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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.

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types., ((C)2001 Elsevier Science.)

We have mapped the binding sites on human papillomavirus (HPV) type 6 for three HPV 6-specific neutralizing monoclonal antibodies (mAbs). The critical binding residues were first identified by making HPV 11-like amino acid substitutions in the HPV 6 major capsid protein L1 and assaying the resulting virus-like particles (VLPs) for reactivity with the mAbs. To confirm the relevance of these residues for mAb binding, we demonstrated that HPV 6 type-specificity could be transferred to HPV 11 VLPs by making the appropriate HPV 6-like amino acid substitutions in the HPV 11 L1. Two binding regions were found. For one mAb, all critical residues are centered at residue 53, while for the other two mAbs, type-specific binding also requires a second site located more than 100 residues distal to the first. Both binding sites coincide with regions of L1 where the sequences of the closely related HPV 6 and 11 diverge. These regions are where the L1 sequences are the least well conserved among all HPV types and they have been implicated in type-specific binding for other HPV types. This suggests that clusters of diverged residues, surrounded by conserved L1 sequences, are presented on the surface of assembled particles and are responsible for eliciting critical humoral immune responses to the virus., (Copyright 2001 Academic Press.)

Characterization of the regions of human papillomaviruses (HPVs) that elicit neutralizing immune responses supports studies on viral infectivity and provides insight for the development and evaluation of prophylactic vaccines. HPV11 is a major etiologic agent of genital warts and a likely vaccine candidate. A conformationally dependent epitope for the binding of three neutralizing monoclonal antibodies (mAbs) has been mapped to residues G(131)T(132) of the L1 major capsid protein. The mAbs bind L1 only when it is assembled into virions or into virus-like particles (VLPs) that mimic the capsid structure. We were interested in identifying other domains of L1 that elicit neutralizing responses. To this end, we have generated a panel of mAbs against VLPs derived from HPV11 L1 harboring a G131S substitution. The new mAbs are unlike the neutralizing mAbs previously mapped to residues G(131)T(132) in that they bind both prototype and HPV11:G131S mutant VLPs. Some of the new mAbs neutralized virus in vitro. We have mapped epitopes for three of these new mAbs, as well as a neutralizing mAb generated against HPV11 virions, by measuring binding to HPV6 VLPs substituted with HPV11-like amino acids. Two regions are critical: one defined by HPV11 L1 residues 263-290 and the other by residues 346-349. mAbs H11.H3 and H11.G131S.G3 bind HPV6 VLPs with substitutions derived from the 346-349 region; in addition, H11.G131S.G3 binds HPV6 VLPs with substitutions derived only from the 263-290 region. Although H11.H3 does not bind HPV6 VLPs with substitutions derived from the 263-290 region, binding to HPV6 VLPs is enhanced when both sets of substitutions are present. mAbs H11.G131S.I1 and H11.G131S.K5 bind HPV6 VLPs with the 263-290 substitutions, but show little binding to HPV6 VLPs with the 346-349 substitutions. However, binding to HPV6 VLPs is enhanced when substitutions at both regions are present. The 346-349 region has not previously been described as eliciting a neutralizing response for any HPV type. In addition, the work demonstrates a complex binding site contributed by two distinct regions of L1., (Copyright 2000 Academic Press.)

A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity.

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.