Journal of General Virology (1991), 72, 2577-2581.
2577
Printed in Great Britain
Comparison of ELISA and Western blotting for human papillomavirus type 16 E7 antibody determination A. Suchfinkovfi, 1. L. Ritterovfi, 1 M . Kr~.m~L 1 V. Krchfifik, 2 J. Vfigner, z I. Jochmus, 3 L. Gisslnann, 3 J. Kafika 4 and V. V o n k a 1 1Department of Experimental Virology, 2Department of Synthetic Peptides, Institute of Sera and Vaccines, Prague, Czechoslovakia, 3Deutsches Krebsforschungszentrum, Heidelberg, Germany and 4Department of Obstetrics and Gynaeeology, Third Medical Faculty, Charles University, Prague, Czechoslovakia
A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35-2 %) than in the other individuals (six of 106, 5.7 %). Ten children's
(1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.
Diagnosis of infection with human papillomaviruses (HPVs) suggested by colposcopical, cytological or histological findings still rests mainly on the demonstration of HPV DNA presence. In the past few years increasing efforts have been made to develop serological tests capable of proving HPV infection. In these tests, three sources of antigens have been utilized: (i) intact or disrupted HPV virions extracted either from HPVinduced lesions (Pfister & zur Hausen, 1978; Baird, 1983; Dillner et al., 1989a; Steele & Gallimore, 1990; Anisimov/t et al., 1990) or from virus-infected xenografts in nude mice (Christensen & Kreider, 1990), (ii) genetically engineered HPV proteins, usually in the form of bacterial fusion proteins (Jenison et al., 1988, 1989; Jochmus-Kudielka et al., 1989) or expressed as part of a capsid protein of genetically modified phage fd (Mfiller et al., 1990) and (iii) synthetic peptides derived from known sequences of different HPV D N A open reading frames (ORFs) (Cason et al., 1989; Dillner, 1990; Dillner et al., 1989b, 1990; Such~.nkov/~ et al., 1990; Miiller et al., 1990). Recently we developed an ELISA to detect typespecific antibody to HPV type 16 E7 protein in human sera (Krchfi~.k et al., 1990). We examined a set of nine overlapping (by 10 amino acids), synthetic peptides
covering the whole amino acid sequence of the HPV-16 E7 protein; of these eight were icosapeptides and one was an octadecapeptide. From this study it was concluded that peptides 16/E7-1 (amino acids 1 to 20), 16/E7-2 (11 to 30), 16/E7-3 (21 to 40) and 16/E7-4 (31 to 50) were type-specific whereas the 16/E7-5 peptide was broadly cross-reactive. In sera from invasive carcinoma patients, positive HPV-16-specific reactions were more frequent with the 16/E7-2 peptide than with the other peptides. This reactivity could be removed by absorption with the 16/E7-2 peptide as well as by the genetically engineered HPV-16 E7 fusion protein but not with the HPV-16 E4 fusion protein. In the present series of experiments we tried to correlate the results obtained in ELISA, using the 16/E72 peptide, and by Western blotting (WB) using the HPV16 E7 fusion protein. A total of 140 serum samples were examined. All of them were collected at the Center of Cervical Cancer Prevention, Third Medical Faculty, Charles University, Prague, from women aged 25 to 53 years. Of these, 85 were free from pathological, colposcopical and cytological findings, 21 suffered from various stages of cervical intraepithelial neoplasia (CIN I to CIN III) and 34 from invasive cervical carcinoma (INCA). Sera from l0 children aged 1 to 3 years were also
0001-0289 © 1991 SGM
2578
Short communication
tested in parallel. All sera were inactivated by heating at 56 °C for 30 rain and kept at - 2 0 °C until investigated. The peptides were prepared and IgG-specific ELISA was performed as described previously (Krchfi/tk et al., 1990). Icosapeptides derived from the HPV-I E7 ORF, denoted 1/E7-1 (amino acids 11 to 30) and 1/E7-2 (amino acids 35 to 54) served as control antigens. Control sera known to be positive or negative for the HPV-16 E7 antibody in ELISA (Krchfifik et al., 1990) were included in each test. All sera were tested in a 1 : 10 dilution. The cutoff value between positivity and negativity was the mean absorbance (A) value plus 3 S.D. of the previously tested group of sera from 44 children aged 1 to 11 years which were considered anti-HPV-16 antibody-negative (Krchfifik et al., 1990). In WB experiments, lysates of genetically engineered Escherichia coli expressing HPV-16 E7 and HPV-16 E4 fusion proteins were used (Jochmus-Kudielka et al., 1989). They were derived from the expression vectors pEX 8 mer-HPV-16 E7 and pEX 12 mer-HPV-16 E4 constructed by Seedorf et al. (1987). They contain the whole HPV-16 E7 ORF [nucleotides (nt) 585 to 855] and HPV-16 E 4 0 R F (nt 3399 to 3617), respectively, fused to the DNA sequence encoding the first 100 amino acids of the bacteriophage MS2 polymerase. Preparation of the expressed fusion protein from bacterial extracts and WB analysis of sera which had been preabsorbed with lysates of the parental bacteria, were performed according to Jochmus-Kudielka et al. (1989); for visualization of the antigen-antibody reaction peroxidase-labelled pig antihuman IgG (SwaHu, Sevac) was used. As a control antigen a protein comprising the first 100 amino acids of the MS2 polymerase was employed (Jochmus-Kudielka et al., 1989). For the WB assay, sera were diluted 1:20. A positively reacting serum (diluted 1:1000) was included as a positive control in each test. If not indicated otherwise each serum sample was tested twice and was scored as strongly (+ + +), intermediately (+ +) and weakly ( + ) positive or negative ( - ) . With very few exceptions mentioned below, the results of the repeated tests were identical. The results are summarized in Tables 1 and 2. Of 106 sera from healthy women and women with CIN, only five (4.7~) were reactive with 16/E7-2 in ELISA. Of these, three reacted also with the HPV-16 E7 fusion protein in WB. In addition, one serum which was negative in ELISA gave a positive reaction in WB. The remaining 100 sera were negative in both tests. Thus, concordant results were obtained in 97-2~ sera. As the measure of agreement of the two methods, Cohen's kappa (Cohen, 1960) was used. The kappa value was 0.6521 _+0.2914, Student's t-test 2.237 (P < 0.05). In the group of 34 sera from INCA patients, positive reactions were more frequent; however, their distribution in
Table 1. HP V-16 E7 antibody presence in sera from women without pathological, colposcopical or cytological findings and from women with CIN, as detected by ELISA using the 16/E7-2 peptide and by WB using the genetically engineered HPV-16 E7 fusion protein ELISA +
-
Total
WB+ WB-
3* 2~
11" 100
4 102
Total
5
101 §
10611
* A values in ELISA: 0-958, 1.438 and 2.215. t A value 0.272. A values 0-992 and 0.820. § Mean A values 0.384; S.D, 0.118. IITwenty-one sera originated from C I N patients; one of these was positive in both ELISA and WB, the remaining sera were negative in both tests.
Table 2. HPV-16 E7 antibody presence in sera from patients with cervical cancer, as detected by ELISA using the 16/E7-2 peptide and by WB using the genetically engineered HPV-16 E7 fusion protein* ELISA + WB + WB Total
91" 1§ 10
-
Total
2~ 22
11 23
2411
34
* Biopsy specimens from 19 patients were examined by dot blot and/or Southern blot hybridization; 10 were positive for HPV-16 D N A and three for HPV-18 D N A . Within the group of HPV-16 D N A positive patients, four sera were reactive in both ELISA and WB whereas the others were non-reactive in either test. None of the three HPV-18 DNA-positive patients possessed antibody reactive in ELISA but two possessed antibody reactive in WB. t A values in ELISA: 2.412, 2.368, 1.674, 2.490, 0.900, 1.147, 0.916, 1.694 and 1.935. A values 0.217 and 0-144. § A value 1.565. II Mean A value 0.262; S.D., 0.104.
ELISA and WB was similar to those in the previous group of patients (Table 2). Ten (29.4~) were reactive with 16/E7-2 in ELISA and of these nine were also positive in WB with HPV-16 E7. Two sera non-reactive in ELISA were clearly positive in WB. Again concordant results were obtained in a great majority of sera (kappa value 0.7935 + 0.2896, Student's t-test 2.740, P < 0.01). Taken together, of 15 sera positive in ELISA, 12 (80~) were also reactive in WB; of 125 sera non-reactive in
Short communication
Table 3. Correlation between reactivity in ELISA and intensity of bands in WB of ELISA- and/or WB-positive sera No. sera with indicated A value WB intensity*
0-8161"
0.817-1.500
> 1.500
--
--
--
+
2:~
2
-
1
++
1
3
-
+ + +
-
1
8
Total
3
6
9
* - , Negative; +, weakly positive; + +, intermediatelypositive; + + +, strongly positive. t Cutoff value. Thesethree sera werescoredin repeated tests as negativeor weakly positive.
ELISA 9 7 . 6 ~ were also negative in WB. Overall concordance of results was 95-7~ (kappa 0.776 + 0-190, Student's t-test 4.083, P < 0-001). In addition, as indicated in Table 3, in most sera there was a good correlation between the strength of the reactions in the two tests. None of the 10 control children's sera was reactive in either ELISA or WB with HPV-16 E7. Only one of the sera positive in ELISA and/or WB with HPV16 E7 antigens was reactive in ELISA with the control l/E7-1 antigen. None of the sera tested reacted with the control MS2 polymerase antigen. The data presented in Tables 1 and 2 indicate a high degree of concordance between the two tests suggesting that both tests are monitoring the same or similar activity and that the major immunoreactive region of the HPV16 E7 protein is covered by the 16/E7-2 peptide. Three sera were positive in WB and negative in ELISA. These sera did not possess antibodies to linear epitopes positioned outside 16/E7-2 as none of them reacted in ELISA with any of the nine overlapping peptides covering the whole amino acid sequence of the HPV-16 E7 protein (not shown). This suggested that epitopes present in the complete protein sequence and not in the synthetic peptides corresponding to parts of the complete sequence were involved. The presence of non-linear epitopes has recently been suggested by the use of a panel of monoclonal antibodies (MAbs) raised against the HPV-16 E7 fusion protein and it has been shown that the respective MAbs were cross-reactive with the HPV-18 E6 protein in both ELISA and WB (Tindle et al., 1990). In this respect it is of interest that two of the discordant sera originated from I N C A patients whose biopsy materials contained HPV-18 D N A (not shown). Thus, the absence of reactivity in ELISA might be due to the fact that these patients did not experience HPV-16 infection and the positive signal in WB could be due to
2579
cross-reacting antibodies. The opposite findings, i.e. positivity in ELISA and negativity in WB, were obtained in three sera. The discrepancy in two of them was apparently associated with their borderline activity; in fact, in repeated ELISA their A values with 16/E7-2 were either just below or just above the cutoff point. Similarly, in one of the three repeated WB assays both sera gave a weakly positive signal. On the other hand, the third serum originating from an I N C A patient exhibited reproducibly the WB- and ELISA-positive pattern. The nature of this discrepancy is not understood. To obtain more information about the location of the reacting epitopes within the HPV-16 E7 protein, absorption experiments were carried out. Five serum samples strongly reactive in both tests were selected. They differed in their pattern of reactivity in ELISA (Krchfifik et al., 1990). Sera nos. 1 and 2 were reactive only with the 16/E7-2 peptide (A values of 1.438 and 2-215, respectively), whereas the other three sera exhibited a broader reactivity. A values for serum no. 3 with 16/E7-2 and 16/E7-3 were 1.147 and 1.087, respectively. A values for serum no. 4 with 16/E7-1, 16/E7-2 and 16/E7-3 were 0.875, 1-674 and 2.507, respectively. The corresponding values for serum no. 5 were 1.373, 2-490 and 1-398, respectively. In absorption experiments the peptides were used either individually or in mixtures. In addition to those already mentioned (see above), the following peptides were also used: 16/E7-5 (amino acids 41 to 60), 16/E7-6 (51 to 70), 16/E7-7 (61 to 80), 16/E7-8 (71 to 90) and 16/E7-9 (81 to 98). The serumpeptide mixtures were incubated for 1 h at 37 °C and overnight at 4 °C and centrifuged for 10 rain at 4000 r.p.m. The supernatants were tested by WB for the presence of antibody. Results obtained with serum no. 1 are shown in Fig. 1. It can be seen that after absorption with the 16/E7-2 peptide alone the reactivity in WB of serum no. 1 was completely removed. It appears that 6 p.g of the peptide was sufficient to bind the antibody completely. On the other hand, 10-fold higher quantities of the other peptides used did not influence the reactivity of serum no. 1 in WB. This serum also reacted with an MS2 HPV-16 E4 fusion protein (Jochmus-Kudielka et al., 1989) in WB and this reactivity was not influenced by treatment with any of the HPV-16 E7 peptides. As expected, the absorbed serum lost its reactivity with the 16/E7-2 peptide in ELISA. Similar results were obtained with serum no. 2 (not shown). Results obtained with serum no. 3 are shown in Fig. 2. Its reactivity clearly differed from that of sera nos. 1 and 2. Although absorption with 60 gg of the 16/E7-2 peptide reduced the WB reactivity markedly, it did not remove it completely; however, treatment with a mixture of 16/E7-1, 16/E7-2 and 16/E7-3 peptides resulted in nearly complete removal of the WB reactivity. After absorption with the
2580
Short communication
3
4
5
6
7
8
9 10 11
i ~e "~
{i
r:,
4?, a
f!:ii ,:.....
MS2E7
{ii:
f
MS2 E4
i
Fig. 1. Absorption of serum no. 1 with peptides derived from HPV-16 E7 and HPV-1 E 7 0 R F s . The following peptides were used for absorption. Lane 1, unabsorbed control sample; lane 2, 6 ~tgof 16/E7-2; lane 3; 6 ~tg of 16/E7-5; lane 4, 6 ~tg of 16/E7-7; lane 5, 6 Ixgof l/E7-1 ; lane 6, 6 ~tgof 1/E7-2; lane 7, 60 lagof 16/E7-2; lane 8, 60 ~tgof 16/E7-5; lane 9, 60 lag of 16/E7-7; lane 10, 60 lag of I/E7-1 ; lane 11, 60 ~tg of 1/E7-2.
123
4
67
8910
1 12 13 14 151617
f o u n d a single m a j o r i m m u n o r e a c t i v e region in the H P V 16 E7 p r o t e i n b e t w e e n a m i n o a c i d s 21 a n d 34. T h i s region is c o n t a i n e d w i t h i n our p e p t i d e s 16/E7-2 a n d 16/E7-3. F r o m the d a t a p r e s e n t e d here it a p p e a r s t h a t m o r e t h a n one e p i t o p e m a y be p r e s e n t in this region. Also, o t h e r e p i t o p e s m a y be p r e s e n t in the N - t e r m i n a l p a r t o f the E7 p r o t e i n as r e p o r t e d by T i n d l e et al. (1990), D i l l n e r (1990) a n d Miiller et al. (1990) a n d as suggested here. In conclusion, the p r e s e n t d a t a seem to i n d i c a t e t h a t sera possessing the a n t i - H P V - 1 6 E7 a n t i b o d y d e t e c t a b l e by b o t h E L I S A a n d W B c a n be d i v i d e d a c c o r d i n g to their r e a c t i v i t y into several groups, i.e. those possessing a n t i b o d i e s to e p i t o p e ( s ) fully c o n t a i n e d w i t h i n the 16/E72 p e p t i d e , a n d those possessing, in a d d i t i o n , a n t i b o d i e s to e p i t o p e s outside the a r e a c o v e r e d by the 16/E7-2 p e p t i d e . T h e l a t t e r c a n p o s s i b l y be f u r t h e r d i v i d e d into those p r e d o m i n a n t l y possessing a n t i b o d i e s to c o n t i n u o u s e p i t o p e s localized w i t h i n the N - t e r m i n a l h a l f o f the p e p t i d e a n d those possessing a n t i b o d i e s to m o r e w i d e l y d i s t r i b u t e d c o n t i n u o u s e p i t o p e s or to d i s c o n t i n u o u s c o n f o r m a t i o n a l e p i t o p e s p r e s e n t in the H P V - 1 6 E7 protein.
:: i
MS2 E7
References
: ,
MS2 E4
:
MS2
ANISlMOV.~,E., BART.I,K,P., VL~EK, D., HIRSCH,I., BI~ICH~EK, B. & VONKA, V. (1990). Presence and type specificity of papillomavirus antibodies demonstrable by immunoelectron microscopy tests in samples from patients with warts. Journal of General Virology 71, 419-422. BAIRD, P. J. (1983). Serological evidence for the association of papiliomavirus and cervical neoplasia. Lancet ii, 17-18. CASON, J., PATEL, D., NAYLOR, J., LUNNEY, D., SHEPHERD, P. S., BEST,
' L
Fig. 2. Absorption of serum no. 3 with peptides derived from HPV-16 E7 and HPV-1 E70RFs. The following peptides were used for absorption. Lanes 1 to 11, the same as in Fig. 1; lane 12, mixture of 16/E7-1, 16/E7-2 and 16/E7-3, 50 lagof each; lane 13, mixture of 16/E74, 16/E7-5 and 16/E7-6, 50 ixg of each; lane 14, mixture of 16/E7-7, 16/E7-8 and 16/E7-9. Lanes 15 to 17, more diluted unabsorbed samples: 1:100 (lane 15), t :200 (lane 16), 1:1000 (lane 17).
16/E7-2 p e p t i d e the r e a c t i v i t y in E L I S A w i t h this p e p t i d e was c o m p l e t e l y r e m o v e d ; it r e m a i n e d unc h a n g e d by a b s o r p t i o n w i t h 16/E7-1 a n d 16/E7-5 p e p t i d e s (not shown). Results o b t a i n e d w i t h sera nos. 4 a n d 5 using the s a m e p e p t i d e s were different; n o n e o f the t r e a t m e n t s resulted in s u b s t a n t i a l r e d u c t i o n o f r e a c t i v i t y in W B (not shown). T h e r e a p p e a r s to be a n a g r e e m e n t o f our p r e v i o u s (Krchfi~k et al., 1990) a n d p r e s e n t d a t a w i t h those p r e s e n t e d r e c e n t l y by J e n i s o n et al. (1991). U s i n g n e s t e d s e t s o f d e l e t e d r e c o m b i n a n t p r o t e i n s these a u t h o r s h a v e
J. M. & MCCANCE, n . J.
(1989). Identification of immunogenic
regions of the major coat protein of human papillomavirus type 16 that contain type-restricted epitopes. Journal of General Virology 70, 2973 2987. CHRISTENSEN, N. D. & KREIDER, J. W. (1990). Antibody-mediated neutralization in vivo of infectious papillomaviruses. Journal of Virology 64, 3151-3156. COHEN, J. 0960). A coefficient of agreement for nominal scales. Educational and Psychological Measurement 20, 37-46. DILLNER, J. (1990). Mapping of linear epitopes of human papillomavirus type 16: the El, E2, E4, E5, E6 and E7 open reading frames. International Journal of Cancer 46, 703-711. DILLNER, L., BEKASSY, L., JONSSON, N., MORENO-LOPEZ, J. &
BLOMBERG,J. (1989a). Detection of IgA antibodies against human papillomavirus in cervical secretions from patients with cervical intraepithelial neoplasia. International Journal of Cancer 43, 36-40. DILLNER, J., DILLNER, L., ROBB, J., WILLENS, J., JONES, I., LANCASTER, W., SMITH, R. & LERNER, R. (1989b). A synthetic peptide defines a
serological IgG response to a human papillomavirus-encoded nuclear antigen expressed in virus-carrying cervical neoplasia. Proceedings of the National Academy of Sciences, U.S.A. 86, 38343841. DILLNER, J., DILLNER, L., UTTER, G., EKLUND, K., ROTOLA, A.,
COSTA, S. & DILUCA, D. (1990). Mapping of linear epitopes of human papillomavirus type 16: the LI and L2 open reading frames. International Journal of Cancer 45, 529-535.
Short communication
JENISON, S. A., FIRZLAFF, J. M., LANGENBERG,A. & GALLOWAY,D. A. (1988). Identification of immunoreactive antigens of human papillomavirus type 6b using Escherichia coli-expressed fusion proteins. Journal of Virology 62, 2115-2123. JENSION, S. A., YU, X. P., VALENTINE, J. M. & GALLOWAY, D. A. (1989). Human antibodies react with an epitope of the human papillomavirus type 6b LI open reading frame which is distinct from the type common epitope. Journal of Virology 63, 809 818. JENISON, S. A., YU, X. P., VALENTINE, J. M. & GALLOWAY, D. A. (1991). Characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18. Journal of Virology" 65, 1208-1218. JOCHMUS-KUDIELKA, I., SCHNEIDER, A., BRAUN, R., KIMMING, R., KOLDOVSKY, V., SCHNEEWEIS, K. E., SEEDORF, g . & GISSMANN, L~ (1989). Antibodies against the human papillomavirus type 16 early proteins in human sera: correlation of anti-E7 reactivity with cervical cancer. Journal of the National Cancer Institute 82, 16981703. KRCH~.~d(, V., V.~.GNER, J., SUCHANKOV.~.,A., KRt~M~.I~,,M., RITTEROV,~, L. & VONKA, V. (1990). Synthetic peptides derived from E7 region of human papillomavirus type 16 used as antigens in ELISA. Journal of General Virology 71, 2719-2724. MULLER, M., GAUSEPOHL, H., DE MARTYNOFF, G., FRANK, R., BRASSEUR, R. & GISSMANN, L. (1990). Identification of seroreactive
2581
regions of the human papillomavirus type 16 proteins E4, E6, E7 and LI. Journal of General Virology 71, 2709-2717. PFISTER, H. & ZUR HAUSEN, H. (1978). Seroepidemiological studies of human papilloma virus (HPV-I) infections. International Journal of Cancer 21, 161-165. SEEDORF, K.,OLTERSDORF, T., K RL~IMER, G. & ROWEKAMP,W. (1987). Identification of early proteins of the human papillomaviruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO Journal 16, 139-144. STEELE, J. C. & GALLIMORE, P. H. (1990). Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1. Virology 174, 388 398. St~CHANKOV/,, A., RIa-rER, O., HIRSCH, I., KRCH~/~, V., KALOL Z., HAMgiKov~,, E., B~.ICrt~EK, B. & VONKA, V. (1990). Presence of antibody reactive with synthetic peptide derived from L2 open reading frame of human papillomavirus types 6b and 11 in human sera. Acta virologica 34, 433-442. TINDLE, R. W., SMITH, J. A., GEYSEN, H. M., SELVEY, L. A. & FRAZER, I. H. (1990). Identification of B epitopes in human papillomavirus type 16 E7 open reading frame protein. Journal of General Virology 71, 1347 1354.
(Received 25 March 1991; Accepted 25 June 1991)
2577
Printed in Great Britain
Comparison of ELISA and Western blotting for human papillomavirus type 16 E7 antibody determination A. Suchfinkovfi, 1. L. Ritterovfi, 1 M . Kr~.m~L 1 V. Krchfifik, 2 J. Vfigner, z I. Jochmus, 3 L. Gisslnann, 3 J. Kafika 4 and V. V o n k a 1 1Department of Experimental Virology, 2Department of Synthetic Peptides, Institute of Sera and Vaccines, Prague, Czechoslovakia, 3Deutsches Krebsforschungszentrum, Heidelberg, Germany and 4Department of Obstetrics and Gynaeeology, Third Medical Faculty, Charles University, Prague, Czechoslovakia
A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35-2 %) than in the other individuals (six of 106, 5.7 %). Ten children's
(1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.
Diagnosis of infection with human papillomaviruses (HPVs) suggested by colposcopical, cytological or histological findings still rests mainly on the demonstration of HPV DNA presence. In the past few years increasing efforts have been made to develop serological tests capable of proving HPV infection. In these tests, three sources of antigens have been utilized: (i) intact or disrupted HPV virions extracted either from HPVinduced lesions (Pfister & zur Hausen, 1978; Baird, 1983; Dillner et al., 1989a; Steele & Gallimore, 1990; Anisimov/t et al., 1990) or from virus-infected xenografts in nude mice (Christensen & Kreider, 1990), (ii) genetically engineered HPV proteins, usually in the form of bacterial fusion proteins (Jenison et al., 1988, 1989; Jochmus-Kudielka et al., 1989) or expressed as part of a capsid protein of genetically modified phage fd (Mfiller et al., 1990) and (iii) synthetic peptides derived from known sequences of different HPV D N A open reading frames (ORFs) (Cason et al., 1989; Dillner, 1990; Dillner et al., 1989b, 1990; Such~.nkov/~ et al., 1990; Miiller et al., 1990). Recently we developed an ELISA to detect typespecific antibody to HPV type 16 E7 protein in human sera (Krchfi~.k et al., 1990). We examined a set of nine overlapping (by 10 amino acids), synthetic peptides
covering the whole amino acid sequence of the HPV-16 E7 protein; of these eight were icosapeptides and one was an octadecapeptide. From this study it was concluded that peptides 16/E7-1 (amino acids 1 to 20), 16/E7-2 (11 to 30), 16/E7-3 (21 to 40) and 16/E7-4 (31 to 50) were type-specific whereas the 16/E7-5 peptide was broadly cross-reactive. In sera from invasive carcinoma patients, positive HPV-16-specific reactions were more frequent with the 16/E7-2 peptide than with the other peptides. This reactivity could be removed by absorption with the 16/E7-2 peptide as well as by the genetically engineered HPV-16 E7 fusion protein but not with the HPV-16 E4 fusion protein. In the present series of experiments we tried to correlate the results obtained in ELISA, using the 16/E72 peptide, and by Western blotting (WB) using the HPV16 E7 fusion protein. A total of 140 serum samples were examined. All of them were collected at the Center of Cervical Cancer Prevention, Third Medical Faculty, Charles University, Prague, from women aged 25 to 53 years. Of these, 85 were free from pathological, colposcopical and cytological findings, 21 suffered from various stages of cervical intraepithelial neoplasia (CIN I to CIN III) and 34 from invasive cervical carcinoma (INCA). Sera from l0 children aged 1 to 3 years were also
0001-0289 © 1991 SGM
2578
Short communication
tested in parallel. All sera were inactivated by heating at 56 °C for 30 rain and kept at - 2 0 °C until investigated. The peptides were prepared and IgG-specific ELISA was performed as described previously (Krchfi/tk et al., 1990). Icosapeptides derived from the HPV-I E7 ORF, denoted 1/E7-1 (amino acids 11 to 30) and 1/E7-2 (amino acids 35 to 54) served as control antigens. Control sera known to be positive or negative for the HPV-16 E7 antibody in ELISA (Krchfifik et al., 1990) were included in each test. All sera were tested in a 1 : 10 dilution. The cutoff value between positivity and negativity was the mean absorbance (A) value plus 3 S.D. of the previously tested group of sera from 44 children aged 1 to 11 years which were considered anti-HPV-16 antibody-negative (Krchfifik et al., 1990). In WB experiments, lysates of genetically engineered Escherichia coli expressing HPV-16 E7 and HPV-16 E4 fusion proteins were used (Jochmus-Kudielka et al., 1989). They were derived from the expression vectors pEX 8 mer-HPV-16 E7 and pEX 12 mer-HPV-16 E4 constructed by Seedorf et al. (1987). They contain the whole HPV-16 E7 ORF [nucleotides (nt) 585 to 855] and HPV-16 E 4 0 R F (nt 3399 to 3617), respectively, fused to the DNA sequence encoding the first 100 amino acids of the bacteriophage MS2 polymerase. Preparation of the expressed fusion protein from bacterial extracts and WB analysis of sera which had been preabsorbed with lysates of the parental bacteria, were performed according to Jochmus-Kudielka et al. (1989); for visualization of the antigen-antibody reaction peroxidase-labelled pig antihuman IgG (SwaHu, Sevac) was used. As a control antigen a protein comprising the first 100 amino acids of the MS2 polymerase was employed (Jochmus-Kudielka et al., 1989). For the WB assay, sera were diluted 1:20. A positively reacting serum (diluted 1:1000) was included as a positive control in each test. If not indicated otherwise each serum sample was tested twice and was scored as strongly (+ + +), intermediately (+ +) and weakly ( + ) positive or negative ( - ) . With very few exceptions mentioned below, the results of the repeated tests were identical. The results are summarized in Tables 1 and 2. Of 106 sera from healthy women and women with CIN, only five (4.7~) were reactive with 16/E7-2 in ELISA. Of these, three reacted also with the HPV-16 E7 fusion protein in WB. In addition, one serum which was negative in ELISA gave a positive reaction in WB. The remaining 100 sera were negative in both tests. Thus, concordant results were obtained in 97-2~ sera. As the measure of agreement of the two methods, Cohen's kappa (Cohen, 1960) was used. The kappa value was 0.6521 _+0.2914, Student's t-test 2.237 (P < 0.05). In the group of 34 sera from INCA patients, positive reactions were more frequent; however, their distribution in
Table 1. HP V-16 E7 antibody presence in sera from women without pathological, colposcopical or cytological findings and from women with CIN, as detected by ELISA using the 16/E7-2 peptide and by WB using the genetically engineered HPV-16 E7 fusion protein ELISA +
-
Total
WB+ WB-
3* 2~
11" 100
4 102
Total
5
101 §
10611
* A values in ELISA: 0-958, 1.438 and 2.215. t A value 0.272. A values 0-992 and 0.820. § Mean A values 0.384; S.D, 0.118. IITwenty-one sera originated from C I N patients; one of these was positive in both ELISA and WB, the remaining sera were negative in both tests.
Table 2. HPV-16 E7 antibody presence in sera from patients with cervical cancer, as detected by ELISA using the 16/E7-2 peptide and by WB using the genetically engineered HPV-16 E7 fusion protein* ELISA + WB + WB Total
91" 1§ 10
-
Total
2~ 22
11 23
2411
34
* Biopsy specimens from 19 patients were examined by dot blot and/or Southern blot hybridization; 10 were positive for HPV-16 D N A and three for HPV-18 D N A . Within the group of HPV-16 D N A positive patients, four sera were reactive in both ELISA and WB whereas the others were non-reactive in either test. None of the three HPV-18 DNA-positive patients possessed antibody reactive in ELISA but two possessed antibody reactive in WB. t A values in ELISA: 2.412, 2.368, 1.674, 2.490, 0.900, 1.147, 0.916, 1.694 and 1.935. A values 0.217 and 0-144. § A value 1.565. II Mean A value 0.262; S.D., 0.104.
ELISA and WB was similar to those in the previous group of patients (Table 2). Ten (29.4~) were reactive with 16/E7-2 in ELISA and of these nine were also positive in WB with HPV-16 E7. Two sera non-reactive in ELISA were clearly positive in WB. Again concordant results were obtained in a great majority of sera (kappa value 0.7935 + 0.2896, Student's t-test 2.740, P < 0.01). Taken together, of 15 sera positive in ELISA, 12 (80~) were also reactive in WB; of 125 sera non-reactive in
Short communication
Table 3. Correlation between reactivity in ELISA and intensity of bands in WB of ELISA- and/or WB-positive sera No. sera with indicated A value WB intensity*
0-8161"
0.817-1.500
> 1.500
--
--
--
+
2:~
2
-
1
++
1
3
-
+ + +
-
1
8
Total
3
6
9
* - , Negative; +, weakly positive; + +, intermediatelypositive; + + +, strongly positive. t Cutoff value. Thesethree sera werescoredin repeated tests as negativeor weakly positive.
ELISA 9 7 . 6 ~ were also negative in WB. Overall concordance of results was 95-7~ (kappa 0.776 + 0-190, Student's t-test 4.083, P < 0-001). In addition, as indicated in Table 3, in most sera there was a good correlation between the strength of the reactions in the two tests. None of the 10 control children's sera was reactive in either ELISA or WB with HPV-16 E7. Only one of the sera positive in ELISA and/or WB with HPV16 E7 antigens was reactive in ELISA with the control l/E7-1 antigen. None of the sera tested reacted with the control MS2 polymerase antigen. The data presented in Tables 1 and 2 indicate a high degree of concordance between the two tests suggesting that both tests are monitoring the same or similar activity and that the major immunoreactive region of the HPV16 E7 protein is covered by the 16/E7-2 peptide. Three sera were positive in WB and negative in ELISA. These sera did not possess antibodies to linear epitopes positioned outside 16/E7-2 as none of them reacted in ELISA with any of the nine overlapping peptides covering the whole amino acid sequence of the HPV-16 E7 protein (not shown). This suggested that epitopes present in the complete protein sequence and not in the synthetic peptides corresponding to parts of the complete sequence were involved. The presence of non-linear epitopes has recently been suggested by the use of a panel of monoclonal antibodies (MAbs) raised against the HPV-16 E7 fusion protein and it has been shown that the respective MAbs were cross-reactive with the HPV-18 E6 protein in both ELISA and WB (Tindle et al., 1990). In this respect it is of interest that two of the discordant sera originated from I N C A patients whose biopsy materials contained HPV-18 D N A (not shown). Thus, the absence of reactivity in ELISA might be due to the fact that these patients did not experience HPV-16 infection and the positive signal in WB could be due to
2579
cross-reacting antibodies. The opposite findings, i.e. positivity in ELISA and negativity in WB, were obtained in three sera. The discrepancy in two of them was apparently associated with their borderline activity; in fact, in repeated ELISA their A values with 16/E7-2 were either just below or just above the cutoff point. Similarly, in one of the three repeated WB assays both sera gave a weakly positive signal. On the other hand, the third serum originating from an I N C A patient exhibited reproducibly the WB- and ELISA-positive pattern. The nature of this discrepancy is not understood. To obtain more information about the location of the reacting epitopes within the HPV-16 E7 protein, absorption experiments were carried out. Five serum samples strongly reactive in both tests were selected. They differed in their pattern of reactivity in ELISA (Krchfifik et al., 1990). Sera nos. 1 and 2 were reactive only with the 16/E7-2 peptide (A values of 1.438 and 2-215, respectively), whereas the other three sera exhibited a broader reactivity. A values for serum no. 3 with 16/E7-2 and 16/E7-3 were 1.147 and 1.087, respectively. A values for serum no. 4 with 16/E7-1, 16/E7-2 and 16/E7-3 were 0.875, 1-674 and 2.507, respectively. The corresponding values for serum no. 5 were 1.373, 2-490 and 1-398, respectively. In absorption experiments the peptides were used either individually or in mixtures. In addition to those already mentioned (see above), the following peptides were also used: 16/E7-5 (amino acids 41 to 60), 16/E7-6 (51 to 70), 16/E7-7 (61 to 80), 16/E7-8 (71 to 90) and 16/E7-9 (81 to 98). The serumpeptide mixtures were incubated for 1 h at 37 °C and overnight at 4 °C and centrifuged for 10 rain at 4000 r.p.m. The supernatants were tested by WB for the presence of antibody. Results obtained with serum no. 1 are shown in Fig. 1. It can be seen that after absorption with the 16/E7-2 peptide alone the reactivity in WB of serum no. 1 was completely removed. It appears that 6 p.g of the peptide was sufficient to bind the antibody completely. On the other hand, 10-fold higher quantities of the other peptides used did not influence the reactivity of serum no. 1 in WB. This serum also reacted with an MS2 HPV-16 E4 fusion protein (Jochmus-Kudielka et al., 1989) in WB and this reactivity was not influenced by treatment with any of the HPV-16 E7 peptides. As expected, the absorbed serum lost its reactivity with the 16/E7-2 peptide in ELISA. Similar results were obtained with serum no. 2 (not shown). Results obtained with serum no. 3 are shown in Fig. 2. Its reactivity clearly differed from that of sera nos. 1 and 2. Although absorption with 60 gg of the 16/E7-2 peptide reduced the WB reactivity markedly, it did not remove it completely; however, treatment with a mixture of 16/E7-1, 16/E7-2 and 16/E7-3 peptides resulted in nearly complete removal of the WB reactivity. After absorption with the
2580
Short communication
3
4
5
6
7
8
9 10 11
i ~e "~
{i
r:,
4?, a
f!:ii ,:.....
MS2E7
{ii:
f
MS2 E4
i
Fig. 1. Absorption of serum no. 1 with peptides derived from HPV-16 E7 and HPV-1 E 7 0 R F s . The following peptides were used for absorption. Lane 1, unabsorbed control sample; lane 2, 6 ~tgof 16/E7-2; lane 3; 6 ~tg of 16/E7-5; lane 4, 6 ~tg of 16/E7-7; lane 5, 6 Ixgof l/E7-1 ; lane 6, 6 ~tgof 1/E7-2; lane 7, 60 lagof 16/E7-2; lane 8, 60 ~tgof 16/E7-5; lane 9, 60 lag of 16/E7-7; lane 10, 60 lag of I/E7-1 ; lane 11, 60 ~tg of 1/E7-2.
123
4
67
8910
1 12 13 14 151617
f o u n d a single m a j o r i m m u n o r e a c t i v e region in the H P V 16 E7 p r o t e i n b e t w e e n a m i n o a c i d s 21 a n d 34. T h i s region is c o n t a i n e d w i t h i n our p e p t i d e s 16/E7-2 a n d 16/E7-3. F r o m the d a t a p r e s e n t e d here it a p p e a r s t h a t m o r e t h a n one e p i t o p e m a y be p r e s e n t in this region. Also, o t h e r e p i t o p e s m a y be p r e s e n t in the N - t e r m i n a l p a r t o f the E7 p r o t e i n as r e p o r t e d by T i n d l e et al. (1990), D i l l n e r (1990) a n d Miiller et al. (1990) a n d as suggested here. In conclusion, the p r e s e n t d a t a seem to i n d i c a t e t h a t sera possessing the a n t i - H P V - 1 6 E7 a n t i b o d y d e t e c t a b l e by b o t h E L I S A a n d W B c a n be d i v i d e d a c c o r d i n g to their r e a c t i v i t y into several groups, i.e. those possessing a n t i b o d i e s to e p i t o p e ( s ) fully c o n t a i n e d w i t h i n the 16/E72 p e p t i d e , a n d those possessing, in a d d i t i o n , a n t i b o d i e s to e p i t o p e s outside the a r e a c o v e r e d by the 16/E7-2 p e p t i d e . T h e l a t t e r c a n p o s s i b l y be f u r t h e r d i v i d e d into those p r e d o m i n a n t l y possessing a n t i b o d i e s to c o n t i n u o u s e p i t o p e s localized w i t h i n the N - t e r m i n a l h a l f o f the p e p t i d e a n d those possessing a n t i b o d i e s to m o r e w i d e l y d i s t r i b u t e d c o n t i n u o u s e p i t o p e s or to d i s c o n t i n u o u s c o n f o r m a t i o n a l e p i t o p e s p r e s e n t in the H P V - 1 6 E7 protein.
:: i
MS2 E7
References
: ,
MS2 E4
:
MS2
ANISlMOV.~,E., BART.I,K,P., VL~EK, D., HIRSCH,I., BI~ICH~EK, B. & VONKA, V. (1990). Presence and type specificity of papillomavirus antibodies demonstrable by immunoelectron microscopy tests in samples from patients with warts. Journal of General Virology 71, 419-422. BAIRD, P. J. (1983). Serological evidence for the association of papiliomavirus and cervical neoplasia. Lancet ii, 17-18. CASON, J., PATEL, D., NAYLOR, J., LUNNEY, D., SHEPHERD, P. S., BEST,
' L
Fig. 2. Absorption of serum no. 3 with peptides derived from HPV-16 E7 and HPV-1 E70RFs. The following peptides were used for absorption. Lanes 1 to 11, the same as in Fig. 1; lane 12, mixture of 16/E7-1, 16/E7-2 and 16/E7-3, 50 lagof each; lane 13, mixture of 16/E74, 16/E7-5 and 16/E7-6, 50 ixg of each; lane 14, mixture of 16/E7-7, 16/E7-8 and 16/E7-9. Lanes 15 to 17, more diluted unabsorbed samples: 1:100 (lane 15), t :200 (lane 16), 1:1000 (lane 17).
16/E7-2 p e p t i d e the r e a c t i v i t y in E L I S A w i t h this p e p t i d e was c o m p l e t e l y r e m o v e d ; it r e m a i n e d unc h a n g e d by a b s o r p t i o n w i t h 16/E7-1 a n d 16/E7-5 p e p t i d e s (not shown). Results o b t a i n e d w i t h sera nos. 4 a n d 5 using the s a m e p e p t i d e s were different; n o n e o f the t r e a t m e n t s resulted in s u b s t a n t i a l r e d u c t i o n o f r e a c t i v i t y in W B (not shown). T h e r e a p p e a r s to be a n a g r e e m e n t o f our p r e v i o u s (Krchfi~k et al., 1990) a n d p r e s e n t d a t a w i t h those p r e s e n t e d r e c e n t l y by J e n i s o n et al. (1991). U s i n g n e s t e d s e t s o f d e l e t e d r e c o m b i n a n t p r o t e i n s these a u t h o r s h a v e
J. M. & MCCANCE, n . J.
(1989). Identification of immunogenic
regions of the major coat protein of human papillomavirus type 16 that contain type-restricted epitopes. Journal of General Virology 70, 2973 2987. CHRISTENSEN, N. D. & KREIDER, J. W. (1990). Antibody-mediated neutralization in vivo of infectious papillomaviruses. Journal of Virology 64, 3151-3156. COHEN, J. 0960). A coefficient of agreement for nominal scales. Educational and Psychological Measurement 20, 37-46. DILLNER, J. (1990). Mapping of linear epitopes of human papillomavirus type 16: the El, E2, E4, E5, E6 and E7 open reading frames. International Journal of Cancer 46, 703-711. DILLNER, L., BEKASSY, L., JONSSON, N., MORENO-LOPEZ, J. &
BLOMBERG,J. (1989a). Detection of IgA antibodies against human papillomavirus in cervical secretions from patients with cervical intraepithelial neoplasia. International Journal of Cancer 43, 36-40. DILLNER, J., DILLNER, L., ROBB, J., WILLENS, J., JONES, I., LANCASTER, W., SMITH, R. & LERNER, R. (1989b). A synthetic peptide defines a
serological IgG response to a human papillomavirus-encoded nuclear antigen expressed in virus-carrying cervical neoplasia. Proceedings of the National Academy of Sciences, U.S.A. 86, 38343841. DILLNER, J., DILLNER, L., UTTER, G., EKLUND, K., ROTOLA, A.,
COSTA, S. & DILUCA, D. (1990). Mapping of linear epitopes of human papillomavirus type 16: the LI and L2 open reading frames. International Journal of Cancer 45, 529-535.
Short communication
JENISON, S. A., FIRZLAFF, J. M., LANGENBERG,A. & GALLOWAY,D. A. (1988). Identification of immunoreactive antigens of human papillomavirus type 6b using Escherichia coli-expressed fusion proteins. Journal of Virology 62, 2115-2123. JENSION, S. A., YU, X. P., VALENTINE, J. M. & GALLOWAY, D. A. (1989). Human antibodies react with an epitope of the human papillomavirus type 6b LI open reading frame which is distinct from the type common epitope. Journal of Virology 63, 809 818. JENISON, S. A., YU, X. P., VALENTINE, J. M. & GALLOWAY, D. A. (1991). Characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18. Journal of Virology" 65, 1208-1218. JOCHMUS-KUDIELKA, I., SCHNEIDER, A., BRAUN, R., KIMMING, R., KOLDOVSKY, V., SCHNEEWEIS, K. E., SEEDORF, g . & GISSMANN, L~ (1989). Antibodies against the human papillomavirus type 16 early proteins in human sera: correlation of anti-E7 reactivity with cervical cancer. Journal of the National Cancer Institute 82, 16981703. KRCH~.~d(, V., V.~.GNER, J., SUCHANKOV.~.,A., KRt~M~.I~,,M., RITTEROV,~, L. & VONKA, V. (1990). Synthetic peptides derived from E7 region of human papillomavirus type 16 used as antigens in ELISA. Journal of General Virology 71, 2719-2724. MULLER, M., GAUSEPOHL, H., DE MARTYNOFF, G., FRANK, R., BRASSEUR, R. & GISSMANN, L. (1990). Identification of seroreactive
2581
regions of the human papillomavirus type 16 proteins E4, E6, E7 and LI. Journal of General Virology 71, 2709-2717. PFISTER, H. & ZUR HAUSEN, H. (1978). Seroepidemiological studies of human papilloma virus (HPV-I) infections. International Journal of Cancer 21, 161-165. SEEDORF, K.,OLTERSDORF, T., K RL~IMER, G. & ROWEKAMP,W. (1987). Identification of early proteins of the human papillomaviruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO Journal 16, 139-144. STEELE, J. C. & GALLIMORE, P. H. (1990). Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1. Virology 174, 388 398. St~CHANKOV/,, A., RIa-rER, O., HIRSCH, I., KRCH~/~, V., KALOL Z., HAMgiKov~,, E., B~.ICrt~EK, B. & VONKA, V. (1990). Presence of antibody reactive with synthetic peptide derived from L2 open reading frame of human papillomavirus types 6b and 11 in human sera. Acta virologica 34, 433-442. TINDLE, R. W., SMITH, J. A., GEYSEN, H. M., SELVEY, L. A. & FRAZER, I. H. (1990). Identification of B epitopes in human papillomavirus type 16 E7 open reading frame protein. Journal of General Virology 71, 1347 1354.
(Received 25 March 1991; Accepted 25 June 1991)
