ANALYSIS OF E2 AMINO ACID VARIANTS OF HUMANPAPILLOMAVIRUS TYPES 16 AND 18 AND THEIR ASSOCIATIONSWITH LESION GRADE AND HLA DR/DQ TYPEGeorge TERRY1,2, Linda HO2 and Jack CUZICK1*1Department of Mathematics, Statistics and Epidemiology, Imperial Cancer Research Fund, London, UK2The Windeyer Institute of Medical Sciences, University College, London, UK

Human papillomavirus (HPV) 16 and HPV18 E2 amino acidvariants and host HLA DR/DQ haplotypes have been identi-fied by direct nucleic acid sequencing from cervical scrapes.HPV16 E2 variants co-segregate with a nucleotide variant atnt350 (in E6), which previously has been associated withpersistent infections. Both HPV16 and HPV18 E2 variantsoccur relatively more frequently in individuals with HLADR/DQ haplotypes 0401/0301 and 1101/0301 but are notrelated to lesion grade. Int. J. Cancer 73:651–655, 1997.r 1997 Wiley-Liss, Inc.

The genomes of human papillomavirus (HPV) types 16 and 18code for 6 early (E) proteins and 2 late (L) proteins.In vitroexperiments show that the HPV16 and HPV18 promoters (P97 andP105, respectively) which regulate the transcription of oncogenesE6 and E7 are themselves regulated by the viral E2 protein. Threefunctional domains have been recognised in the E2 protein. TheN-terminus, containing the transactivation domain and the aminoacid residues (156–159) known to co-operate with E1 in directingthe synthesis of HPV16 DNA, is linked to a C-terminal DNA-binding domainvia a flexible hinge region (Giri and Yaniv, 1988;Picciniet al.,1995). Reaction of E2 peptides with hyperimmune E2anti-sera have identified multiple antigenic sites in or adjacent tothe hinge region (in HPV16, amino acids 191–287; in HPV18,amino acids 226–265) (Dillner, 1990). Analysis of the expressionof HPV16 genes in cervical cancer cells in culture has shown thatintegration of the viral genome can result in continuous expressionof oncoproteins E6 and E7 and that this frequently is accompaniedby disruption of the E2 open-reading frame (ORF). However,high-level expression of E2 protein suppresses the P97 E7 pro-moter in cells transiently transfected and transformed by HPV16E7. These experimental observations suggest an inverse relation-ship between the levels of expression of E2 and E7 oncoproteins.

The functional role of E2 in the pathogenesis of disease isunclear. An intact E2 gene frequently is detectable in HPV16- orHPV18-positive cervical cancers (Matsukuraet al.,1989; Cullenetal., 1991), though its physical state (episomal and/or integrated) isusually unknown. In at least some cases, the E2 gene is expressedsince sera from women with cervical cancer more frequently arefound to contain antibody to synthetic or bio-engineered HPV16 orHPV18 E2 peptides derived from either the hinge region or theadjacent DNA-binding domain (Dillner, 1990) than sera fromcontrols.

Nucleic acid sequencing data show that many natural HPVvariants exist (Yamadaet al., 1997), and some of these areassociated with amino acid changes in functional and/or antigenicdomains and are liable to introduce unique biological and immuno-genic properties, conferring differences in behaviour from eachother and from the prototype. Hechtet al.(1995) identified HPV18E2 nucleotide variants and associated them with different lesiongrades. We report on HPV16 E2 amino acid variants which hithertohave not been described but whose existence was assumed sinceHPV16 long control region (LCR), E6, E7, L1 and L2 variants havebeen described (Yamadaet al.,1997). It is conceivable that aminoacid variations in E2, a viral protein with major regulatory andantigenic functions, may alter the outcome of HPV infections.Therefore, we have determined, by nucleic acid sequencing, thecomplete E2 gene sequences of individual HPV16 and HPV18

isolates and have related variations found to the source lesiongrade. Since there is increasing evidence to suggest that theoutcome of an HPV infection also may be influenced by a specificmatch between the HPV type (and/or variant) and the host immunesystem (Appleet al.,1994; Elliset al.,1995), we have determinedthe major histocompatibility complex (MHC) class II DR/DQhaplotypes from the same women and assessed these parameterstogether in relation to the severity of cervical disease.

MATERIAL AND METHODS

Clinical material

DNA extracts from 34 HPV16-positive cervical scrapes and 29HPV18-positive cervical scrapes were included initially in thisstudy. The HPV16 lesions had been diagnosed as invasive cancer(n 5 16), CIN2/3 (n5 5), CIN1 or lesser abnormality (n5 2) ornormal (or no biopsy, presumed normal) (n5 11). HPV18 lesionsincluded invasive cancer (n5 8), CIN2 (n5 1), CIN1 or less(n 5 3) or normal (or no biopsy, presumed normal) (n5 17). Allsamples histologically graded as CIN3 or less were obtained in thecourse of previous studies (Cuzicket al.,1995; Londesboroughetal., 1996). Cancer scrapes were collected from major gynaecologi-cal oncology centres in the UK (see ‘‘Acknowledgements’’), andtheir diagnoses were based on routine cytology and histology.

Sequence analysisTemplates were generated by PCR and sequenced by cycle

sequencing using Thermo Sequenase (Amersham, Aylesbury, UK).The primers used are summarised in Table I. HPV sequences andbase positions are numbered according to the HPV16r and HPV18sequences in the HPV sequence database (Los Alamos NationalLaboratory, NM).

All PCR analyses were carried out in 25 µl volumes for 35 cycleswith 1.5 mM Mg21 and an annealing temperature of 55°C. In thecase of each HLA gene, all forward primers and all reverse primerswere present at 0.4 µM each. HPV primers were used at 0.25 µM.The PCR product (5 µl) was electrophoresed in agarose and thetemplate quality-assessed visually after ethidium-bromide staining.The remainder was purified using a DNA-purification kit (Wizard;Promega, Madison, WI).

Templates were sequenced in both directions using Cy-5-labelled primers. Reactions were analysed using an automatedsequencer (ALFexpress; Pharmacia, Uppsala, Sweden). For thisinvestigation, nucleotide changes were accepted only when con-firmed by repeat PCRs and sequencing from both directions. HLAalleles were assigned by comparison of the exon 2 sequencesobtained with the DRB1 and DQB1 master alignment sets providedwith the HLA SequiTyper software (Pharmacia).

*Correspondence to: Imperial Cancer Research Fund, 61, Lincoln’s InnFields, London WC2A 3PX, UK. Fax: 44(0)171-269-3429. E-mail:cuzick@icrf.icnet.uk

Received 12 May 1997; Revised 25 July 1997

Int. J. Cancer:73,651–655 (1997)

r 1997 Wiley-Liss, Inc.

Publication of the International Union Against CancerPublication de l’Union Internationale Contre le Cancer

RESULTS

Identification of HPV16 variants

HPV16 isolates were initially subtyped by the E6 nucleotidesequence according to the classification of Yamadaet al. (1997).Thirty-two were found to be of the European prototype, 1 AAsubtype and 1 AF1 subtype. The single AA and AF1 cancer isolateswere not studied further.

DNA from the 32 HPV16-positive European prototype isolateswas amplified in 2 overlapping segments (nt2611–3523 andnt3315–4341) covering the entire E2 gene (nt2726–3853) andsequenced. Under the conditions used here, both E2 segmentscould be amplified in 12 of 14 cancers (86%) and in all lower-gradelesions. Neither segment was amplified in the remaining 2 cancers.Base changes were observed in 10 positions, but the overalldeduced amino acid sequence was highly conserved. Four aminoacid variants were observed, v1 (substitution at amino acids 142and 165), v2 (substitution at amino acid 219), v3 (substitution atamino acids 210 and 219) and v4 (substitution at amino acids 219and 261) (Table II). Within the E2 antigenic hinge region, variantsv2, v3 and v4 were found to share a common base change from C toT at nucleotide position 3409, resulting in an amino acid change atposition 219 (proline= serine), and only these variants werereferred to as the HPV16 ‘‘E2 variants’’ in this investigation. Ourresults show that E2 variants were not specifically prevalent in anyindividual lesion grade (p 5 0.71) but were associated frequentlywith an E6 nucleotide variation at nt350 (p 5 0.009) previouslyfound to represent a marker for persistent HPV16 infection.

Identification of HPV18 variantsDNA was amplified again in 2 overlapping fragments (nt2644–

3428 and nt3345–4005) covering the entire E2 gene (nt2817–3914)and the E2/E5 intergenic region. Only 4 of 8 cancers were found to

contain a complete set of E2 gene sequences (both segmentsamplified, 50%). No PCR products were obtained from theremaining 4. Nucleotide re-arrangements were common and in-cluded base changes at 17 distinct positions, 1 in-frame intragenicdeletion (nt3624–3629) and 1 intergenic deletion (nt3916–3937).Amino acid sequences were, however, highly conserved. Withinthe antigenic hinge region, 2 amino acid substitutions wereobserved (amino acid 240, tyrosine= histidine and amino acid257, lysine= threonine), resulting from nucleotide changes atnt3534 and nt3586, respectively (Table II). Only these (v4) werereferred to as the HPV18 ‘‘E2 variants’’ in this investigation andwere not found to be associated with high lesion grade (p 5 1.00,2-sided exact test).

Association of HPV16 and HPV18 variants with DR/DQhaplotypes in cervical disease

The potential biological significance of the amino acid variationsobserved in the antigenic domains of HPV16 and HPV18 isolateswas evaluated by examining their association with MHC class IIDR/DQ haplotypes in different lesion grades (Tables III, IV). Inthis small data set, no specific combination of HPV16 or HPV18variants and HLA DR/DQ haplotypes could be related to severityof cervical disease. HPV16 E2 variants showed some associationwith DR alleles 0401 and 1101 [x2(3) 5 10.4,p 5 0.015], whichhave been related to susceptibility to HPV infection, but not withany individual DQ alleles. An apparent association between E2variants and previously identified at-risk DR/DQ haplotypes 1501/0602, 0401/0301 and 1101/0301 (Appleet al.,1994; Odunsiet al.,1996) is difficult to assess statistically because of the number ofpossible haplotypes.

TABLE I – SUMMARY OF PCR AND SEQUENCING PRIMERS USED

GenePrimers

Number Type1 Geneposition Sequence (58–38)

HPV16 E2 1 PCR fwd 2611 TGGTGGTGTTTACATTTC2 PCR rev 3525 CAACAACTTAGTGGTGTG3 PCR fwd 3315 TGCGGGTGGTCAGGTAAT4 PCR rev 4341 CCTTAGGTATAATGTCAGGTG5 Sequencing fwd 2732 ACGAGGACGAGGACA6 Sequencing rev 3496 GGTGTCTGGCTCTGA7 Sequencing fwd 3353 TTTAGCAGCAACGAA8 Sequencing rev 3893 AAGCACGCCAGTAAT

HPV16 E6 1 PCR fwd 99 CTGCAATGTTTCAGGACCC2 PCR rev 818 GTGCCCATTAACAGGTCTTC3 Sequencing rev 437 GACACAGTGGCTTTTGACAG

HPV18 E2 1 PCR fwd 2605 AAAATGTCCTCCAATACTACTAAC2 PCR rev 3500 ATACGGTGAGGGGGTGTG3 PCR fwd 3265 GGGACAAAACCGCTACCT4 PCR rev 4028 ATACGCACACATACAGACAGAT5 Sequencing fwd 2644 AGCAAAGGATAATAG6 Sequencing rev 3428 CATAGAGTCATTACA7 Sequencing fwd 3345 AAAAGTGAATGTGAA8 Sequencing rev 4005 GCAAAAGCGGGACAT

HLA DR 1 PCR fwd TTGTGGCAGCTTAAGTTTGAAT2 PCR fwd TTCCTGTGGCAGCCTAAGAGG3 PCR fwd GTTTCTTGGAGTACTCTACGT4 PCR fwd GTTTCTTGGAGCAGGTTAAAC5 PCR fwd AGTACTCTACGGGTGAGTGTT6 PCR fwd CCTGTGGCAGGGTAAGTATA7 PCR fwd CAGACCACGTTTCTTGGAGG8 PCR fwd GTTTCTTGAAGCAGGATAAGTT9 PCR rev CTCGCCGCTGCACTGTGAAG

10 Sequencing fwd GAGTGTCATTTCTTCAAHLA DQ 1 PCR fwd GCATGTGCTACTTCACCAACG

2 PCR rev CAGGATCCCGCGGTACGCCA3 PCR rev CCGCTGCAAGGTCGTGCGG4 Sequencing rev CAGGTAGTTGTGTCTGCACAC

1Fwd, forward primer; rev, reverse primer.

652 TERRYET AL.

DISCUSSION

HPV16 and HPV18 DNA are demonstrable in 60–90% ofcervical cancers (Boschet al., 1993) and at least 5–10% oflow-grade viral infections, which usually regress spontaneously(Cuzick et al., 1995). The risk factors which predispose aninfection to development into a high-grade lesion are not clear.Invitro studies suggest that the viral E2 gene may play a pivotal role.

In the clinical specimens analysed in this study, complete E2sequences could be amplified from 86% of HPV16 cancers and50% of HPV18 cancers. Although a complete sequence derivedfrom 2 overlapping PCR fragments does not necessarily imply anintact gene, the rate of E2 detection observed here is similar to thatreported by Southern blotting for the intact E2 gene (Matsukuraetal., 1989; Cullenet al., 1991). From this, we conclude that adisrupted E2 ORF, through either integration or mutation, is not aprerequisite for the development of cervical cancer. Amino acidchanges were rare in both the transactivation and the DNA-bindingdomains (Table II).

The presence of an intact E2 ORF is consistent with the frequentdetection of E2 antibodies in women with high-grade CIN andcancer (Strickleret al.,1994; Dillneret al.,1995). In an analysis of66 women with cervical cancer, Lenneret al. (1995) showed thatincreased levels and positivity rates for IgA antibody to immunore-active HPV16 or HPV18 E2 epitopes before treatment werecorrelated with the absence of a complete response to therapy and areduced survival. Surprisingly, the presence of antibody to HPV16E2 correlates poorly with HPV16 DNA status (Hamsikovaet al.,1994). This observation was extended by a study involving 1,087women from Colombia and Spain, where more than 50% of womenwith HPV16 DNA-positive cancers had no detectable E2 antibodyand a similar proportion of women with HPV16 DNA-negativecancers were positive for E2 antibody (De Sanjoseet al., 1996).This suggests that characterisation of E2 variants may contribute toan understanding of the contradiction observed.

Our analysis shows that the amino acid sequence at or adjacentto the immunoreactive hinge region of HPV16 E2 is highlyconserved. A substitution at amino acid 219 (nucleotides 3410and/or 3409) was found to be common among the HPV16 variants.This amino acid is close to, but outside, the antigenic domainpreviously identified by Gauthieret al. (1991), and no amino acidvariation was found in any other described antigenic domains(HPV16 amino acids 226–275 and HPV16 or HPV18 amino acids328–345) (Table II). No correlation was found between theoccurrence of either HPV16 or HPV18 variants with lesion grade.For these reasons, the E2 amino acid variation observed in thisstudy cannot be assigned specific biological or immunologicalfunctions. The nucleotide and amino acid variations observed inour HPV18 isolates confirm those reported by Hechtet al. (1995).However, the 2 amino acid changes in positions 240 and 257,previously identified as being associated with low-grade lesions,were identified in 1 of 4 HPV18-positive cancers in this study(Table II). Since the HPV16 E2 variant co-segregates with the E6nucleic acid variant nt350, which we previously reported torepresent a marker for increased risk of persistent viral infection(Londesboroughet al.,1996), it can be considered as an additionalmarker of disease.

Variations in immune response to a particular viral isolate alsocan be regarded as a function of the specificity of the host immunemechanism. Previously, we showed that increased susceptibility toHPV16, 18, 31 and 33 infections in a Caucasian population in theUK is associated with the MHC class II DR/DQ haplotypes0401/0301 and 1101/0301, whereas 0101/0501 may be protective(Odunsiet al.,1996). In a Hispanic population from New Mexico,relative susceptibility to, and protection from, HPV16-positivecancer have been associated with MHC II DR/DQ haplotypes1501/0602 and 1302/0604, respectively (Appleet al., 1994). AnHPV16 variant with a nucleotide change at position 131 andconsequent amino acid change from glycine to valine in an MHCclass I-restricted E6 epitope has been found to bind less effectively

TAB

LEII

–N

UC

LEO

TID

EA

ND

DE

DU

CE

DA

MIN

OA

CID

VAR

IAT

ION

SIN

HP

V16

AN

DH

PV

18E

2G

EN

S

Type

base

don

spec

ific

PC

R

Sub

type

base

don

E6

sequ

ence1

Num

ber

ofte

mpl

ates

sequ

ence

dD

esig

natio

nC

hang

esin

E2

Lesi

ongr

ades

Nuc

leot

ide

Am

ino

acid

3180

142

3181

143

3222

157

3248

165

3337

208

3386

210

3409

219

3448

232

3536

/726

137

7734

137

8634

4C

aC

IN2/

3#

CIN

1N

orm

alor

nobi

opsy

HP

V16

Eur

opea

npr

otot

ype

15P

roto

type

Glu

Ala

Leu

Arg

Pro

IleP

roG

luS

erT

rpA

sp5

42

4E

urop

ean

prot

otyp

e1

v1A

sp.

.P

ro.

..

..

..

01

00

Eur

opea

npr

otot

ype

12v2

..

..

..

Ser

..

..

50

07

Eur

opea

npr

otot

ype

1E

2va

riant=

v3.

..

..

Thr

Ser

.1

00

0E

urop

ean

prot

otyp

e1

v4.

..

..

.S

er.

Leu

..

10

00

Type

base

don

spec

ific

PC

R

Sub

type

base

don

E2

sequ

ence2

Num

ber

ofte

mpl

ates

sequ

ence

dD

esig

natio

nC

hang

esin

E2

Nuc

leot

ide

Am

ino

acid

3534

240

3586

257

3624

–362

627

036

27–3

629

271

3630

,363

227

238

2933

8

HP

V18

Pro

toty

pe4

Pro

toty

peTy

rLy

sA

laT

hrP

roH

is1

11

11

v1.

..

..

Leu

00

01

15v2

..

..

Ala

.2

02

111

v3.

..

.A

laLe

u0

00

1

Varia

nt2

4E

2va

riant=

v4H

is3

Thr

3de

l.de

l..

.1

00

3

Am

ino

acid

sun

derli

ned

are

asso

ciat

edw

ithde

sign

ated

antig

enic

dom

ains

.–1 Y

amad

aeta

l.(1

995)

.–2H

echt

eta

l.(1

995)

.–3A

min

oac

idch

ange

sas

soci

ated

with

low

-gra

dele

sion

s.

653HPV16 AND HPV18 E2 VARIANTS

TABLE IV – HLA TYPING OF HPV18-POSITIVE ISOLATES

HPV18 E2HLA haplotypes

Histological diagnosisDR1 DR2 DQ1 DQ2

Prototype1 v1 1v2 1 v3

150111101

150111303

06020301

06020301

CancerCancer

03011 03011 0201/02 0202 Cancer0701 1101 0301 03032 CIN20404 15011 0302 0602 CIN10101 1201 0301 0501 CIN103011 1401 05031 0201/02 Atypia0101 0401 0301 0501 No biopsy, persistent HPV180101 15011 0501 0602 NAD, normal cytology on FU03011 0701 0201/02 0201/02 NAD, normal cytology on FU0101 0101 0501 0501 NAD0301 15011 0201/02 0602 No biopsy0101 15011 0501 0602 No biopsy0103 15011 0501 0602 No biopsy0301 1501 0201/02 0602 No biopsy0301 0701 0201/02 0201/02 No biopsy0305 0413 0201/02 0301 No biopsy1114 1419 0302 0604 No biopsy0301 0401 0201/02 0302 No biopsy0912 1401 03032 05031 No biopsy09012 15022 0302 0302 No biopsy

Variant (v4) 0401 1104 0301 0301 Cancer1101 1105 0301 0301 NAD0401 0413 0302 0302 NAD0101 0701 0201/02 0501 No biopsy

Cancer risk HLA DR/DQ type 1501/0602 is underlined. Susceptibility risk HLA DR/DQ types0401/0301 and 1101/0301 are shown in bold. NAD, no abnormality detected; FU, Follow-up.

TABLE III – HLA TYPING OF HPV 16-POSITIVE ISOLATES

HPV16 HLA haplotypesHistological diagnosis

E2 aa219 E6 nt350 DR1 DR2 DQ1 DQ2

Prototype1 v1 TT

07011501

150115021

0201/020301

06020602

CancerCancer

T 1405 1501 05031 0602 CancerT 03011 03011 0201/02 0201/02 CancerT 08031/32 1312/13 0301 06011/12 CancerG 03011 15011 0201/02 0602 CIN2T 0403 11041/42 0302 0304 CIN3T 0402 0404 0302 0302 CIN3T 0101 0701 0201/02 0501 CIN3T 0401 1401 0301 05031 CIN2G 1501 1505 0301 0602 NADT 0413 1501 0301 0602 NADT 0302 0701 0201/02 0402 NADG 0401 1302 0301 0604 No biopsy, normal

cytology on FUT 03011 0401 0301 0602/03 Atypia, regressed on FUT 041 141 0301 05031 NAD

Variants (v2, v3, v4) GG

04010401

07011101

0201/020301

03010301

CancerCancer

G 0701 0401 0201/02 0301 CancerG 111 141 0301 05031 CancerG 0701 15021 0201/02 0602 CancerT 0805 1101 0301 0302/04 CancerT 0401 1301/02 0302 0605 CancerT 0401 1301/02 0302 0604 Inflammation, regressed

on FUG 1101 1501 0301 0602 Inflammation, regressed

on FUT 0401 1101 0301 0304 NADG 1101 1307 0301 0501 NADG 0301 0701 0201/02 0602 NADG 1001 1201 0301 0501 NADG 1301/02 1301/02 0603 0604 NAD

Cancer risk HLA DR/DQ type 1501/0602 is underlined. Susceptibility risk HLA DR/DQ types0401/0301 and 1101/0301 are shown in bold. NAD, no abnormality detected; FU, follow-up.–1Cannot befurther typed.

654 TERRYET AL.

to the peptide transporter encoded by the HLA B9 haplotype insome cervical cancer cell lines (Elliset al., 1995). The resultsreported here suggest that both HPV16 and HPV18 E2 variants aremore frequent in women with HLA DR/DQ haplotypes 0401/0301or 1101/0301, who are at greater risk of infection, whereas HPV16E2 prototype may be associated more frequently with women withHLA DR/DQ haplotype 1501/0602, who are those at greater risk ofHPV16-positive cervical cancer.

Although the number of isolates we examined is small, ourresults suggest that infection with HPV16 or HPV18 E2 variantsmay be related to the ability of the individual host to respond. Ourresults show that E2 variants are associated with ‘‘at-risk’’ HLADR/DQ alleles, but their identification in a given individual is notindicative of disease. This complex relationship between viral andhost risk markers can be analysed by direct nucleic acid sequencing

on a single sample, and such an approach has a good potential indefining more precisely the risk factors which may predisposeindividuals to oncogenic HPV infection.

ACKNOWLEDGEMENTS

We thank Drs A. Chan and B. Vallins (Pharmacia Biotech) fortechnical advice on ALFexpress nucleic acid sequencing and Mr J.Monaghan and Mr A. Lopez (Queen Elizabeth Hospital, Gates-head, UK), Mr P. Clarkson (Mayday Hospital, Thornton Heath,UK), Dr P. Trott (Department of Cytopathology, Royal MarsdenHospital, London, UK), Mr I. Duncan (Ninewells Hospital, Dun-dee, UK) and Mr A. McIndoe (Hammersmith Hospital, London,UK) for cancer biopsy specimens.

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655HPV16 AND HPV18 E2 VARIANTS