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1 Department of Genome Modifications and Cancer, Infection and Cancer Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
2 Departments of Pathology and Microbiology and Immunology, The Jake Gittlen Cancer Research Foundation, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA
Correspondence
Michael Pawlita
M.Pawlita{at}dkfz.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Joint DKFZ-EMBL Chemical Biology Core Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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So far, more than 100 HPV types have been fully characterized by cloning and complete sequencing of their genomes. Classification of HPV is based on the major capsid protein L1 open reading frame nucleotide sequence: HPV of the same genus show at least 60 % sequence identity, those of the same species at least 70 %, those of the same type at least 90 % and those of the same subtype (variants) at least 98 % (de Villiers et al., 2004
). HPV of the genus alpha (15 species) mostly infect anogenital and oral mucosa, some can additionally (species 2 and 8) or exclusively (species 4) infect the skin. Many alpha HPV can induce benign genital or common skin warts, while so called high-risk types (most frequently HPV types 16 and 18) (Munoz et al., 2003) belonging to species 5, 6, 7, 9 and 11, can induce intraepithelial neoplasia, the precursor of cervical cancer (Bosch & de Sanjose, 2003; Clifford et al., 2003; de Villiers et al., 2004).
Upon infection with HPV, serum antibodies to L1 protein can develop (Dillner, 1999; van Doornum et al., 1998; Wang et al., 1996; Wideroff et al., 1999). This immune response is highly type-specific (Carter et al., 1996, 2000; Giroglou et al., 2001; Wang et al., 1997) and persists for years (Carter et al., 2000; Dillner, 1999). L1 antibodies are considered markers for current and past infection (Carter et al., 1996; Carter & Galloway, 1997; Kirnbauer, 1996) and are weakly associated with cervical cancer (van Doornum et al., 2003). In contrast, antibodies to the early oncoproteins E6 and E7 that are consistently expressed in HPV-transformed cells are strongly associated with cervical carcinoma (Lehtinen et al., 2003; Meschede et al., 1998; Silins et al., 2002; Zumbach et al., 2000).
Analysis of HPV humoral immune responses faces some limitations due to the large number of HPV types associated with the different diseases and to difficulties in producing sufficient quantities of infectious virus particles. As an alternative to virion production, efforts were made to reproduce the antigenic properties of virions by virus-like particles (VLP). VLP are formed spontaneously after overexpressing L1 by vaccinia virus (Hagensee et al., 1993), plasmid vectors (Pastrana et al., 2004) or baculovirus (Kirnbauer et al., 1992) in yeast (Sasagawa et al., 1995), mammalian, or insect cells (Le Cann et al., 1994), respectively. VLP resemble papillomavirus virions in morphology and display conformational and neutralizing epitopes (Christensen et al., 1996a, b, 2001; Le Cann et al., 1994). VLP-based ELISA is the standard technique in HPV serology. It was developed to screen for antibodies to HPV1 (Carter et al., 1993), HPV16 (Kirnbauer et al., 1994), canine oral PV (CoPV) (Suzich et al., 1995), cottontail rabbit PV (CRPV) and bovine PV1 (BPV1) (Christensen et al., 1996a), BPV4 (Kirnbauer et al., 1996), HPV45 (Touze et al., 1996), HPV6 and 11 (Touze et al., 1998), HPV5 (Favre et al., 1998), HPV8 (Bouwes Bavinck et al., 2000), HPV31, 33, 35, 18, 39 (Giroglou et al., 2001), HPV59 (Combita et al., 2002) and HPV15, 20 and 24 (Feltkamp et al., 2003).
L1 of HPV11 and HPV16 expressed as fusion proteins with glutathione S-transferase (GST) in E. coli spontaneously form homogeneous capsomeres (Chen et al., 2000, 2001; Li et al., 1997). Capsomeres generated from bacterially expressed L1 of HPV11 and CoPV display neutralizing, linear and conformational epitopes, and induce neutralizing antibodies upon experimental immunization (Rose et al., 1998; Yuan et al., 2001).
Recently, GST–HPV L1 fusion protein-based antibody detection systems have been developed for many HPV types (Karagas et al., 2006; Sehr et al., 2002; Waterboer et al., 2005).
Monoclonal antibodies (mAb) have been generated against VLP of different HPV including types 6, 11, 16, 18, 31, 33, 35 and 45 (Christensen et al., 1996a, b; Combita et al., 2002; Ludmerer et al., 2000). They are used to define HPV capsid epitopes.
Epitopes on HPV capsids can be experimentally classified as conformational (present on intact VLP and capsomeres) or linear (displayed by synthetic peptides and denatured L1) (Christensen et al., 1996a, b). Neutralizing epitopes have also been identified in the L1 major capsid protein (Christensen et al., 1994b; Giroglou et al., 2001; Rose et al., 1998; White et al., 1998, 1999). To analyse HPV neutralization properties, many systems have been established using athymic mouse xenograft (Kreider et al., 1987), raft culture systems (Meyers & Laimins, 1994) or production of infectious HPV pseudovirions in vitro (Roden et al., 1996; Touze & Coursaget, 1998; Unckell et al., 1997). Pastrana and colleagues (Buck et al., 2005; Pastrana et al., 2004) have developed an in vitro neutralization assay utilizing HPV pseudovirions carrying a secreted alkaline phosphatase (SEAP) reporter plasmid.
This study aimed, first, to evaluate GST–L1 fusion proteins as ELISA antigens for detecting capsid-specific antibodies against alpha papillomaviruses, and second, to use GST–L1 proteins of 15 different alpha HPV types to determine type specificity and cross-reactivity of 92 mAb recognizing HPV capsid epitopes. We show that GST–L1 fusion proteins display almost all epitopes previously defined on VLP, including conformational, linear and neutralizing epitopes. Additionally, we describe a series of intra- and/or inter-species cross-reactive epitopes and show that cross-reactivity only loosely follows phylogenetic relationships. We also show that neutralizing epitopes are always conformational and mostly, but not always, type-specific.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Similarly, the complete L1 open reading frames (ORF) of HPV2, 3, 10, 11, 31, 32, 33, 35, 45, 52, 57 and 58 were expressed as double fusion proteins with N-terminal GST and a C-terminal undecapeptide (tag) of the SV40 large T-antigen. These constructs and characterization of the fusion proteins will be described in detail elsewhere (manuscript in preparation).
Transformation of the modified pGEX plasmids into E. coli strain BL21 Rosetta DE3 (Invitrogen) and further expression and treatment of the fusion proteins was performed as previously described (Sehr et al., 2002).
Anti-HPV VLP monoclonal antibodies.
Altogether, 89 tissue culture supernatants and two ascites preparations of 91 mAb were analysed (Tables 1, 2). Yet unpublished mAb were generated and characterized as previously described (Christensen et al., 1990, 1994a, b, 1996a, b; Muller et al., 1997; Sehr et al., 2002). Seventy-three mAb were raised against VLP of nine different HPV types (HPV6, 16, 11, 18, 45, 31, 33, 52 and 35). Six mAb (the H263 series) were generated against a hybrid containing residues 1–168 of HPV11 and residues 172–505 of HPV16. Another 12 mAb were generated against HPV11 VLP containing a G131S substitution (Tables 1, 2). All antibody preparations used had an IgG concentration of at least 800 ng ml–1 (Easy-Titer IgG kit; Pierce).
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GST–L1 capture ELISA.
Ninety-six-well PolySorp plates (Nunc) were coated overnight at 4 °C with glutathione-casein and blocked the next day as previously described (Sehr et al., 2001). The plates were incubated (room temperature, 1 h) with the cleared GST–L1 fusion protein lysates diluted in casein blocking buffer to 0.25 µg µl–1 total protein (saturating concentration) and washed. As a negative control, GST-tag lysate was used in parallel. For titrations (each mAb against the immunogen HPV L1), plates were incubated (room temperature, 1 h) with threefold dilutions (starting concentration 1 : 10) of the mAb in casein blocking buffer (Sehr et al., 2001). For cross-reactivity analyses, mAb were incubated (room temperature, 1 h) with the 15 GST–L1. Plates were washed, incubated (room temperature, 1 h) with biotin-conjugated goat anti-mouse Ig, incubated (room temperature, 30 min) with streptavidin–poly-HRP (horseradish peroxidase), washed again and the colour reaction was developed as previously described (Sehr et al., 2001). For 28 HPV16 mAb analysed on two separate days, the coefficient of determination of the linear regression of the absorption values was R2=0.78.
HPV16 VLP-capture ELISA.
HPV16 VLP-capture ELISA was performed as previously described (Muller et al., 1997). Briefly, plates (Nunc) were coated overnight at 4 °C with rabbit anti-HPV16 VLP polyclonal antibody diluted 1 : 200 in PBS and blocked the next day with 5 % milk, PBS, 0.05 % Tween 20 (blocking buffer). As antigen, HPV16 VLP (3.5 µg ml–1 in blocking buffer) generated from the same parental plasmid as GST 16L1 and purified by two gradient centrifugations as previously described (Muller et al., 1997) were used. Further assay procedures were as described above for GST–L1 capture ELISA.
Neutralization assay.
In vitro neutralization assays based on HPV pseudovirions carrying a SEAP reporter gene were done as previously described (Buck et al., 2005; Pastrana et al., 2004). Briefly, 300 000 293TT cells ml–1 (293 cells transfected with an expression plasmid encoding a cDNA for SV40 large T-antigen) were seeded, infected after 5 h with a mixture of serial mAb dilutions (starting concentration 1 : 50) with the pseudovirions of the immunogen HPV type and incubated (5 days, 37 °C). Cell supernatants were assayed for SEAP using a chemiluminescent SEAP Reporter Gene Assay (Roche Diagnostics). Pseudovirions of HPV16, 18, 45, 6 and 11 were available. For cross-neutralization analyses, all neutralizing mAb were investigated at 1 : 50 dilution with the HPV pseudovirions of the other types.
Data analysis and statistics.
In all ELISA binding experiments, lysate from bacteria expressing GST-tag alone was analysed in parallel to define the reaction background. The measured absorbances (A) at 450 nm were expressed in milliunits (mAU). The net A450 value of a mAb was obtained by subtracting the background reactivity from the absorbance with the GST–L1-tag fusion protein. Monoclonal antibodies were arbitrarily classified as reactive when the A450 value at 1 : 90 dilution was equal to or greater than 110 mAU. The antibody titre was defined as the last dilution yielding readings equal to or greater than 110 mAU. For cross-reactivity experiments, a mAb concentration close to saturation (mean 79 % of Amax with the immunogen HPV type; SD 15 %) was used. All mAb were measured in duplicate wells and the mean of the specific reactivity of the duplicate values was taken as the final readout.
In SEAP neutralization assays, net relative light units (RLU) were calculated by subtracting RLU of cells without mAb and pseudovirions (background) from RLU of cells treated with the mAb/pseudovirion mixture. The end-point neutralization titre was defined as the last dilution yielding 
70 % reduction in the SEAP activity in comparison to the reactivity of the pseudovirions added without antibody. For cross-neutralization, all mAb were analysed in duplicate cultures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2). The six mAb (H263.A2, H263.C3, H263.F4, H263.G2, H263.H9 and H263.D1) generated against a hybrid VLP containing residues 1–168 of HPV11 and residues 172–505 of HPV16 were analysed with both HPV16 and HPV11 GST–L1. H263.A2 reacted only with HPV16 while H263.G2 reacted only with HPV11 (Table 1). The other four mAb reacted more strongly with HPV11 than with HPV16 (Table 2). All 12 mAb generated against VLP of HPV11 L1 mutant G131S reacted with HPV11 GST–L1.
Of the 89 reactive mAb, 82 showed Amax values above 1500 mAU with log end-point dilution titres (the last dilution giving a signal 110 mAU) varying from 3.3 to 6.3 for tissue culture supernatants and up to 7.3 for ascites as antibody source (Tables 1, 2). The titration curves of these mAb showed steep slopes, i.e. A450 values decreasing from 80 to 20 % of Amax within 10-fold dilution. For the remaining seven mAb (H45.N5, H35.Q8, H35.H9, H35.N6, H33.E12, H33.B6 and H18.A7), the Amax values were below 1100 mAU, log titres were between 2.0 and 4.3 and the titration curves were flat, i.e. A450 values decreasing from 80 to 20 % of Amax only within more than 100-fold dilution. Fig. 1 shows six representative examples of mAb titration curves.
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) and were not analysed further. For H6.C6 and H16.L4, the L1 N terminus is known to be an essential part of the epitope (Christensen et al., 1996b), and the HPV6 and 16 GST–L1 proteins used here lack the 10 N-terminal amino acid residues (Sehr et al., 2002). The reason for the very weak reactivity of mAb H35.O3 remains unclear. However, the reactivity of the other three mAb generated against HPV35 VLP with HPV35 GST–L1 was rather weak with low Amax and flat titration curves (Table 1, Fig. 1).
HPV type specificity of mAb
HPV type specificity of the 89 reactive mAb was investigated with GST–L1 fusion proteins of 15 HPV types representing six alpha papillomavirus species (
1, 2, 4, 7, 9 and 10). To allow cross-reactivity analysis in similar conditions for all antibodies, we used a mAb concentration below but close to saturation for the immunogen HPV type (Fig. 1).
Under these conditions, 46 (52 %) of the mAb reacted only with the GST–L1 fusion protein of the HPV type used for immunization (Table 1), while the other 43 mAb (48 %) showed different levels of intra- and/or inter-species cross-reactivity (Table 2).
Of the 43 cross-reactive mAb, 23 (54 %) showed strong (A450 relative to the type used for immunization >80 %), 11 (26 %) showed intermediate (relative A450 50–80 %) and 9 (20 %) showed weak (relative A450 < 50 % but absolute A450 > 110 mAU) cross-reactivity with at least one other HPV type. Of the 153 cross-reactions, 66 (43 %) were strong, 34 (22 %) were intermediate and 53 (37 %) were weak. This suggests that the cross-reactive epitopes recognized by about half of the cross-reactive antibodies and in about half of all cross-reactions are very similar or even identical to the specific epitope.
All mAb reactive with HPV31 (n=1), HPV35 (n=3) and HPV52 (n=4) were monotypic.
Of the 27 mAb generated against HPV16 VLP, 16 (59 %) were cross-reactive. Two mAb showed strictly intra-species and four showed strictly inter-species cross-reactivity. The other 10 mAb showed mixed intra/inter-species cross-reactivity. Most frequent intra-species cross-reactions were with HPV35 (n=10), HPV31 and HPV58 (both n=9), followed by HPV33 (n=7). HPV52 L1 (n=4) showed the least frequent cross-reactivity. On the other hand, the most frequent inter-species cross-reactions were with HPV18 (n=12), followed by HPV45 (n=8), HPV11 (n=7) and HPV6 (n=6). Cross-reactivity was less frequent with HPV2 and HPV32 (both n=4), HPV10 (n=3), and HPV57 and HPV3 (both n=2). Five HPV16 mAb cross-reacted also with skin alpha papillomavirus types; in this group, cross-reactivity was weak or absent with low-risk mucosal HPV types 6, 11 and 32, but strong or intermediate with the high-risk HPV types 18 and 45. Antibody H16.11B, which recognized a conformational epitope, showed the broadest cross-reactivity. It reacted with all 15 alpha papillomaviruses analysed, strongly with 12 HPV types and weakly with types 6, 11 and 32.
From the four HPV33 mAb, only one cross-reacted strongly with HPV52 and HPV58 and weakly with HPV31, all from its own 9 species. In addition, a strong but isolated cross-reaction was seen with the phylogenetically distantly related HPV32.
Of the 11 HPV18 mAb, 6 (55 %) were cross-reactive. They all showed mixed intra- and inter-species cross-reactivity and reacted with the closely related HPV type 45. Five of them reacted also with skin HPV types, i.e. HPV2 and HPV57, and four also with HPV3 and HPV10. Fourteen out of the 18 cross-reactions with skin types were strong (n=11) or intermediate. Antibody H18.A7, which reacted only weakly with HPV18, showed very broad although mostly weak (n=7) or intermediate (n=6) cross-reactions with all other HPV tested. The other five mAb cross-reacted with neither the mucosal high-risk HPV of the 9 nor the low-risk HPV of the 10 and 1 species, which is in contrast to the frequent cross-reactions of HPV16 mAb with HPV18.
Of the five HPV45 mAb, two showed strong and intermediate intra-species cross-reactivity while inter-species cross-reactivity was absent.
HPV6 and 11 are very closely related low-risk types of the 10 species. Of the 34 mAb raised against these two types, 18 (53 %) were cross-reactive; all recognized both HPV types. Inter-species cross-reactivity was rare, with only four mAb additionally reacting weakly or intermediately with one or two types of the high-risk HPV.
Direct comparison of mAb reactivity with HPV16 GST–L1 and VLP
In a direct experimental comparison, 32 mAb, encompassing 28 raised against HPV16 and the four generated against other HPV types but cross-reactive with HPV16 GST–L1, were also analysed by HPV16 VLP ELISA. All HPV16-specific mAb reacted in VLP-ELISA, including H16.L4, which was non-reactive in GST–L1 ELISA; this mAb is known to detect an epitope located within the N terminus of L1 (Christensen et al., 1996a; Christensen, unpublished) that is deleted in HPV16 GST–L1. GST–L1 and VLP titres were similar (GST–L1 to VLP titre ratio: median 2.9, range 0.04–27) for the 14 neutralizing mAb, but GST–L1 titres were much higher for the 13 non-neutralizing mAb (222, 3–6000). Also the 11 monospecific mAb had similar GST–L1 and VLP titres (1, 0.05–10), whereas the GST–L1 titres of the 16 cross-reactive mAb were always higher (50, 2.5–6500). Finally, the GST–L1 to VLP titre ratios were higher for the 9 mAb recognizing linear (27, 8–6000) than for those 9 mAb recognizing conformational epitopes (3, 0.04–800). In summary, GST–L1 display neutralizing, monospecific and conformational epitopes like VLP, but GST–L1 appears to display non-neutralizing, cross-reactive and linear epitopes in larger quantity than highly purified, antibody-captured VLP.
Neutralization activity of VLP-specific mAb
Seventy four mAb were analysed for neutralization of HPV6, 11, 16, 18 and 45 (Tables 1, 2). Thirty four (46 %) mAb neutralized pseudovirions of the immunogen HPV type. End-point neutralization log titres ranged from 1.7 to 5.6. Thirty three of the neutralizing mAb recognized a conformational epitope and one (MM07) recognized a linear epitope. Among mAb recognizing monospecific epitopes, 28 out of 42 (67 %) were neutralizing, in contrast to only five out of 29 (17 %) mAb recognizing cross-reactive epitopes.
All neutralizing mAb were further investigated at a 1 : 50 dilution for cross-neutralization with pseudovirion preparations of HPV6 and 11 (10), 16 (9), as well as 18 and 45 (7). Cross-neutralization was observed only once; mAb H6.L12 neutralized both HPV6 and the most closely related HPV11 pseudovirions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The majority of published HPV serology data has been established using VLP-based ELISA assays. Here, we used HPV L1 antibody assays based on bacterially expressed, affinity-purified glutathione S-transferase L1 (GST–L1) fusion proteins as antigens (Sehr et al., 2002). This assay has been previously validated for HPV16 and 18 with human and experimental mouse sera by comparison with VLP-ELISA data (Davidson et al., 2003; Sehr et al., 2002). The bacterial expression system allowed analysing nine mucosal HPV types that had been available before as VLP and six HPV types, i.e. skin HPV2, 3, 10 and 57 as well as the mucosal high-risk HPV type 58 and low-risk HPV type 32 that had not been available before as VLP. These numbers reflect the ease with which GST–L1 fusion proteins can be generated, purified and applied in antibody assays.
The binding of 89 (97 %) of the 92 mAb indicates that GST–L1 fusion proteins (with the exception of HPV35) present most (if not all) of the antigenic epitopes presented by VLP. Only two mAb reacted extremely weakly and one did not react with the GST–L1 protein of the respective HPV type. H6.C6 and H16.L4 detect an epitope located on the N terminus of L1 (Christensen et al., 1996a, b), and GST–L1 preparations of HPV6 and 16 lack aa 1–10. Fusion of GST to the L1 N terminus apparently did not inhibit binding of any other mAb, indicating that all of them are either targeting epitopes elsewhere or that GST fusion does not affect N-terminal epitope conformation (Sehr et al., 2002).
Four HPV35 specific mAb reacted very weakly, suggesting that the HPV35 GST–L1 preparation lacked most of the VLP-displayed epitopes for yet unknown reasons. However, the presence of three conformational cross-reactive epitopes (H16.8B, H16.11B and H263.C3) indicates at least partially correct conformation of HPV35 GST–L1. H18.R5, reported to recognize a conformational epitope on HPV18 and HPV45 VLP, reacted only with HPV18 GST–L1, suggesting a subtle epitope difference of HPV45 GST L1.
Our results show that GST–L1 fusion proteins have the epitope repertoire of intact VLP (conformational), but also display epitopes presented by denatured VLP (linear). Linear epitopes are also present on intact VLP preparations either because they are surface-exposed on capsids or because the VLP preparation also contains some denatured L1, but reactions with intact VLP are usually weaker than with denatured proteins (Christensen et al., 1996a). Our direct comparison using twofold gradient-purified and antibody-captured HPV16 L1 VLP as ELISA targets also demonstrated the presence of all linear and all cross-reactive epitopes in VLP ELISA, but in clearly lower density than in GST–L1 ELISA.
Of the 89 mAb reacting with GST–L1, 71 are known to recognize conformational epitopes, including 34 neutralizing epitopes, and 14 are known to recognize linear epitopes, of which eight were reported also to react with some intact VLP preparations (see Tables 1 and 2 for mAb and references). These findings suggest that the GST–L1 fusion protein preparations display neutralizing and conformational as well as linear epitopes. Two mAb recognizing linear epitopes (H16.B20, H16.S1) did not react with GST–L1 of HPV33 or 31, respectively, in contrast to their reported cross-reactivity with denatured VLP of these types, indicating that the GST–L1 preparations of these types presented these linear epitopes in insufficient amounts.
The titres of the different mAb preparations used in this study varied by several orders of magnitude. Choosing of the first dilution below saturation for cross-reactivity experiments allowed comparison of the strength of the cross-reactivity signals and their classification into three categories, strong, intermediate or weak. In a previous, smaller analysis of VLP cross-reactivity of some of the mAb used here, only a uniform dilution of 1 : 50 of tissue culture supernatants irrespective of the effective antibody concentration had been applied (Christensen et al., 1996a).
Among the 89 reactive mAb, 46 reacted only with GST–L1 proteins of the specific HPV type used for immunization, indicating that GST–L1 fusion proteins are displaying type-specific epitopes. Although we analysed cross-reactivity with 15 alpha papillomavirus types, as compared to a maximum of nine types in previous VLP studies, we cannot exclude that some of the mAb classified here as monotypic might turn out to be cross-reactive when analysed with additional types. Previously published data suggested that conformation-dependent mAb H16.11B and H16.8B might be specific for HPV16 alone (Christensen et al., 2001), but here we describe strong cross-reactivity to many other mucosal and skin alpha papillomaviruses. In contrast, the cross-reactivity to HPV11 GST–L1, which was not seen with HPV11 VLP, was very weak. For mAb H16.7E we confirm the absence of cross-reactivity to HPV11 (Christensen et al., 2001), but observed a weak cross-reactivity to HPV35. However, we did not see any reactivity to the four other HPV types in the same species (9). For mAb H16.2F we confirm the absence of cross-reactivity to HPV11 (Christensen et al., 2001), but we additionally found weak cross-reactivity with HPV18 and HPV45 (both 7).
We describe new cross-reactivity for 12 additional antibodies, including five raised against HPV16, one against HPV18, two against HPV45, one against HPV33 and three against the HPV11 G131S L1 mutant. Moreover, we extend the cross-reactivity pattern for 12 mAb. Altogether we have data for 43 cross-reactive monoclonal antibodies, encompassing a total of 153 individual cross-reactions. This provides a comprehensive description of some features of cross-reactive epitopes among human papillomaviruses.
Cross-reactivity follows the phylogenetic grouping only loosely. Among very closely related HPV types, i.e. HPV6 and 11 (>92 % aa identity in L1) as well as 18 and 45 (88 %), cross-reactivity is frequent (Table 3). Since no other HPV types from species 10 and 7 were analysed, it is unclear whether there might be more intra-species cross-reactivity among these antibodies. Only two of the 17 cross-reactive mAb raised against a HPV type of the 9 species were strictly intra-species cross-reactive. These two mAb both recognized only a subset of the six HPV types analysed from this species, suggesting that species-specific epitopes are rare or even do not exist. Furthermore, most mAb with inter-species cross-reactivity did not react with all members of the species of the HPV type against which they were raised, indicating again that HPV species may not share common epitopes.
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9 but instead reacted selectively with HPV18 L1. The weak cross-reaction suggests that the epitope recognized on HPV18 is not identical to that on HPV16. Antibody H16.H5 has been described to bind HPV16 L1 peptide aa 174–185 (Christensen et al., 1996a). HPV18 has seven mismatches in the homologous sequence, suggesting that weak cross-reactivity is not mediated by this sequence. On the other hand, two other HPV16 mAb (Ritti01 and MM07) reacted with five of the six 9 HPV types and very selectively also with HPV45. One of them reacted marginally even with skin HPV2, but not with closely related skin HPV57. Monoclonal antibody H16.S1 cross-reacted with mucosal HPV types, including HPV35 (9), HPV18 and 45 (7) and additionally with skin HPV types 3 and 10 (2). It has been described to bind HPV16 L1 peptide aa 111–130 (Christensen et al., 1996a). Leucine residues 122 and 126 appear to be critical since all non-reactive HPV types have replaced at least one of them by an aromatic residue and the weakly cross-reactive HPV10 differs at the neighbouring position (D127E). While HPV18 was the type most frequently recognized by cross-reactive HPV16 mAb (12 out of 16), only one (pan-reactive H18.A7) of the six cross-reactive HPV18 mAb recognized HPV16. Most frequently observed inter-species cross-reactivity was directed against skin HPV3, 10 and/or 2, 57. Antibody H18.Q2 cross-reacted with HPV types 45, 2 and 57.
Of 14 mAb detecting a linear epitope, 13 were inter-species cross-reactive and one (H16.15G) monotypic (Table 4). Of 71 mAb detecting conformational epitopes, 29 (41 %) were cross-reactive. While 16 of these recognized only closely related HPV types (6/11 and 18/45), three showed weak and four strong inter-species cross-reactions, even including skin HPV types. In conclusion, the cross-reactivity observed here suggests that distantly related mucosal and skin alpha HPV share conformational epitopes and that the phylogenetic L1-based species definition may not define a serological unit since no species-specific epitope was found.
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, whereas in our experiments this antibody showed no HPV16 neutralization.
For five of the six cross-reactive neutralizing mAb, cross-neutralization could be assessed. H6.L12 was strongly cross-reactive with the very closely related HPV11 and neutralized HPV11 with a similar titre as HPV6, which is in agreement with previously published data (Christensen et al., 1996b). Antibodies H16.2F, H6.F62, Ritti01 and MM07 were weakly cross-reactive with HPV18/45, HPV11, and HPV45 (Ritti01 and MM07), respectively, but showed no cross-neutralization with these types. The weak cross-reaction indicates lower affinity of the mAb to the cross-reactive epitope, which might be insufficient to induce neutralization on the cross-reactive epitope; alternatively, the absence of cross-neutralization might indicate that the cross-reactive epitope is functionally different, i.e. that antibody binding does not lead to neutralization.
GST–L1 ELISA titres were always higher than neutralization titres, as has been described previously (Combita et al., 2002; White et al., 1999) (Tables 1, 2). ELISA log titres on average were similar among monospecific (median 5.3, range 3.8–6.2) and cross-reactive mAb (5.0, 2.0–6.2). However, cross-reactive mAb rather consistently showed lower neutralization log titres (2.5, 1.7–3.8) than the monospecific mAb (3.5, 2.3–5.6). Thus, the ratio of ELISA to neutralization titres was on average about 100-fold higher among the cross-reactive (log median 3.3, range 2.4–3.6) than among the monospecific mAb (1.5, 0.2–2.9). This may indicate that neutralization epitopes are highly type-specific. The cross-reactive antibodies that are monospecific in neutralization might recognize the L1 surface involved in neutralization (functional structure) only partially because they also recognize neighbouring, non-functional structures shared by other types; thus their ability to induce a structural change or to block a surface essential for the infection process is reduced, but could be overcome in higher concentrations, resulting in lower neutralization titres.
In conclusion, HPV capsid epitopes defining neutralizing sites are always conformational and most of the mAb detecting monospecific conformational epitopes are neutralizing. Monoclonal antibodies binding to conformational cross-reactive epitopes are rarely neutralizing, and if neutralizing they are rarely cross-neutralizing. Monospecific mAb neutralize more efficiently than cross-reactive mAb.
In summary, our data indicate that bacterially expressed GST–L1 fusion proteins display the same epitopes as VLP and therefore fulfil the essential requirements of antigens for HPV serology. They are useful tools to define and to recognize complex patterns of conformational and linear cross-reactive epitopes. However, when extrapolating these data to the analysis of human sera, it should be kept in mind that the experimentally produced mAb analysed here and human antibodies generated by natural HPV infection may differ due to differences in kind of antigen (recombinantly produced and purified VLP versus native virions), host producing the antibodies (mouse versus human), and site, dose and kinetics of immunization (long-term and low dose in natural infection versus short-term boosting with high doses in experimental animals).
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ACKNOWLEDGEMENTS |
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Received 7 May 2007;
accepted 23 August 2007.
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