 1990), we synthesized an oligonucleotide 5’-AAGCTTGATGGCTGATCCTGCAGCAGCAACGAAGTATCCTCTCCTGAAATTATTAGGCAGCACTTGG-3’ (DNA Synthesizer 381 A; Applied Biosystems, Foster City, CA), which contains a HindIll site at the 5’end, an initiation codon ATG [corresponding ATG from nucleotides (nt) 865 to 867 of HPV 16 DNA (Seedorf et al., 1985)], the splice junction GCAGI CAGC (nt 880/3358), and a 5’-half of Ball site at the 3’ end (nt 3401). The oligonucleotide was hybridized with a complementary synthetic oligonucleotide. The double-stranded synthetic DNA was ligated to the HPV 16 Ball-&t1 fragment (nt 3402 to 3697) and then a HindIll linker was added to the 3’ end of the ligated DNA. The resultant DNA fragment (encoding 92 amino acid El/ E4 protein) was inserted into pUCl8 or pSRcu-0 at its HindIll site. DNA sequences of the insert and surrounding junctions were determined by the dideoxy chain termination method (Sanger et al., 1977) to verify the expected structure. Construction of pUCl9-E7 and pUCl9-E6 has been described (Kanda eta/., 1991a,b).

726

Purification

KANDA

of antigens

Protein lac-El/E4 was purified by successive gel filtration and ion exchange column chromatography. YA2 1 transformed with pUC 18-lac-El /E4 was grown overnight in L-Broth (Maniatis eta/., 1982) containing 1 rnn/l isopropyl-P-o-(-)-thiogalactopyranoside, washed once with 10 mM Tris-HCI (pH 7.5) 50 mM NaCI, 1 mM phenylmethylsulfonylfluoride (PMSF), sonicated in the same buffer, and then centrifuged at 10,000 g for 30 min. The pellet (PlO fraction) was suspended in A buffer [8 M urea, 10 mM acetate (pH 5), 2 mM EDTA, 5% 2-mercaptoethanol (2ME)] and loaded onto a AcA44 (IBF Biotechnics, Villeneuve-la-Garenne, France) column equilibrated in A buffer. Protein was collected and loaded onto an SP-TOYOPEARL650C (Tosoh Corp., Tokyo, Japan) column equilibrated in A buffer. Fractions eluted with 0.3 M NaCl in A buffer were dialyzed against PBS. The final material formed a single band by SDS-polyactylamide gel electrophoresis. Partial amino acid sequence of the purified fusion protein was determined with an automated gas phase protein sequencer (PSQ-1; Shimadzu Corp., Kyoto, Japan), and the N-terminal 33 AAs were found to agree with those expected from the DNA sequence. Nonfusion and full-length E6 and E7 proteins were expressed in YA21 and purified as described previously (Kanda et a/., 1991a,b). Preparation

of antisera

against

HPV 16 proteins

Anti-HPV 16 El/E4 rabbit serum (anti-lac-El/E4) was raised against electrophoretically purified lacElIE protein by immunizing rabbits subcutaneously. AntiHPV 16 E7 rabbit serum (anti-/ac-E7) and anti-E6 monoclonal antibody (MAb 618) were prepared and characterized previously (Sato et al., 1989; Kanda et a/., 1991a,b). Enzyme-linked

immunosorbent

ET AL.

with 0.01% H,O, and 1 mg/ml o-phenylenediamine in 0.1 M citrate (pH 4.7). The absorbance (A) at 450 nm was measured after incubation at room temperature for 15 min in an automatic ELISA reader. Since some samples reacted with gelatin that we used for the blocking, specific A for E7, El/E4, or E6 protein was calculated by subtracting A for corresponding well of mock. Western

blot assay

Purified lac-El/E4 or E7 protein was electrophoresed (1 pgllane) and blotted to a nitrocellulose membrane. The membrane was coated by incubation in 1% BSA in Tris-buffered saline (TBS) (pH 7.4) overnight at 4’, reacted with human sera (1: 10 dilution), rabbit antilac-El/E4 or anti-/ac-E7 serum (1:200 to 1:128,000), that were preabsorbed twice with YA21 lysate in PBS containing 10% gelatin for 120 min at room temperature and washed three times with TBS containing 0.05% Tween 20. The membrane was incubated with a peroxidase-labeled goat anti-human or anti-rabbit IgG antibody (1 :lOOO dilution in TBS containing 1% BSA) for 60 min at room temperature, washed twice with TBS containing 0.05% Tween 20 and twice with PBS, and then stained with Konica lmmunostain HRP (Konica Corp., Tokyo, Japan). lmmunofluorescence

assay

COS-1 cell (Gluzman, 1981) on cover slips were transfected with pSRa-El/E4 plasmid DNA (1 pg of DNA per cover slip) by the DEAE-dextran method (McCutchan and Pagano, 1968). Cells were fixed with cold acetone after 48 hr incubation, air dried, and reacted with human sera at a 1:5 dilution or with antilac-El/E4 rabbit serum for 40 min at 37” and then reacted with fluorescein-conjugated anti-human IgG or anti-rabbit IgG (Cappel-Organin Teknika Corp., West Chester, PA) for 30 min at 37”.

assay

Microtiter plates (Immuno Assay-Plate, Titertek, The Netherlands) were coated with 50 ~1 of purified E7 protein (1 pg/ml), /ac-El/E4 protein (1 vg/ml), E6 protein (1 pg/ml), or mock in 0.05 M Na,CO, (pH 9.6) at 4”, for 14 hr, then blocked with 250 ~1 of 0.2% gelatin in PBS at 4” for overnight. Each well of a microtiter plate received 45 ~1 of a sample and was incubated for 120 min at room temperature, washed with PBS containing 0.05% Tween 20 and 0.05% Nonidet-P40, and received 50 ~1 of peroxidase-conjugated goat antihuman IgG, anti-rabbit IgG, or anti-mouse IgG antisera (Cappel-Organin Teknika Corp., West Chester, PA) at a 1 :lOOO dilution in PBS containing 1% BSA, incubated for 60 min at room temperature, washed, and stained

Statistical

analysis

Difference between proportions of positive sera in age-matched different groups were evaluated with the use of the x-square test. A P value below 0.05 was considered to indicate a significant difference. RESULTS Specificity

of ELISA

We tested specificity of the ELISA system used in this study, with anti-/ac-El/E4 rabbit serum, anti-lac-E7 rabbit serum, and normal rabbit serum. Antibodies were measured by ELISA with sera serially diluted twofold from 1: 100 to 1:5 12,000. Anti-lac-El /E4 gave spe-

ANTIBODIES

AGAINST

160

80

-0.1

0

0.1

Specific

0.2

0.3

0.4

0.5

absorbance

FIG. 1. Detection of IgG antibodies against HPV 16 El/E4 protein (A) and E7 protein (B). Four hundred and twenty-one sera, including 140 from healthy women and 108 from cervical cancer patients, were examined by ELISA and the number of samples are plotted against intervals of specific A. Arrows indicate the cut-off values determined statistically.

cific As over 0.100 at a 1:32,000 dilution for lac-El/E4 protein and specific As less than 0.05 at a 1: 100 dilution for E7 protein. Anti-&-E7 gave specific As less than 0.05 at a 1:lOO dilution for lac-El/E4 protein and specific As over 0.100 at 1:64,000 dilution for E7 protein. Normal rabbit serum showed specific As less than 0.050 at a 1:lOO dilution for both lac-El/E4 and E7 proteins. The relation between serum concentration and specific A was linear in the range of specific As from 0.01 to 0.5. From the lack of cross-reaction and from the positive reaction given by the highly diluted sera, we concluded that the ELISA system was sufficiently specific and sensitive to detect a low level of the respective antibody. Antibodies against HPV 16 El/E4 and E7 proteins in human sera We examined 421 human sera (including 108 from the patients with cervical cancer) by ELISA for anti-El/ E4 and anti-E7 IgG antibodies and summarized the results in the histograms shown in Fig. 1A and 1B. For the two antibodies, the frequency distributions of the great majority of the samples appeared to be normal

HPV16

El/E4

AND

E7

727

and to peak around specific A zero. We statistically determined arbitrary cut-off values, assuming that those distributed around specific A zero were seronegative. First, the mean and standard deviation (SD) were calculated for the 42 1 samples, and those with specific As higher than the mean + 4 X SD were considered to be seropositive. Second, the mean and SD were calculated again for those with specific As lower than the initial mean + 4 x SD, and those higher than the second mean + 4 x SD were considered to be positive. The calculation was repeated until there were none to be excluded for the subsequent calculation, and the last mean + 4 x SD was chosen as a cut-off value. Thus, we determined 15 and 22 samples to be positive for anti-El/E4 and anti-E7, respectively, because they showed specific As higher than the statistical cut-off values of specific As 0.045 and 0.079 for El/E4 and E7, respectively (Fig. 1). Figure 2 shows the correlation of anti-El/E4 and anti-E7 titers obtained with the 421 serum samples. None had the two antibodies, although one sample (214 from a patient with cervical cancer) had antibodies against the El/E4 and E7 proteins. Table 1 lists the samples tested in this study and those found to be positive by ELISA in various populations. Anti-El/E4 or anti-E7 positives were not found in the 140 healthy females consisting of 20 each of the 1O-year interval groups (0 to over 65 years) or in young pregnant women (17 to 24 years). Eleven of 15 anti-El/ E4 positives and all of the 22 anti-E7 positives were from the patients with cervical cancer (25 to 93 years old, average 55.3 years old). The proportions of positive sera from patients with cervical cancer were compared with those of age-matched controls by the Xsquare test. The antibody prevalences in cervical cancer patients were significantly higher than those in 60 age-matched healthy females (40 to 88 years old,

E

0.5

E

0.4

$

0.3

jj

0.2

;

0.1

q 0 u 4w -0.1 d

$ 8

1 -f

-0.1

0 Specific

FIG. 2. Correlation E7 antibody. Specific plotted against each

0.1 absorbance

0.2

0.3 for

0.4

El/E4

of positivities for anti-El/E4 antibody and antiAs for each protein summarized in Fig. 1 are other for respective samples.

728

KANDA

average 61.1 years; no anti-El/E4 and anti-E7 positives; P < 0.01 for anti-El/E4 and P < 0.01 for anti-E7) and those in 57 patients with genital diseases except cervical cancer (28 ovarian cancers, 6 endometrial cancers, 3 choriocarcinomas, 1 vulvar cancer, 2 vaginal intraepithelial neoplasias, 1 vulvar intraepithelial neoplasia, 15 condyloma acuminatas; 24 to 80 years old, average 50.0 years; one anti-El/E4 positive and no anti-E7 positive; P < 0.05 for anti-El/E4 and P < 0.01 for anti-E7). Therefore, we concluded that antibodies against El/E4 and E7 were associated specifically with cervical cancer. Among the cervical cancer cases with histological records, all of the 10 anti-El/E4 positives and 18 of 19 anti-E7 positives had been diagnosed as squamous cell carcinoma. None of patients with cetvical or vaginal intraepithelial neoplasia were positive for anti-El /E4 or for anti-E7. The anti-E l/E4-positive cases not related to cervical cancer consisted of one ovarian carcinoma, one bowenoid papulosis, and two prostitutes. The ELISA titers obtained with the prostitutes were low, as compared with those obtained with the cancer patients (Table 2). Detection of antibodies blot assay

in human

TABLE

Sample 214 177 188 280 178 281 344 249 387 393 208 204 180 202 223 a Cut-off-specific

AND E7 ANTIBODIES

Populations

Cases

Healthy women Pregnant women Prostitutes Patients with Cervical cancer Squamous cell carcinoma Adeno/squamous cell carcinoma Adenocarcinoma Unknown Ovarian cancer Endometrial cancer Choriocarcinoma Vulvar cancer Cervical intraepithelial neoplasia Vaginal intraepithelial neoplasia Vulvar intraepithelial neoplasia Condyloma acuminata Bowenoid papulosis Wart

140 53 30 108 80

Specific

A”

Diagnosis

0.367 0.356 0.340 0.193 0.188 0.184 0.118 0.078 0.075 0.073 0.072 0.070 0.060 0.059 0.051

Cervical Cervical Cervical Cervical Bowenoid Cervical Ovarian Cervical Prostitute Prostitute Cervical Cervical Cervical Cervical Cervical

cancer cancer cancer cancer papulosis cancer cancer cancer

cancer cancer cancer cancer cancer

A: 0.045.

1:64,000 could visualize a lac-El/E4 protein band and anti-/ac-E7 serum at a 1:32,000 dilution could recognize a E7 protein band. Under these conditions, selected human serum samples were examined by Western blot method (Table 3). The lowest level of detection by Western blot method seems to be for those with specific A around 0.12 for El/E4 and around 0.13 for E7. Apparently, the ELISA was more sensitive for detection of human anti-El/E4 and anti-E7 antibodies than the Western blot method.

sera by Western

1

HPV 16 El/E4

2

CASES POSITIVE FOR ANTI-E 1 /E4 ANTIBODY

We compared the sensitivity of ELISA with that of Western blot method for anti-El/E4 and anti-E7. In Western blot assay, anti&c-El/E4 serum at a

TABLE

ET AL

IN VARIOUS POPULATIONS Anti-E 1 /E4 (O/o) Anti-E7 0 0 2 11 (10.2) 10

(%)

0 0 0 22 (20.4) 18

5 12 11 28 6 3 1

0 0 1 1 0 0 0

0 1 3 0 0 0 0

12

0

0

3 1 33 1 2

0 0 0 1 0

0 0 0 0 0

Recognition of eucaryotic El/E4 and human serum

El/E4

protein

by anti-lac-

We tested whether rabbit anti-lac-El/E4 and human serum positive against lac-El/E4 can recognize native HPV 16 El/E4 protein transiently expressed in COS-1 cells. COS-1 cells were transfected with pSRa(-El/E4 and were stained with anti&c-El/E4 or a human sample (177) showing specific A of 0.356 for lac-El/E4 in ELISA assay. In both cases immunofluorescence-positive cells were found among the majority of unstained cells in the background (Fig. 3). The positive cells showed granular immunofluorescent dots distributed in cytoplasm occasionally with faint nuclear immunofluorescence. Although immunofluorescence staining was less intense with human serum than with rabbit anti-lac-El/E4, the results clearly show that the human antibody detected by ELISA was capable of reacting with the native HPV 16 El/E4 protein. To test the specificity of recognition, we attempted to stain with the above two sera the COS-1 cells transfected with the pSR plasmid capable of expressing the

ANTIBODIES TABLE DETECTION OF ANTIBODIES

ELISAa

HPV16

El/E4

AND

E7

729

3 BY WESTERN BLOT ASSAY

anti-El/E4 Sample

AGAINST

anti-E7 Westernb

Sample

ELISA”

Westernb

160 143

-0.024 -0.010

-

344 228

-0.017 -0.010

-

262 159 307 286 308

-0.004 -0.001 0.002 0.007 0.013

-

178 307 308 235 281

-0.009 0.000 0.018 0.020 0.025

-

235 198

0.015 0.025

-

143 117

0.026 0.053

-

273 245 223 202

0.027 0.035 0.051 0.059

-

287 198 161 197

0.067 0.069 0.077 0.101

-

180 204 208 249 344 281 178 214

0.060 0.070 0.072 0.078 0.118 0.184 0.188 0.367

+ t * t

290 233 297 160 159 214 286 262

0.104 0.113 0.126 0.134 0.253 0.260 0.379 0.407

2 + t t +

a Cut-off-specific A for anti-El/E4 antibody; 0.045. b The test was repeated several times. The constant positives and the negatives were scored as + and -, respectively. The sample which showed positive signal inconstantly was scored as +. ’ Cut-off-specific A for anti-E7 antibody: 0.079.

HPV 1 El/E4, HPV 6 El/E4, or HPV 58 E4 protein. The two sera failed to stain any of these COS-1 cells. Production of HPV 6 El/E4 and HPV 58 E4 in the transfected COS-1 cells had been detected by immunofluorescence staining with anti-lac-HP6El/E4 rabbit serum and with human serum from a patient with HPV 58 DNA-positive cervical cancer, respectively. Construction of expression plasmids for HPVs 1, 6, and 58 E4s, preparation of anti-lac-HP6El/E4, and characterization of anti-HP58E4 positive human serum will be described elsewhere. The results indicate that the immunofluorescence staining shown in Fig. 3 was specific for the HPV type. ELISA for anti-E6 antibody with cervical carcinoma

FIG. 3. lmmunofluorescence stainrng of monkey COS-1 cells expressing HPV 16 El/E4 protein. Cells transfected with ~SRLU-El/E4 were fixed with cold acetone and stained with anti-lacElIE rabbit serum (A and B) or human serum 177 (C and D).

in the cell lines from cervical cancer (Smotkin and Wettstein, 1986; Androphy et al., 1987; Smotkin eta/., 1989). Specificity of ELISA was tested with anti-E6

of sera from patients

We tested the sera from the patients with cervical cancer (108 samples) and the patients with cervical intraepithelial neoplasia (12 samples) for antibody against HPV 16 E6 protein by ELBA (Fig. 4) because it is known that the HPV 16 E6 and E7 ORFs are together conserved (zur Hausen, 1989) and are often expressed

Specific

absorbance

FIG. 4. Detection of IgG antibody against HPV 16 E6 protein. One hundred and twenty sera from patients with cervical cancer or patients with cewical intraepithelial neoplasia were examined by ELISA. The number of samples are plotted against intervals of specific A. Arrow indicates a statistical cut-off A value.

730

KANDA

monoclonal antibody (MAb 6 18). We determined a statistical cut-off value for anti-E6 as we did for anti-El/E4 and anti-E7 (Fig. 1). We found three positives, specific As of which were marginally higher than the cut-off value. The three were from the patients with cervical carcinoma; one was positive for anti-El/E4 and the other two were positive for anti-E7. No sera showed high reactivity against the E6 protein.

DISCUSSION In this study we found by ELISA 15 and 22 seropositives for El/E4 and E7 of HPV 16, respectively, in 108 patients with cervical cancer, but none in 140 healthy women from 0 to over 65 years of age. There was no correlation of seropositivity between the two antibodies, except for one patient who had both antibodies. We showed that the ELISA system was specific and sensitive to detect the antibodies in human sera, and that the human serum positive by ELISA for bacterial &-El/E4 was capable of specifically recognizing the native HPV 16 El/E4 protein in an immunofluorescence assay. Specificity of detection of human anti-E7 has been established in our previous study (Hashido et al., 1991) in which 500 human sera were examined for anti-E7 of HPV 16 with partially purified lac-E7 and some of the selected were examined by immunoprecipitation and immunofluorescence with the nonfusion E7 expressed in COS-1 cells. The statistical analysis of the frequency of seropositives in sera from the patients with cervical cancer and those of age-matched controls indicates that occurrence of the anti-El/E4 and anti-E7 antibodies is specifically but independently associated with cervical cancer. The data indicating specific association of anti-E7 with cervical cancer agree with our previous report (Hashido et a/., 1991) and are essentially consistent with those of Jochmus-Kudielka et al, (1989) Mann et a/. (1990) Kochel et al. (199la,b), and Mllller et al. (1992). Because the E7 protein is the most abundant HPV protein in cervical cancer cells (Smotkin and Wettstein, 1987) it would be reasonable to expect prevalence of anti-E7 in some of the patients with cervical cancer. Despite the high sensitivity of ELISA, we found no anti-E7 positives in the healthy control females, including children. However, low levels of anti-E7 antibodies have been reported by the others (Jochmus-Kudielka, et al., 1989; Mann et al., 1990; Kochel et a/., 1991 a,b; MUller et al., 1992). Furthermore, Jenison et a/. (I 990) reported that prevalence of anti-E7 positive in the children (12%) is not significantly different from that in the patients with sexually transmitted diseases (13%). Perhaps the minor and major differences

ET AL.

among our data and these reports are ascribable to the difference in type, source, and purity of the antigens. Our data concerning anti-El/E4 do not agree with those of Jochmus-Kudielka et al. (1989) who showed that the anti-E4 antibody is prevalent among young normal females and not associated with cervical cancer. The data in this study, however, are consistent with those of Kochel et al. (1991a,b), who found antiE4, anti-E7, or both in sera from patients with cervical cancer but not in those from control females. This discrepancy would not be accounted for by the difference in sensitivity of the assay system, because both the Western blot assay used by Kochel et al. (1991 a) and the ELISA in this study appear to be more sensitive than the Western blot assay by Jochmus-Kudielka et al. (1989). It remains to be examined whether the use of different antigens is responsible for the discrepancy. Elucidation of natural history of HPV 16 and of its role in cancer development would require search for markers of the HPV 16 replication. Besides sera from the cancer patients, a few samples were positive for anti-El/E4. Serum from a patient with bowenoid papulosis, which is known to be associated with HPV infection (Ikenberg et a/., 1983) was positive for anti-El/E4 antibody (specific A; 0.188) but negative for anti-E7 antibody. A patient with ovarian cancer and two prostitutes were positive for anti-El/E4 antibody. The significance of these results is unclear at present, but would be clarified by further extensive studies. We found three weak positives for anti-E6 antibody in sera from the cervical cancer patients. Mijller et al. (1992) also reported that antibody against HPV16 E6 protein is associated with invasive cervical cancer and that prevalence of anti-E6 positives is less frequent than anti-E7 antibody. Lack of high-titer anti-E6 in the human sera probably reflects the low yield of the mRNA encoding E6 protein, as compared with that encoding E7 protein, in the cervical cancer cells (Smotkin et a/., 1989). Little is known about the El/E4 function of papillomaviruses and, therefore, it is difficult to infer the role of HPV 16 El/E4 protein in carcinogenesis. It remains to be determined whether the anti-El/E4 antibody in the cancer patients is produced by the El/E4 protein expressed continuously in some of the cancer cells or by a large amount of El/E4 protein transiently expressed in lytic infection prior to the development of tumors. It will be important to determine whether biopsies from the cancer contain the transcripts encoding EllE4, as well as those encoding E7, and to define the functions of El/E4 in the life cycle of HPV 16. The independent association of the anti-E4 and antiE7 antibodies to cervical cancer and the low incidence

ANTIBODIES

AGAINST

of the double infection of high-risk HPVs (Lorincz eta/., 1992) suggest that both anti-El/E4 and anti-E7 may serve as independent markers of carcinogenesis by HPV 16. However, the combined positivity of anti-El/ E4 and anti-E7 in sera from patients with cervical carcinoma in this study (30.6%) is still lower than the HPV 16 DNA positivity for cervical cancer (47.1 O/o)(Lorincz eta/., 1992). The sera from cervical carcinoma patients used in this study included five serum samples from the patients with the cervical cancer that were positive for HPV 16 DNA in Southern blot assay. With these five samples we could detect neither anti-El/E4 nor antiE7 antibody. The discrepancy between the antibody and DNA data may be explained, at least partly, by the difference in sensitivity between the two assays. Another possible explanation, however, is that not all the cervical cancers harboring HPV 16 DNA are producing HPV proteins to stimulate antibody production. The second possibility is supported by the reports showing that the integrated HPV 16 DNA in cervical cancer biopsies is not always transcriptionally active (Lehn el al., 1985) and that in some carcinomas only the antisense HPV RNA are transcribed (Higgins et al., 1991). Systematic studies are required to correlate epidemiologic data on DNA and antibodies.

ACKNOWLEDGMENTS This work was supported by a grant-in-aid from the Ministry of Health and Welfare for the Comprehensive lo-Year Strategy for Cancer Control, by a grant from the Japan Health Sciences Foundation, by a research grant for aging and health, the Ministry of Health and Welfare, and by a cancer research grant from the Ministry of Education, Science, and Culture. We are indebted to Dr. Kikuko Miyamura, National and WHO Serum Reference Bank, Central Virus Diagnostic Laboratory, National Institute of Health, MusashiMurayama, for supplying 140 sera from healthy women. We thank Dr. Madoka Hashido for her help in compiling records of the patients. T. Onda and T. Yasugi were fellows of the Japan Health Sciences Foundation.

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