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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ABSTRACT |
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MAIN TEXT |
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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 270290 and 340345. 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 4954 (region I, Fig. 1
a) 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 169178 (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 BC 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. 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 4560 (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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Received 1 October 2002;
accepted 30 January 2003.