Int. J . Cancer: 45, 52%535 (1990) 0 1990 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication ds I’Union Internationale Contre 18 Cancer
MAPPING OF LINEAR EPITOPES OF HUMAN PAPILLOMAVIRUS TYPE 16: THE L1 AND L2 OPEN READING FRAMES Joakim DILLNER’’~, Lena DILLNER’,Goran UTTER’,Carina EKLUND’,Antonella ROTOLA~, Silvano COSTA3 and Dario DILUCA~ ‘Department of Virology, Karolimh Institute, S-10521 Stockholm, Sweden;2Department of Microbiology, University of Ferrara, Ferrara; and 3Department of Gynecology, University of Bologna, Bologna, Italy. Certain types of human papillomavirus (HPV), notably HPV type 16, are associated with flat or inverted proliferative lesions of the cervix uteri that can progress to malignancy. As a first step towards the serological study of the epidemiology of HPV, we have synthesized the entire amino acid sequences of the 2 major viral capsid proteins of HPV type 16, LI and U, as a set of 66 synthetic 20-residue peptides with an overlap of 5 amino acids. The peptides were tested for reactivity with IgA, IgG and IgM antibodies in the sera of 30 patients with HPV-16-carrying cervical neoplasms. Both IgG and IgM antibody responses were detected, but most of the reactivity found was of the IgA class. The most immunoreactive peptides were futher analyzed for reactivity with sera from 22 patients with parotid gland tumors and with sera from 38 healthy individuals. The U-encoded protein contained only one major linear epitope, which was not specific for HPV16-carrying neoplasms. In contrast, the LI -encoded protein contained several epitopes that were regularly immunoreactive with antibodies present in the sera of patients with HPV16-carryingcervical neoplasms, but only rarely so in the sera of patients with other tumors or of healthy individuals.
abundant (Komly et al., 1986; Doorbar and Gallimore, 1987; Banks et al., 1987; Tomita et al., 19876). The L2 protein has been reported to contain type-specific epitopes (Komly et al., 1986). We have recently reported that the IgA response to linear epitopes of HPV-encoded nuclear antigens (J. Dillner et al., 1989) and capsid proteins (L. Dillner et al., 1989) predominates over the IgG response to these proteins in patients with CIN, suggesting that IgA-specific immunoassays using linear epitopes of HPV proteins could be used to study the epidemiology of HPV in relation to CIN (L. Dillner et al., 1989; J. Dillner et al., 1989). In order to obtain a map of the linear epitopes of the HPV 16 major capsid proteins, we synthesized the entire amino acid sequences of these 2 proteins as a set of 20 amino-acid synthetic peptides with overlap of 5 amino acids. The positions of the sequence-specific epitopes that reacted with IgA, IgG or IgM antibodies in the sera of patients with HPV- 16-carrying cervical neoplasms are reported. MATERIAL AND METHODS
The human papillomaviruses are a widely distributed family of viruses that cause various proliferative diseases in the in- Peptide synthesis fected epithelium (Pfkter, 1984). Different types of HPV are Sixty-six 20 amino-acid peptides, with a 5 amino-acid overassociated with different diseases and different anatomical sites lap, representing the deduced amino acid sequence of the L1 of infection (Pfister, 1984, 1987). Several types, notably HPVs and L2 OFWs of HPV 16 (Seedorf et al., 1985) were synthe6, 11, 16, 18, 31, 33, 35, and 52, infect the genital tract sized using t-Boc amino acids (Bachem, Bubendorf, Switzer(Pfkter, 1987; Shah and Buscema, 1988). HPV types 6 and 11 land) and p-methylbenzhydrylamine resin (Fluka, Buchs, Switare mainly associated with elevated, pointed, wart-like lesions, zerland) according to the multiple solid-phase peptide synthesis condyloma acuminata (Gissman et al., 1983; Shah and method of Houghten (1985). Removal of the protecting groups Buscema, 1988). In contrast, HPV types 16, 18, 31, 33, 35, from the formyl-tryptophane and methionine sulfoxide residues and 52 are mainly associated with flat or “inverted” incon- was achieved by cleavage with 25% hydrogen fluoride (Tam et spicuous lesions, designated cervical intra-epithelial neoplasms al., 1983). The peptides were then cleaved from the resin with (CIN) (Crum et al., 1985; Durst et al., 1983; Boshart et al., liquid hydrogen fluoride using a multi-vessel apparatus 1984; Shah and Buscema, 1988). These lesions can progress, (Houghten et al., 1986). In order to denote the position in the albeit at a low frequency, to cervical carcinoma (zur Hausen, protein, the putative initiation codon was assigned to be amino 1989). More than 90% of cervical carcinomas carry some type acid number 1. Synthetic peptide number 1 corresponds to of HPV, with HPV 16 alone being found in about 60% of amino acids 2-21 in the L1 OW, peptide number 2 to amino tumors (Durst et al., 1983; Boshart et al., 1984; Crum et al., acids 17-36, number 3 to amino acids 32 to 51 and so on up to 1984; DiLuca et al., 1986). the L1 carboxyterminal peptide (number 35) which correAll HPV genomes have at least 8 potentially protein- sponds to amino acids 5 12-53 1. Peptide number 36 correencoding regions, open reading frames (ORFs) (Pfkter, 1987; sponds to the amino terminus of the L2 ORF (amino acids Baker, 1987). Two ORFs, L1 and L2, have been shown to 2-21), number 37 is amino acids 17-36 and so on. The 2 encode viral capsid proteins. The L1 protein is an abundant, peptides at the L2 carboxyterminus (numhers 65 and 66) were approximately 54-kDa capsid protein (Li et al., 1987; Doorbar synthesized as 21-residue peptides. Thus, their amino acid poand Gallimore, 1987) that is regularly immunogenic (Li et al., sitions are 437-457 and 453473, respectively. The amino acid 1987; Banks et al., 1987; Jenison et al., 1989) for both IgG sequences of the most immunoreactive peptides are listed in and IgA antibodies (L. Dillner et al., 1989). Antibodies to the Table I. L1 protein have virus-neutralizing activity, at least for the bovine papillomavirus (BPV) (Pilacinski et al., 1984). Several Sera studies using monoclonal antibodies (MAbs) (Nakai et al., Sixty-four sera from patients with either carcinoma in situ 1986; Pate1 et al., 1989) or bacterially expressed fusion pro- (CIN grade 3) or invasive cervical carcinoma were collected at teins (Li et al., 1987; Strike et al., 1989; Jenison et al., 1988; the Department of Gynecology, University of Bologna, Italy. Tomita et al., 1987a) have shown that the L1 protein has epitopes in common for all types of papillomavirus ( P V t t h e so-called group-specific epitopes-as well as epitopes specific for each PV type. One group-specific and one type-specific 4To whom reprint requests should be addressed. epitope of L1 in HPV 6 have been mapped using a set of small fusion proteins (Jenison et al., 1989). The L2 ORF encodes an Received: October 5 , 1989. approximately 70-kDa capsid protein that is considerably less
530
DILLNER ET A L .
TABLE I - DETF,CTION OF SIGNIFICANTLY ELEVATED ANTIBODY TITERS AGAINST HPV 16 SYNTHETIC PEPTIDES AMONG SERA FROM 30 PATIENTS WITH HPV-16 CARRYING CERVICAL NEOPLASMS AS COMPARED TO A CONTROL GROUP OF 60 SERA FROM PATIENTS WITH OTHER TUMORS OR FROM HEALTHY W N O R S
Peptide number
8 12 13 14
16 17
24
31 49
IgA % > 0.1'
Peptide sequence
SCC(%)
COY%)
NKFGFPDTSFYNPDTORLVW VDNRECISMDYKQTQLCLIG LCLIGCKPPIGEWGKGSPC KGSPCTNVAVNPGDCPPLEL VHTGFGAMDFITLQANKSEV NKSEVPLDICTSICKYPDYI NGICWGNQLFVTVVDTTRST ACOKHTPPAPKEDDPLKKYT PML~TFIVSTNPNTVTSSTPI
57 53 17 60 53 63 37 33 57
17 7 13 7 8 25 42 27 78
p
IgG % > 0.1
valueZ
SCC(%)
CO(%)
43 40 17 50 20 30 53 10 73
2 2 5 5 2 18 55 8 68
Publication of the International Union Against Cancer Publication ds I’Union Internationale Contre 18 Cancer
MAPPING OF LINEAR EPITOPES OF HUMAN PAPILLOMAVIRUS TYPE 16: THE L1 AND L2 OPEN READING FRAMES Joakim DILLNER’’~, Lena DILLNER’,Goran UTTER’,Carina EKLUND’,Antonella ROTOLA~, Silvano COSTA3 and Dario DILUCA~ ‘Department of Virology, Karolimh Institute, S-10521 Stockholm, Sweden;2Department of Microbiology, University of Ferrara, Ferrara; and 3Department of Gynecology, University of Bologna, Bologna, Italy. Certain types of human papillomavirus (HPV), notably HPV type 16, are associated with flat or inverted proliferative lesions of the cervix uteri that can progress to malignancy. As a first step towards the serological study of the epidemiology of HPV, we have synthesized the entire amino acid sequences of the 2 major viral capsid proteins of HPV type 16, LI and U, as a set of 66 synthetic 20-residue peptides with an overlap of 5 amino acids. The peptides were tested for reactivity with IgA, IgG and IgM antibodies in the sera of 30 patients with HPV-16-carrying cervical neoplasms. Both IgG and IgM antibody responses were detected, but most of the reactivity found was of the IgA class. The most immunoreactive peptides were futher analyzed for reactivity with sera from 22 patients with parotid gland tumors and with sera from 38 healthy individuals. The U-encoded protein contained only one major linear epitope, which was not specific for HPV16-carrying neoplasms. In contrast, the LI -encoded protein contained several epitopes that were regularly immunoreactive with antibodies present in the sera of patients with HPV16-carryingcervical neoplasms, but only rarely so in the sera of patients with other tumors or of healthy individuals.
abundant (Komly et al., 1986; Doorbar and Gallimore, 1987; Banks et al., 1987; Tomita et al., 19876). The L2 protein has been reported to contain type-specific epitopes (Komly et al., 1986). We have recently reported that the IgA response to linear epitopes of HPV-encoded nuclear antigens (J. Dillner et al., 1989) and capsid proteins (L. Dillner et al., 1989) predominates over the IgG response to these proteins in patients with CIN, suggesting that IgA-specific immunoassays using linear epitopes of HPV proteins could be used to study the epidemiology of HPV in relation to CIN (L. Dillner et al., 1989; J. Dillner et al., 1989). In order to obtain a map of the linear epitopes of the HPV 16 major capsid proteins, we synthesized the entire amino acid sequences of these 2 proteins as a set of 20 amino-acid synthetic peptides with overlap of 5 amino acids. The positions of the sequence-specific epitopes that reacted with IgA, IgG or IgM antibodies in the sera of patients with HPV- 16-carrying cervical neoplasms are reported. MATERIAL AND METHODS
The human papillomaviruses are a widely distributed family of viruses that cause various proliferative diseases in the in- Peptide synthesis fected epithelium (Pfkter, 1984). Different types of HPV are Sixty-six 20 amino-acid peptides, with a 5 amino-acid overassociated with different diseases and different anatomical sites lap, representing the deduced amino acid sequence of the L1 of infection (Pfister, 1984, 1987). Several types, notably HPVs and L2 OFWs of HPV 16 (Seedorf et al., 1985) were synthe6, 11, 16, 18, 31, 33, 35, and 52, infect the genital tract sized using t-Boc amino acids (Bachem, Bubendorf, Switzer(Pfkter, 1987; Shah and Buscema, 1988). HPV types 6 and 11 land) and p-methylbenzhydrylamine resin (Fluka, Buchs, Switare mainly associated with elevated, pointed, wart-like lesions, zerland) according to the multiple solid-phase peptide synthesis condyloma acuminata (Gissman et al., 1983; Shah and method of Houghten (1985). Removal of the protecting groups Buscema, 1988). In contrast, HPV types 16, 18, 31, 33, 35, from the formyl-tryptophane and methionine sulfoxide residues and 52 are mainly associated with flat or “inverted” incon- was achieved by cleavage with 25% hydrogen fluoride (Tam et spicuous lesions, designated cervical intra-epithelial neoplasms al., 1983). The peptides were then cleaved from the resin with (CIN) (Crum et al., 1985; Durst et al., 1983; Boshart et al., liquid hydrogen fluoride using a multi-vessel apparatus 1984; Shah and Buscema, 1988). These lesions can progress, (Houghten et al., 1986). In order to denote the position in the albeit at a low frequency, to cervical carcinoma (zur Hausen, protein, the putative initiation codon was assigned to be amino 1989). More than 90% of cervical carcinomas carry some type acid number 1. Synthetic peptide number 1 corresponds to of HPV, with HPV 16 alone being found in about 60% of amino acids 2-21 in the L1 OW, peptide number 2 to amino tumors (Durst et al., 1983; Boshart et al., 1984; Crum et al., acids 17-36, number 3 to amino acids 32 to 51 and so on up to 1984; DiLuca et al., 1986). the L1 carboxyterminal peptide (number 35) which correAll HPV genomes have at least 8 potentially protein- sponds to amino acids 5 12-53 1. Peptide number 36 correencoding regions, open reading frames (ORFs) (Pfkter, 1987; sponds to the amino terminus of the L2 ORF (amino acids Baker, 1987). Two ORFs, L1 and L2, have been shown to 2-21), number 37 is amino acids 17-36 and so on. The 2 encode viral capsid proteins. The L1 protein is an abundant, peptides at the L2 carboxyterminus (numhers 65 and 66) were approximately 54-kDa capsid protein (Li et al., 1987; Doorbar synthesized as 21-residue peptides. Thus, their amino acid poand Gallimore, 1987) that is regularly immunogenic (Li et al., sitions are 437-457 and 453473, respectively. The amino acid 1987; Banks et al., 1987; Jenison et al., 1989) for both IgG sequences of the most immunoreactive peptides are listed in and IgA antibodies (L. Dillner et al., 1989). Antibodies to the Table I. L1 protein have virus-neutralizing activity, at least for the bovine papillomavirus (BPV) (Pilacinski et al., 1984). Several Sera studies using monoclonal antibodies (MAbs) (Nakai et al., Sixty-four sera from patients with either carcinoma in situ 1986; Pate1 et al., 1989) or bacterially expressed fusion pro- (CIN grade 3) or invasive cervical carcinoma were collected at teins (Li et al., 1987; Strike et al., 1989; Jenison et al., 1988; the Department of Gynecology, University of Bologna, Italy. Tomita et al., 1987a) have shown that the L1 protein has epitopes in common for all types of papillomavirus ( P V t t h e so-called group-specific epitopes-as well as epitopes specific for each PV type. One group-specific and one type-specific 4To whom reprint requests should be addressed. epitope of L1 in HPV 6 have been mapped using a set of small fusion proteins (Jenison et al., 1989). The L2 ORF encodes an Received: October 5 , 1989. approximately 70-kDa capsid protein that is considerably less
530
DILLNER ET A L .
TABLE I - DETF,CTION OF SIGNIFICANTLY ELEVATED ANTIBODY TITERS AGAINST HPV 16 SYNTHETIC PEPTIDES AMONG SERA FROM 30 PATIENTS WITH HPV-16 CARRYING CERVICAL NEOPLASMS AS COMPARED TO A CONTROL GROUP OF 60 SERA FROM PATIENTS WITH OTHER TUMORS OR FROM HEALTHY W N O R S
Peptide number
8 12 13 14
16 17
24
31 49
IgA % > 0.1'
Peptide sequence
SCC(%)
COY%)
NKFGFPDTSFYNPDTORLVW VDNRECISMDYKQTQLCLIG LCLIGCKPPIGEWGKGSPC KGSPCTNVAVNPGDCPPLEL VHTGFGAMDFITLQANKSEV NKSEVPLDICTSICKYPDYI NGICWGNQLFVTVVDTTRST ACOKHTPPAPKEDDPLKKYT PML~TFIVSTNPNTVTSSTPI
57 53 17 60 53 63 37 33 57
17 7 13 7 8 25 42 27 78
p
IgG % > 0.1
valueZ
SCC(%)
CO(%)
43 40 17 50 20 30 53 10 73
2 2 5 5 2 18 55 8 68
