Int. J. Cancer: 50,349-355 (1992) 0 1992 Wiley-Liss, Inc.

Publication of the InternationalUnion Against Cancer Publication de I'Union Internationale Contre le Cancer

DETECTION OF ANTIBODIES TO A LINEAR EPITOPE ON THE MAJOR COAT PROTEIN (Ll) OF HUMAN PAPILLOMAVIRUS TYPE-16 (HPV-16) IN SERA FROM PATIENTS WITH CERVICAL INTRAEPITHELIAL NEOPLASIA AND CHILDREN John CASONlB3, Parminder K. KAMBO',Jennifer M. BEST' and Dennis J. MCCANCE"' 'The Richard Dimbleby Laboratory of Cancer Virology, The Rayne Institute, St Thomas's Hospital, Lambeth Palace Road, London, SEI 7EH, UK; and 'Departmentof Microbiology and Immunology, University of Rochester, Box 672, 601 Elmwood Avenue, Rochester, Ny 14642, USA. Antibodies to the major (LI) coat protein of human papillomavirus type 16 (HPV-16) in sera from patients with cervical intra-epithelialneoplasia(CIN) have been investigatedby means of recombinant proteins and synthetic peptides. When LIHPV-I6 fusion proteins were used in immunoblot assays, no antibody reactivity was found in sera from 52 patients with CIN or from 2I unrelated children. Amino-acid sequence analyses indicated that L I-HPV- I 6 amino acids 473 to 492 may contain an HPV- 16 type-restricted epitope since the greatest diversity occurs in this region. In the ELISA, seropositivity to peptides 473 to 492 was more common among CIN patients whose biopsies contained HPV- I 6 DNA (9IYo, 2 I of 23) than among their children (24%, 5 of 2 I ;p < 0.00 I) or other CIN patients with HPV- I 6 DNA-negative biopsies (66Y0, I9 of 29; p < 0.05), but was unrelated to the severity of the CIN lesion. Antibodies to LI-HPV-16 peptide 473 to 492 among seropositive CIN patients cross-reacted with the analagous LI-HPV-33, but not with the LI-HPV-6b peptide, and were predominantly IgM. In contrast, antibodies which recognized a less variable region of L I -HPV- I 6 (amino acids 279 to 293) showed no association with HPV- I 6 DNA status. Seropositivity to the L I -HPV-6b (amino acids 473-492) was less frequent (33%) among CIN patients and unassociated with HPV- I 6 DNA status (p > 0. I); however 5 I Yo (37 of 72) of patients with genital warts had antibodies to this peptide.

Human papillomaviruses (HPVs) are small, naked, icosahedral DNA viruses which usually cause benign epithelial cell proliferations. HPV-6 and -11 cause genital warts, but other genital HPVs (HPV-16, HPV-18, -31, -33, -34, -35, -42, -43, -44, -45 and -52) are associated with cervical intra-epithelial neoplasia (CIN) which may progress to malignancy (de Villiers, 1990). HPV-16 DNA is detected most frequently (about 60%) when biopsies from cervical carcinomas are tested in dot-blot and Southern-blot hybridization assays (Durst et al., 1983; Lorincz et al., 1987). In contrast to HPV-6 and HPV-11, the prevalence of HPV-16 DNA in CIN increases with the severity of the lesion (Syrjanen et al., 1985), suggesting that the association between HPV-16 infection and malignant disease of the cervix may be more than casual (zur Hausen and Schneider, 1987). At present, differentiation between patients infected with high-cancer-risk genital HPVs, such as HPV-16, and those infected with low-risk HPVs, such as HPV-6 and HPV-11, relies upon DNA-DNA hybridization assays or the polymerase chain reaction. A serological test specific for HPV-16 infection may be of value for diagnosis and for sero-epidemiological studies. However, HPV-16 virions have not yet been propagated in conventional cell cultures (Taichman et al., 1984) and lesions from patients with CIN contain few HPV-16 capsid proteins. Thus, to obtain sufficient quantities of HPV-16 antigens for serological studies, it is necessary to produce synthetic constructs by the techniques of molecular biology or organic chemistry. Papillomavirus capsids consist of a major (Ll) and a minor (L2) protein. The former appears to be of the greatest immunological interest: Jenison et af. (1988, 1989) have shown

that for HPV-6 it is immunodominant, while Pilacinski et al. (1986) have demonstrated that the L1 protein of bovine papillomavirus type 1 (BPV-1) contains a neutralizing epitope. However, L1 proteins from different papillomaviruses have highly conserved amino-acid sequences (Baker, 1987) which makes it difficult to detect HPV-type-specific antibodies. Nevertheless, we have identified HPV-type-restricted linear epitopes on L1-HPV-16, using monoclonal antibodies (MAbs) and synthetic peptides (Cason et al., 1989). Antibodies that recognize L1-HPV-16 peptides have also been detected in sera from patients with CIN (Dillner et al., 1990). In the present study, recombinant proteins and synthetic peptides have been used as antigens to determine the incidence of antibodies specific to HPV-16 amongst CIN patients of known HPV-16 DNA status. MATERIAL AND METHODS

Recombinant proteins A PATH-I1 plasmid [containing the entire L1-HPV-16, Bst-N1 (nucleotide 5529) to Nsi-1 (nucleotide 253)] in Escherichia coli (strain HB101) (from Dr. D.A. Galloway, Fred Hutchinson Cancer Center, Seattle, WA) and a pEX 1plasmid [containing L1-HPV-16 BamHl (nucleotide 6150) to Pst-1 (nucleotide 6787), i.e. amino acids 172 to 375: Patel et al., 19891 in E. coli pop 2136 were used. Induction of recombinant proteins resulted in a tryptophan E synthetase/Ll-HPV-16 (TES/ L1-HPV-16) and a P-galactosidase /L1-HPV-16 (B-gal/ Ll-HPV-16) fusion protein, respectively. Peptide antigens To identify possible HPV-16 type-restricted B-cell epitopes, a hydrophilicity chart (Hopp and Woods, 1981) for L1-HPV-16 and a Kabut and Wu amino-acid variability plot (Roitt et al., 1985) [calculated from aligned amino-acid L1 sequences of HPV-la, HPV-6b, HPV-16, BPV-1 and cotton-tail rabbit papillomavirus (CRPV): Baker, 19871 were produced. Other sequence alignments of the L1 proteins of HPV-8, HPV-11, HPV-18, HPV-33, BPV-2, BPV-4 and deer papillomavirus (DPV) (from the Genebank data-base) were performed with the help of Drs. J. Thornton and H. Stirk (Birkbeck College, London, UK). The variability plot indicated that a region near the carboxylic terminus of Ll-HPV-16 (residues 473 to 492: numbered from the second methionine of L1-HPV-16) is very poorly conserved (Fig. 1, Table I). This region is also adjacent to the major hydrophilic peak of L1-HPV-16 (Fig. 1) suggesting that it may also be an immunodominant B-cell epitope. Thus, a peptide corresponding to L1-HPV-16 amino acids 473 to 492 (GLKAKPKFTLGKRKATPTTS), and its counterparts to L1-HPV-6b (GYRGRSSIRTGVKRPAVSKA), L1-HPV-11 (GYRGRTSARTGIKRPAVSK), L1-HPV-33 (GLKAKPK'To whom correspondence and reprint requests should be sent.

Received: May 28,1991 and in revised form September 17,1991.







FIGURE 1 - Top: hypervariability plot for the full-length (residues 1 to 505) L1-HPV-16 protein. Beneath this (unmarked) are positions of HPV-16IHPV-6b sequence homology. Below: a hydrophilicity plot (Hopp and Woods algorithm [ 19811). TABLE I -ALIGNED AMINO-ACID SEQUENCES OF Ll PROTEINS FROM OTHER PAPILLOMAVIRUSES CORRESPONDING TO THE Ll-HPV-16 PEPTIDES OF INTEREST ~~




HPV-16 -18 -33 -6b -11 -la -8 BPV-1 -2 -4








....................... GLKAKPKFTLGKRKATPTTS. . . . . . . . . . . . . (46%) . . . . . . . . . . . . .GLRRKPT IGPRKRSAPSATT. . . . . . . . (46%) . . . . . . . . . . . . .GLKAKPK L L R A i L P T - S . . . . . 60’7 . . . . 40%) ............ .GYRGRSSIRTGVKRPAVSKA ... (10%)

. . . GG-.”-RSSVLSSIWH . . . . [33%) . . . . . . . . . . . ..GYRGRTSARTGIKRPAVSKP . . . (15%) . . . A D A E P R T T L L S C V Y S P . . . . (33%) . . . . . . . . . . . . .GMT QRTATSSTTKRKTVRVS . . .(5%) . . . GGQDQSQKDIGdAMYFP . . (27%) . . . . . . . . . . . . GLQQTTV NG-TKSISRGS . . . . (20%) . . . NNKGDATLLKIPS-VH . . . . . . 27% . . . . . . . . . . . . .GAGCS-TVRKRRISQKT . . . . . 15% . . . NNKGDATLKIPS-VH . . . . . . 27% . . . . . . . . . . . . .GAGCS-TVRUVATRN . . . . 30% . . . PATGRGPELPSS-VY . . . . . . . . 27% . . . . . . . . . . . . .KRSMK_TVTFENTEGKKA. .. . . . RTQMQGEANANIATDNY-C . . 27%) . . . . . . . . . . . . .GLQRIGTKRPAPAPVSIVX . . . . 10% . . . PP-GEMELLKMPS-VF. . . . . . . $20%i . . . . . . . . . . . . .LLPQKDHFTYPEKRYKRHMR. . 20%

I{ A

Figures in parentheses indicate the degree of (best fit) homology of the given sequence with the corresponding human papillomavirus (HPV) type-I6 sequence. A, HPVs associated with pre-malignant/malignant genital lesions: B, HPVs associated with genital warts: C, HPVs associated with cutaneous lesions: D, various animal papillomaviruses, BPV, bovine papillomavirus; CRPV, cottontail rabbit papillomavirus; DPV, deer papillamovirus.

LKRAAPTSTRTSS) and a less variable region of LI-HPV-16 (amino acids 279 to 293: GSGSTANLASSNYFP) were synthesized by f-moc chemistry and purified by high-pressure liquid chromatography. As peptides bind unpredictably to immunoassay plates (Geerligs et aL, 1988) we conjugated them to BSA before use, using l-(3-dimethylamino propyl)-3-ethyl carbodiimide-hydrochloride (Doolittle, 1986). Patients Sera from 52 patients with CIN were obtained from Dr. J. Cuzick (ICRF, London, UK). Biopsies from 20 of these 52 patients contained HPV-16 DNA when tested in Southern blot assays (data not shown). Of 20 biopsies investigated for HPV-6 DNA, 2 contained HPV-6 DNA (one of these also had HPV-16 DNA) and 18 contained no HPV-6 DNA (14 were negative for both HPV-6 and -16 DNA: 4 contained HPV-16, but not HPV-6, DNA). All of 16 biopsies also tested for HPV-11 DNA were negative. All sera were pre-adsorbed with an equal volume of the appropriate uninduced bacteria at 1 g bacterial proteinil (for TES/LI-HPV-16) or induced (pEx minus the L1-HPV-16 insert) as appropriate, for 2 hr at room temperature prior to use in immunoblot assays. Seventy-two sera from 23 male patients attending St Thomas’s Hospital for genital warts were also studied: their mean age was 25.3 (range 1 9 4 3 ) years, and 32 had a history of current or recurrent disease exceeding 3 months (up to 2 years). Sera from 21 children (12 males), with a mean age of 2.4 years (range: 3 months to 10 years), attending hospital for surgery, for conditions other than cancer or genital warts, were also tested. No biopsies for DNA studies were available from the patients with genital warts or from children.

Immunoblot assays Fifty microliters/well of fusion-protein-containing celllysate (1 g of bacterial protein/l) were loaded into wells of 12% polyacrylamide gels, electrophoresed, then transferred to Hybond-N nylon membranes (Amersham, Aylesbury, UK). Membranes were blocked with 10% (v/v) dried skimmed milk (Marvel, Cadbury, London, UK) in Tris-buffered saline (TBS) overnight at 4”C, then strips 5-mm wide were exposed to a 1/50 dilution of (adsorbed) test sera in TBS for 12 hr at room temperature. Membranes were washed in TBS and then immersed in biotinylated sheep anti-human (or, for MAbs, rabbit anti-mouse) immunoglobulin (Ig; 1/500 in TBS) for 20 min at room temperature, then washed again. Strips were exposed to streptavidin-alkaline-phosphatase conjugate ( I / 300 in TBS) for 20 min, washed and immersed for 30 min in diethanolamine buffer containing nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Amersham). Nylon strips were then washed in distilled water. Reactivities of test sera were compared to that of a 1/50 dilution of MAb 1C6 [which recognizes L1-HPV-16 (Cason et al., 1989)l. Transfer of proteins to the nylon strips was confirmed by co-electrophoresis, and transfer of human Ig (Amersham), was detected by means of biotin-labelled anti-human Ig conjugate. These assay conditions have previously been used to identify antibodies to a B-gallLl-HPV-6b fusion protein at serum dilutions of up to 1/250 in 44% of patients with genital warts (data not shown). Enzyme-linked immunosorbent assays (ELISAs) Immunoassay plates were coated with BSA-peptide conjugates (50 kl/well) at 9.1 X lo-’ M/I PBS, for 1 hr at room temperature, and control wells were coated with 1 g/l BSA in


PBS. Non-specific binding sites were blocked with 1g/l BSA in PBS (90 p,l/well) for 1 hr. Plates were washed in PBS and (unadsorbed) test sera (50 p,l/well) were incubated overnight at 4°C (at 1/20-1/2,560 as triplicates in PBS containing 1 g/l BSA). After PBS washes, antibody was detected with 50 p,l/well of horse-radish peroxidase (HRP)-labelled rabbit anti-human Ig, or anti-human IgM, IgG or IgA (Dako, Copenhagen, Denmark: at 1/500 in PBS containing 1 g/l BSA). Plates were washed again and bound antibody was visualized with 50 p,l/well of ortho-phenylene diamine substrate (Cason et al., 1989): reactions were stopped after 15 min with 25 p,l/well of 2 M sulphuric acid and A,,, values were determined. Results are expressed as peptide-attributable A,* values, i.e. A,,, of antisera tested against peptide minus A,,, of the same sera tested against BSA-coated control wells. This method of immunoassay was validated by using a wellcharacterized MAb (5A4) and its L1-HPV-16 peptide epitope (amino acids 269-284 ENVPDDLYIKGSGSTA) prior to use. For investigations of the specificity of antibodies to L1HPV-16 peptide 473-492, sera were tested as described above at 1/50 (in PBS) before and after preincubation (2 hr at room temperature) with L1-HPV-16 473 to 492, or its L1-HPV-6b and L1-HPV-33 peptide analogues (at 1 g/l in PBS), using ELISA plates coated with the L1-HPV-16 473 to 492 peptide. Statistical tests Student’s paired and unpaired t-tests, x2 tests and linear regression analyses were used to assist the interpretation of data from ELISA. RESULTS

Immunoblot reactivity of sera with LI-HPV-16fusion proteins None of the sera from 21 children or 52 patients with CIN reacted with TES/Ll-HPV-16 (amino acids 1 to 505) or B-gal/Ll-HPV-16 (amino acids 172 to 375) fusion proteins (data not shown). In contrast, MAb 1C6 [raised against B-gal/Ll-HPV-16 (amino acids 172 to 375)J reacted well with both fusion proteins as reported previously (Patel et al., 1989). Similarly, human Ig, which was electrophoresed and transferred in parallel with the fusion proteins, was detected on all immunoblots. Reactivity of sera from CINpatients to L1 peptides Mean A,,, values of sera from CIN patients were greater than those of children’s sera at all serum dilutions with each of the 3 peptide antigens used (Fig. 2). Statistical analyses (Student’s unpaired t-tests) revealed that, at dilutions of 1/20 and 1/40, sera from CIN patients gave significantly higher mean A,* values than those from children when tested against L1-HPV-16 peptide 279 to 293 (p < 0.005), L1-HPV-16 peptide 473 to 492 (p < 0.001) and their corresponding L1HPV-6b (473 to 492) analogue (p < 0.001). When an arbitrary value of 0.3 A,,, units was introduced to differentiate between positive ( > 0.3) and negative sera ( < 0.3), 38.5% of sera from CIN patients and 19% of children’s sera (diluted 1/20) were positive to L1-HPV-16 279 to 293 (Table 11), but this difference was not significant (p > 0.1, xz test). However, more sera from CIN patients (77%) than from children (24%) were positive (p < 0.001) when the L1HPV-16 473 to 492 peptide was used as an antigen. Of the 5 children seropositive to L1-HPV-16 473 to 492,3 were concordantly reactive with L1-HPV-16 peptide 279 to 293. While 19 of 20 CIN patients who were seropositive to L1-HPV-16 peptide 279 to 293 were also positive to L1-HPV-16 473 to 492, antibodies to L1-HPV-16 peptide 473 to 492 were most common in sera from CIN patients with HPV-16-DNAcontaining lesions (91%) than from those with HPV-16-DNAnegative lesions (66%: p < 0.05): this association was not observed when L1-HPV-16 peptide 279 to 293 was used as antigen (p > 0.1). Seropositivity to L1-HPV-16 peptide 473 to


492 was unrelated to the severity of CIN lesions. Seven of 10 (70%) patients with mild lesions (CIN-I, CIN-1/11), 8 of 10 (80%) with moderate lesions (CIN-11) and 21 of 25 (84%) with severe lesions (CIN-II/III and CIN-111) were seropositive. Also, the magnitudes of mean A,,, ELISA values to this peptide did not differ significantly (p > 0.1) between these groups (X = 0.4, SD = t0.17; X = 0.42, SD = k0.17; X = 0.45, SD = k0.19 respectively). To determine whether antibodies which reacted with L1HPV-16 peptide 473 to 492 were HPV-16 type-specific, 39 of the 40 sera from CIN patients found to be seropositive to this peptide were examined in inhibition ELISAs. Pre-incubation of these sera with L1-HPV-16 473 to 492 caused a significant reduction in the mean A,,, value detected with plates coated with L1-HPV-16 473 to 492 (t = 4.4, p < 0.001: by Student’s paired t-test) as did pre-incubation with the corresponding L1-HPV-33 peptide (t = 2.4, p < 0.025). In contrast, preincubation with the equivalent L1-HPV-6b peptide caused no inhibition of the mean A,,, reading (t = 0 . 9 9 5 , ~> 0.3). When these data were normalized (so that the A4,, of each sera to L1-HPV-16 473-492 = loo%), there were significant reductions in percentage absorbance values when sera were preincubated with the L1-HPV-16 or L1-HPV-33 peptides as compared to L1-HPV-6b (Ll-HPV-16 versus L1-HPV-33: t = 2.25, p < 0.05; L1-HPV-16 VS. L1-HPV-6b: t = 6.68, p < 0.001, Fig. 3). Thus, the degree of inhibition of immunoglobulin binding to L1-HPV-16 peptide 473 to 492 reflected the amino-acid homology of the inhibiting peptide with this region of L1-HPV-16 (Table I). Seropositivity to L1-HPV-6b peptide 473 to 492 was also higher among patients with CIN than among children (p < 0.001: Table 11). However, there was no significant relationship between seropositivity to the L1-HPV-6b peptide and HPV-16 DNA status of CIN patients’ lesions (p > 0.1: Table 11). Both CIN patients whose lesions contained HPV-6b DNA were seropositive, and 13 of 18 HPV-6b-DNA-negative CIN patients were seronegative to the L1-HPV-6b peptide. When sera from 72 patients with genital warts and from 26 CIN patients (previously seronegative to the L1-HPV-6b peptide, i.e., values 3-month history of genital warts, 21 (66%) were seropositive to the L1-HPV-6b peptide, whereas, of 40 patients with genital warts for less than 3 months, 16 (40%) were seropositive. The specificity of antibodies to the HPV-6b peptide in 72 sera from patients with genital warts was investigated by comparing A,* values obtained when immunoassay plates were coated with either the L1-HPV-6b peptide or its L1HPV-11 counterpart. Linear regression analysis of these data indicated that there was a significant (R = 0.469, p < 0.001) linear relationship between the A,,, values obtained when these 2 antigens were used; furthermore, pre-incubation of sera with the HPV-11 peptide caused a significant reduction in AdWvalues when they were tested against plates coated with the HPV-6b peptide and vice versa (data not shown). This cross-reactivity of antibodies to HPVdb and HPV-11 peptides is not surprising as they differ by only 4 amino acids (Table I).

Ig class of antibodies to LI-HPV-16peptide 473 to 492 Sera from the 40 L1-HPV-16 473 to 492 seropositive CIN patients were tested simultaneously (at 1/40 dilution) for



















0 c crn

2 56 0




c crn



c cin










c cia


Reciprocal serum d i l u t i o n

FIGURE 2 - Titration curves for sera from children (c) or patients with cervical intra-epithelial neoplasia (CIN) tested against: (a) Ll-HPV-16 peptide 279 to 293; (b) L1-HPV-16 peptide 473 to 492; and (c) L1-HPV-6b peptide 473 to 492 in ELISA tests for Ig (all classes). Results are expressed as A4w(BSA/Ll-HPV-16 473 to 492) minus A,g,, (BSA and, for clarity, zero absorbance values are not shown. Arrows indicate absorbance values of 0.3, the arbitrary cut-off value used to de ne seropositivity. Bars indicate mean values.





Children CIN CIN DNA 16 + CIN DNA 16 CIN DNA 6 + CIN DNA 6 Genital warts


L1-HPV-16 279-293

21 52 23 29 2 18 72

4 (19% 20 (38%))



LI-HPV-6b 473-492

21 91% 19 [66%] 2 100%) 12 (66%) 13 (18%)

0 0%) 17 f33%) 9 (39% 8 (28%{ 2 100%) 5 (28%) 37 (51%)

Analysis of data in Figure 3, using an arbitrary criterion for defining seropositivity (A490 > 0.3). Sera from patients with warts were considered positive when their A490 value exceeded that for seronegativeCIN patients. Data are presented as the number of positive sera and, in parentheses, the percentage of positive sera in each group. CIN DNA 16 + CIN patients with lesions containing HPV-16 D N A CIN 16 -, CIN patients in whom HPV-16 DNA could not he detected; CIN 6 + and CIN 6-, CIN patients with or without detectable HPV-6 DNA, number, number of patients; ND, not determined. %





a 0

E rd P



0 W


4 60









0 HPV-16



Inhibitor peptide FIGURE3 - Inhibition of antibodv reactivitv to L1-HPV-16 peptide 473492 by prior incubation df 39 sera from CIN patients with L1-HPV-16 473-492 or its L1-HPV-33 and L1-HPV-6 analogues. Data expressed as percentage of response detected when no inhibitory peptide was added. Mean and standard errors of the mean values are shown.

peptide-specific IgM, IgG and IgA antibodies. IgM antibodies were detected in all 40 sera; only 7 and 11 respectively of the 40 sera contained low levels of IgG or IgA antibodies to this peptide. Mean IgM A4wvalues were greatest, and significantly exceeded absorbance values detected for IgG (p < 0.001: by Student’s paired t-test) or IgA (p < 0.001; Fig. 5). DISCUSSION

Antibodies to a predicted type-specific epitope (amino acids 473 to 492) on the major capsid protein of HPV-16 were detected significantly more frequently by ELISA among patients with CIN (77%) than among children (24%,p < 0.001) and in 91% of CIN patients whose lesions contained HPV-16

FIGURE4 - (a) Detection of antibodies (all classes of Ig) that react with HPV-6b peptide 473 to 492 in sera (1/20) from 72 patients with genital warts compared to 26 sera from CIN patients previously observed to be seronegative to this peptide (X). (b) Antibodies (all classes of Ig) to L1-HPV-16 peptide 473 to 492 in the sera (1/20) of patients with genital warts compared to 10 sera from CIN patients. Previous observations had shown that 5 of these CIN patients were seronegative (Y) and 5 were seropositive (Z),to this peptide. Results are expressed as A4%(BSAipeptide conjugate) minus A490 (BSA) and, for clarity, zero absorbance values are not shown.

DNA. The fact that 12 of 29 (66%) HPV-16-DNA-negative CIN patients were seropositive to L1-HPV-16 473 to 492 may indicate that the amount of HPV-16 DNA in some biopsies was below the limits of detection for Southern blots, or that HPV-16 infection was present at another site, as HPV-16 infections can be multifocal (McCance et al., 1985). Furthermore, it could be anticipated that overall seropositivity would be more frequently detected than the presence of viral DNA, due to persistence of antibodies after the virus has been eliminated by the immune system. Our preliminary studies showed variable degrees of antibody reactivity among sera from CIN patients when 17 other L1-HPV-16 peptides were used as antigens; nevertheless, in all instances antibody levels in sera from patients with CIN to these L1-HPV-16 peptides were greater than those for children (data not shown). However, other regions of L1-HPV-16 are more highly conserved than region 472 to 493 and are thus more likely to be recognized by cross-reactive antibodies. For





a2 0


cs 6 P I4

0 P

0.3 .

... . -..... *..














FIGURE 5 - Class of Ig reactive with L1-HPV-16 peptide 473 to 492. Forty sera (at 1/40 in PBS) from L1-HPV-16 473 to 492 seropositive (Ig of all classes) CIN patients were tested against this peptide [as a bovine serum albumin (BSA)/peptide conjugate] and bound antibody detected in a single assay using HRP-labelled antisera to human IgM, IgG or IgA. Results are ex ressed as A,,, (BSA/Ll-HPV-16 473 to 492) minus (BSAY. Absorbance values of zero have been omitted. example, in ELISAs using L1-HPV-16 peptide 279 to 293, antibodies were detected in sera from CIN patients, but did not correlate with their HPV-16-DNA status. In contrast, antibodies which recognized L1-HPV-16 amino acids 473 to 492 appeared to be restricted in their reactivity to analogous HPV L1 peptides in that: (i) 28 of 45 of L1-HPV-16 473 to 492 positive sera (62%) were seronegative to the corresponding Ll-HPV-6b peptide; (ii) while seropositivity to the L1-HPV-6b peptide was more common amongst CIN patients than among children, there was no association between seropositivity and HPV-16 DNA status; and (iii) pre-incubation of sera with the HPVdb peptide did not inhibit reactivity to the HPV-16 peptide. There was further evidence for the HPV-type-restricted nature of region 473 to 492 of L1-HPV-16 when sera from patients with genital warts (usually caused by HPV-6 or HPV-11: de Villiers, 1990) were tested. Antibodies to the L1-HPV-6b peptide 413 to 492 were detected in 37 of 72 sera from patients with genital warts (51%). Of these samples, only 8 of 37 (22%) were concordantly positive to L1-HPV-16 (amino acids 473 to 492). This low prevalence of reactivity to equivalent HPV-6b and HPV-16 peptides could reflect HPV6b/HPV-16 co-infections, rather than antibody cross-reactivity, since patients can be infected with more than one HPV type (Young et al., 1989). However, when the specificity of the antibodies which recognized the HPV-6b peptide was investigated, they were found to cross-react with the equivalent HPV-11 peptide. This result is not surprising since the HPV-6b and HPV-11 peptides have an 87% amino-acid homology (Table I). Thus, region 473 to 492 of papillomavirus L1 proteins probably encodes an HPV-group-specific (eg. HPV-16 and -33, or HPVdb and HPV-ll), rather than an HPV-typespecific, epitope. The relatively high incidence (24%) of seropositivity to HPV-16 among children has been noted by others using fusion proteins (Li et al., 1987; Jenison et al., 1990) and may be

explained by HPV-16 infection resulting from sexual abuse, HPV-16 infection at birth, or some other mode of transmission. Only 2 of the 5 children seropositive to L1-HPV-16 peptide 473 to 492 were very young (3-6 months) and may have had maternal antibodies. Antibodies to L1-HPV-16 473 to 492 in sera from patients with CIN were predominantly IgM, which is indicative of recent or persistent infections (Roitt et al., 1985). While Dillner et al. (1990) found that IgA antibodies to HPV-16 peptides were more frequently detected than IgM or IgG, their results may well reflect the use of differing peptides, the absence of a carrier protein to attach peptides to immunoassay plates, or the variable assay conditions used to detect IgM, IgG and IgA antibodies. None of the sera tested reacted with L1-HPV-16 fusion proteins in immunoblot assays. Similarly, Jenison et al. (1990) found that only 2 of 91 sera from CIN patients recognized the TES/Ll-HPV-16 construct, whereas antibodies to a TES/L2HPV-16 fusion protein were more frequently detected. Such observations conflict with studies of HPV-6b that have shown that the L1 protein is immunodominant (Jenison et al., 1988). The failure to detect antibodies to L1-HPV-16 in immunoblot assays may be due to stereochemical blocking of important HPV-16 epitopes by the irrelevant moieties (€3-gal or TES), although no evidence for such an effect was found in our previous studies with h4Abs (Pate1 et af., 1989). Alternatively, the immunoblot assay may lack the sensitivity required to detect antibodies to HPVs (Strike et al., 1989). As L1-HPV-16 fusion proteins were expressed in bacteria, they are not post-transcriptionally modified and may not react with antibodies to conformation-dependent epitopes. Indeed, while a comparative study of the antigenicity of HPV-6 and HPV-11 L2 proteins expressed in baculovirus or in E. coli found that such differences may not be important (Rose et al., 1990), our preliminary observations of a baculovirus-expressed L1HPV-16 protein indicate that it may be post-transcriptionally modified (data not shown). As L1 protein expression in vivo is usually restricted to low-grade CIN lesions, seropositivity to L1-HPV-16 epitopes is unlikely to have a prognostic value with respect to malignant progression. Indeed, no relationship between the severity of the CIN lesion and seropositivity to peptide 473 to 492 was detected in this study. In contrast to HPV-16 capsid proteins, E7 protein persists in cervical cancers (Chow et al., 1987; Seedorf et al., 1987; Smotkin and Wettstein, 1986) and may be a useful antigen for serological investigations. Indeed, Dillner (1990) and Jochmus-Kudielka et al. (1989) have found that antibodies to E7-HPV-16 peptides and fusion proteins may be up to 14 times more common among patients than among controls. As in these studies the antibodies to E7 were predominantly detected in severe lesions, they are unlikely to be of value for determining the incidence of HPV-16 exposure per se in study populations. Thus, detection of antibodies to HPV-16 capsid protein epitopes, as shown in the present report, may be of value for sero-epidemiological studies of HPV-16 exposure and for the early detection of HPV-16 infections. ACKNOWLEDGEMENTS

Financial support for J.C. was provided by the Cancer Research Campaign, the Special Trustees of St Thomas’s Hospital and the Richard Dimbleby Cancer Fund.


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Detection of antibodies to a linear epitope on the major coat protein (L1) of human papillomavirus type-16 (HPV-16) in sera from patients with cervical intraepithelial neoplasia and children.

Antibodies to the major (L1) coat protein of human papillomavirus type 16 (HPV-16) in sera from patients with cervical intra-epithelial neoplasia (CIN...
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