Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33

Abstract

Background: Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs), type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33) with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1.

Results: Reactivities of monoclonal antibodies (mAbs) H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa) 51-58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132-140) and FGb (aa 282-291), were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260-270), in addition to loops DE and FGb.

Conclusion: These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines.

Figure 1

Type-specificity of HPV-reactive antibodies. A) Infection of human 293TT cells with HPV16 and HPV33 pseudovirions in the presence of type-specific neutralizing antibodies. Infectious events unaffected by HPV16-specific (mAb H16.56E) or HPV33-specific (mAbs H33.B6, H33.J3) monoclonal antibodies were monitored 72 hrs post infection. B) Interaction of type-specific antibodies with HPV16 and HPV33 virus-like particles (VLPs) in a Heparin-BSA ELISA assay. All three antibodies displayed type-specificity. Although background binding of mAb H33.E12 is significantly increased, specific binding is also restricted to particles of HPV type 33.

Figure 2

Determinants of type-specificity. Alignment of amino acid sequences in surface-exposed loops of capsid proteins L1 of HPV16 and HPV33. Divergent amino acids are listed; identical amino acids are marked by asterisks. On the right, localization of these hyper variable loops in the L1 monomer is shown. Modeling by RasMol was based on the monomeric structure of the HPV16 capsid protein L1.

Figure 3

Epitope mapping of type-specific antibodies. A) Elimination of HPV33-specific epitopes by loop exchanges in capsid protein L1. Recombinant HPV L1 capsid proteins expressed in HUTK^- cells were tested by immunofluorescence analysis for the presence of epitopes for antibodies H16.56E, H33.E12, H33.J3 and H33.B6. Loss of reactivity is marked by (-), gain of antibody reactivity by (+). B) Functional transfer of HPV33-specific epitopes to HPV 16 by loop swapping, leading to reactivity (+) with the respective HPV33-specific antibody. Note that the correct presentation of corresponding epitopes is also influenced by neighboring loops.

Figure 4

Heparin-BSA ELISA. Analysis of epitope expression on wild type (HPV33) and chimeric (HPV33:16BC and HPV16:33BC) VLPs bound to Heparin-coated ELISA plates using type-specific antibodies H16.56E, H33.J3 and H33.B6. Exchange of aa 51–58 (BC-loop of capsid protein L1) results in the loss or gain of reactivity with antibody H33.J3.

Figure 5

Neutralization of HPV pseudovirus infection of 293TT cells by type-specific antibodies. In contrast to wt HPV16 and HPV33 pseudovirions, HPV16:33BC pseudovirions are neutralized by the HPV16-specific H16.56E as well as the HPV33-specific H33.J3 antibodies. Infection was monitored 72 h post infection.

Figure 6

Epitopes of HPV33-specific antibodies on the pentameric L1 capsomere. RasMol pictures showing the epitope patterns for mAb H33.J3 (A), mAb H33.B6 (B) and mAb H33.E12 (C). Variations in the complexity of the epitopes (D-F), ranging from a single loop (D; H33.J3 epitope), two neighboring loops (E, H33.B6 epitope), to at least three loops (F; H33.E12 epitope). Type-specific amino acids are shown in yellow, conserved amino acids in red).