Human papillomavirus type 6 virus-like particles present overlapping yet distinct conformational epitopes

Xin-Min Wang1, James C. Cook1, Jessica C. Lee1, Kathrin U. Jansen1, Neil D. Christensen2, Steven W. Ludmerer1 and William L. McClements1

1 Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
2 The Jake Gittlen Cancer Research Institute, Department of Pathology, Penn State University, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA

Correspondence
William McClements
william_mcclements{at}merck.com


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The epitope for a human papillomavirus (HPV) type 6 conformation-dependent, neutralizing monoclonal antibody (mAb) was partially mapped using HPV L1 recombinant virus-like particles (VLPs). The mAb H6.J54 is cross-reactive with the closely related HPV types 6 and 11. By making HPV-6-like amino acid substitutions in the cottontail rabbit papillomavirus (CRPV) major capsid protein L1, we were able to transfer H6.J54 binding activity into a CRPV/HPV-6 hybrid L1 protein. Full binding activity was achieved with only nine amino acid changes and identified a region centred on the HPV-6 residues 49–54. This region has previously been shown to be a critical part of HPV-6 type-specific epitopes. Fine mapping of the region by scanning a series of alanine substitution mutations showed that in HPV-6 VLPs this type-common epitope overlaps HPV-6 type-specific epitopes.


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Human papillomavirus (HPV) infections are among the most common sexually transmitted diseases (Bauer et al., 1991; Ho et al., 1998; Moscicki et al., 2001) and are implicated in both benign and malignant disease of the genital epithelia (Bosch et al., 1995; Galloway, 1994; Gissmann et al., 1983; Munoz, 2000; Pisani et al., 1993). To meet medical needs imposed by HPV infection, several experimental vaccines have been developed and are currently in human clinical trials (Brown et al., 2001; Evans et al., 2001; Harro et al., 2001), and one has been shown to be effective in preventing persistent viral infection (Koutsky et al., 2002). These vaccines are based on virus-like particles (VLPs), which assemble spontaneously when the papillomavirus major capsid protein, L1, is recombinantly expressed either alone or together with L2, the minor capsid protein (Hofmann et al., 1995; Kirnbauer et al., 1992, 1993; Neeper et al., 1996; Rose et al., 1993; Volpers et al., 1994; Zhou et al., 1991). VLPs are morphologically and immunologically similar to native virions (Christensen et al., 1994; Kirnbauer et al., 1992; Neeper et al., 1996; Rose et al., 1993), can induce neutralizing antibody responses (Bryan et al., 1997; Christensen et al., 1994, 1996b; Lowe et al., 1997; Ludmerer et al., 2000; Pastrana et al., 2001; Roden et al., 1997; Rose et al., 1994; Yeager et al., 2000) and in animal models, can protect against viral disease (Breitburd et al., 1995; Christensen et al., 1996c; Jansen et al., 1995; Kirnbauer et al., 1996; Suzich et al., 1995).

The amino acid sequences of L1 proteins are similar among papillomaviruses of all species and are well conserved among HPV genotypes. Alignment of HPV L1 proteins reveals that within these well-conserved proteins there are localized regions of amino acid sequence divergence. These regions contain determinants of type-specificity (Ludmerer & McClements, 1999). We and others have shown that the epitopes recognized by HPV neutralizing monoclonal antibodies (mAbs), which are generally type-specific and conformation-dependent, map to these regions (Christensen et al., 2001; Ludmerer et al., 1996, 1997, 2000; McClements et al., 2001; Roden et al., 1997). Furthermore, structural studies of HPV capsomers and VLPs have shown that these divergent sequence regions are surface-exposed (Chen et al., 2000), and other studies have shown that substitution of these regions with non-L1 sequences results in the presentation of foreign epitopes on VLPs (Chackerian et al., 1999; Slupetzky et al., 2001).

Previously, we mapped type-specific neutralizing epitopes for the closely related HPV types 6 and 11 by demonstrating that HPV-11-like amino acid substitutions in HPV-6 L1 transferred type 11-specific epitopes to VLPs assembled from mutant type 6 L1 proteins and that reciprocal experiments transferred HPV-6-specific epitopes to type 11 VLPs (Ludmerer et al., 1996, 1997, 2000; McClements et al., 2001). These studies mapped the HPV-11 immunodominant neutralizing epitope to a sequence of approximately 20 amino acids centred on residue Y132 and found that a second subdominant neutralizing epitope comprised residues 270–290 and 340–345. For HPV-6, neutralizing epitopes are bipartite and comprise two regions of sequence divergence, both of which are distinct from the regions identified in HPV-11. The principal region is centred on residues 49–54 (region I, Fig. 1a) and contains determinants for H6.B10.5, H6.N8 and H6.M48, the three HPV-6-specific conformation-dependent mAbs (Christensen et al., 1996b) that are neutralizing in an HPV-6 pseudovirion assay (Yeager et al., 2000). The second region is centred on residues 169–178 (region II, Fig. 1a) and modulates binding to region I. Monoclonals H6.M48 and H6.B10.5 require region II as well as region I to achieve full binding to VLPs (McClements et al., 2001). The physical association of regions I and II is consistent with the crystal structure of HPV-16 VLPs (Chen et al., 2000) where HPV-16 L1 sequences analogous to regions I and II map to loop B–C and segment G1, respectively, and are predicted to be in close proximity in capsomers and VLPs. Type-specific bipartite epitopes have also been described for HPV types 11 and 16 (Christensen et al., 2001; Ludmerer et al., 2000). Preliminary studies on H6.J54 (Christensen et al., 1996b), a conformation-dependent mAb that is cross-reactive with HPV types 6 and 11, suggested that it also recognizes a bipartite epitope, with one part mapping in the N-terminal region of L1 (N. D. Christensen, unpublished observations). This raised the possibility that, in HPV-6, type-common and type-specific epitopes may overlap and could interfere with precise serological characterization.



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Fig. 1. Amino acid sequence alignments of HPV-6, HPV-11 and CRPV L1 proteins. Alignment of the three (complete) amino acid sequences was carried out using Vector NTI software. (a) Aligned N termini of HPV-6 and CRPV. Regions I and II of HPV-6 are indicated. Arrowheads indicate residues in CRPV replaced with HPV-6-like residues in the CR/H6 hybrids; shaded areas denote the extent of amino acid identity with HPV-6 in hybrid L1 molecules. (b) Alignment of HPV-11 region I with HPV-6 region I. Residues in HPV-11 identical to those in HPV-6 are indicated by dots; the three differences (at residues 49, 53 and 54) that confer type 6 specificity are shown. Residues of HPV-6 replaced with alanine in the scanning study are underlined.

 
To extend our mapping studies to address the question of epitope interference and to generate novel reagents for monitoring immune responses to HPV infection or immunization with experimental HPV vaccines, we attempted to transfer the HPV-6-specific epitopes to a less closely related papillomavirus VLP, i.e. one that should have fewer epitopes in common with HPV-6 (Fig. 1a). We chose cottontail rabbit papillomavirus (CRPV) for the L1 scaffold. The CRPV and HPV-6 L1 molecules are nearly identical in length and CRPV VLPs are structurally similar to HPV VLPs, but the CRPV L1 amino acid sequence (SWISS-PROT accession no. P03102) is only about 50 % identical with that of HPV-6. Using PCR mutagenesis, we transferred the HPV-6-specific residues of regions I and II into the CRPV L1. The CRPV/HPV-6 hybrid L1, CR/H6:I, had nine HPV-6-like substitutions in region I making that sequence identical to HPV-6 over a 20-residue sequence (CRPV L1 aa 45–64). In addition to the region I changes, CR/H6:I-II was generated, which also had nine HPV-6-like substitutions in region II making that sequence identical to HPV-6 for the 22 residues (aa 166–187). The alignment of HPV-6 and CRPV L1 sequences in Fig. 1(a) highlights these changes and also shows the extended similarity between the two sequences. All constructions were confirmed by complete sequencing of the L1 genes. These hybrid proteins, as well as those from wild-type CPRV, HPV-6 and HPV-11, were expressed using the baculovirus vector pVL1393 (Stratagene). Extracts were prepared from transfected Spodoptera frugiperda cells (Sf9; Invitrogen) and assayed by ELISA for binding activity to conformation-dependent mAbs as described previously (Benincasa et al., 1996; Ludmerer et al., 1996; McClements et al., 2001).

Fig. 2 shows the results of the binding activity assay. With wild-type L1 proteins, type specificity of mAbs H6.B10.5, H6.N8 and H6.M48 was clearly evident. Also evident was the failure to transfer quantitatively HPV-6-specific binding activity to the CRPV backbone. Lack of binding was not due to the failure of the hybrid L1 proteins to form high-order structures including capsomers and VLPs; CRPV-5A, a conformation-dependent CRPV-specific mAb (Christensen & Kreider, 1991) bound both hybrid proteins well. Of the three type 6-specific mAbs, only H6.N8 showed any binding to a hybrid L1, and while the signal was well above background, binding was clearly impaired. The surprising result was that H6.J54, a neutralizing conformation-dependent HPV-6 and -11 cross-reactive mAb (Christensen et al., 1996b), bound CR/H6:I as well as it bound wild-type HPV-6 or -11 VLPs. This indicated that at least part of the J54 epitope was located in region I. However, when the second HPV-6-like substitution at region II was made, the resultant hybrid L1 protein was not recognized by H6.J54 or H6.N8. This result was in contrast to earlier data from HPV-6/11 hybrid VLPs where we found that sequences in region II could stabilize binding of the HPV-6 type-specific mAb interactions at region I (McClements et al., 2001). However, it was consistent with H6.J54 binding studies on HPV-11/16 chimeric VLPs, which suggested that part of the J54 epitope maps to HPV-11 residues 45–60 (N. D. Christensen, unpublished observations). H6.J54 binding is most likely independent of region II, and because regions that are well separated in the linear L1 sequence can affect the secondary and tertiary structure of assembled VLPs, introduction of HPV-6-like amino acids at this site may distort one or more components of the J54 epitope.



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Fig. 2. Binding of HPV-6 mAbs to CRPV VLPs bearing HPV-6-like amino acid substitutions. Baculovirus-expressed VLPs and control extracts (Sf9) were assayed by ELISA as previously described. Assays were carried out in duplicate and the results are the average of assays of two independently derived clones for each mutant. Error bars denote standard deviation. H6 wt, wild-type HPV-6; H11 wt, wild-type HPV-11; CRPV wt, wild-type CRPV; CR/H6:I, CRPV with HPV-6-like substitutions in region I; CR/H6:I–II, CRPV with HPV-6-like substitutions in regions I and II.

 
These results suggested that the J54 type-common epitope overlapped the HPV-6-specific epitope and might share determinants. To map the region I portion of J54 more precisely, we next made a series of alanine substitution mutants that scanned a 22-residue stretch centred on the critical binding region I (Fig. 1b). Because H6.J54 is cross-reactive with HPV-11, we selected charged or hydrophilic residues that were common to both HPV types and likely to contribute to antibody binding. The mutant L1 proteins – R40A, S50A, K52A, N55A, T57A, P60A and K61A – were expressed and assayed for binding to H6.J54, the type-specific mAbs H6.B10.5 and H6.N8, and to H6.C6, which recognizes a distal linear epitope common to both type 6 and 11 (Ludmerer et al., 1996). Binding results are shown in Fig. 3. The signals with the conformation-dependent mAbs indicated that all mutants formed ordered structures and that region I was intact, although reduced binding of mAb H6.B10.5 to the R40A, K52A and K61A mutants was observed. While the relative binding of the four antibodies varied across the mutant set, the results from one mutation – N55A – stood out. Binding to H6.J54 was completely lost but with little impairment of H6.B10.5 or H6.N8 binding. To assess the assembly state of this critical mutant, partially purified material from the N55A extract was characterized by transmission electron microscopy and was found to be indistinguishable from preparations of either wild-type or the T57A mutant; each showed the presence of 55 nm particles with the characteristic appearance of VLPs (data not shown). We consider it likely that the N55A mutation has a direct effect and that residue 55 asparagine is critical for H6.J54 binding. We infer this because both H6.B10.5 and H6.N8 recognized this mutant VLP well, indicating that region I was not grossly distorted. The reduced binding of H6.B10.5 seen with the R40A, K52A and K61A mutants suggests that these residues contribute to the region I type-specific epitope. Taken together with the J54 epitope transfer to CRPV described above, these data confirm the overlap of the type-specific and type-common conformation-dependent epitopes.



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Fig. 3. Binding of HPV-6 mAbs to mutant HPV-6 VLPs. VLPs from alanine scanning mutants of HPV-6 region I and control extracts (Sf9) were assayed by ELISA for binding to HPV-6-specific mAbs. For the S50A, K52A, N55A and K61A mutants, the results are averages obtained from assays of two independently derived clones. Error bars denote standard deviation. H6 wt, wild-type HPV-6; alanine mutants as indicated.

 
These observations extend an earlier study describing the theoretical overlap of a linear epitope with a conformation-dependent one (Ludmerer et al., 1997). The linear epitope, mapped to residues 108–127 of HPV-16 (Christensen et al., 1996a; Heino et al., 1995), was transferred into HPV-11 VLPs by making HPV-16-like substitutions (V123L, V126T) at the two residues in that region where the HPV-16 and -11 L1 proteins differed. In this hybrid VLP context, the linear HPV-16 epitope overlapped the HPV-11 major neutralizing epitope, which mapped to an approximately 20 amino acid sequence centred on residue 132. Residue 123 was critical for each epitope but because it differed between the two types (L in HPV-16, Y in HPV-11) simultaneous presentation of both epitopes could not occur. The present results are of greater significance because the epitopes exist simultaneously on the VLP and both are conformation-dependent, yet have distinguishable binding properties. H6.J54 binding can be eliminated from HPV-6 without affecting the binding of the type-specific mAbs and it can be transferred into CRPV VLPs without reconstituting the epitopes for the other antibodies. This suggests that alteration of epitope structure can be significant in impact, yet highly localized in effect. This has important implications for HPV serology. If cross-reactive responses between two closely related HPV types, for example types 16 and 31 or 18 and 45, hinder precise serological evaluation, hybrid VLPs can be made in which the cross-reactive responses are reduced or eliminated, thus creating serological typing reagents with improved sensitivity and selectivity.


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Received 1 October 2002; accepted 30 January 2003.