Abstract
The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr135 and Val141 on the DEa loop were shown to be critical residues for 26D1 binding via site-directed mutagenesis. Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection.
Highlights
(a) Binding (EC50: 66 ng/ml) of 26D1 to HPV16 L1 virus-like particles (VLPs) in an antibody dose-dependent manner. (b) Neutralizing titers (NT50: 0.585 ng/ml) of 26D1 as measured by blocking HPV16 PsVs entry
The KD value of 26D1 binding to HPV16 VLPs as measured by Biacore was 6.2 × 10−9 M. These results suggest that the human monoclonal antibody 26D1 possesses a high affinity to HPV16 VLPs and potent viral neutralization activity
The primary objective of a prophylactic HPV16 L1 VLP-based vaccine is to develop protective immunity against viral infection, which in large part depends on elicitation of neutralizing antibodies[40]
Summary
(a) Binding (EC50: 66 ng/ml) of 26D1 to HPV16 L1 VLPs in an antibody dose-dependent manner. (b) Neutralizing titers (NT50: 0.585 ng/ml) of 26D1 as measured by blocking HPV16 PsVs entry. Well-characterized HPV16 murine neutralizing antibody, and its binding to HPV16 VLPs completely blocks the reactivity of more than 75% of human antisera[23]. Identification of immunodominant regions of L1 protein is critical for designing cross-reactive hybrid VLPs. For antigenic determinants of HPV16 L1 VLP, because the reconstituted epitopes of HPV16 VLPs are generally identified by murine monoclonal antibodies such as HPV16.V5, epitope mapping on HPV16 virus has been hampered by limited antibody sources and a lack of structural information of the antibody-antigen complexes. Essential surface loops of the L1 proteins for 26D1 binding were identified by employing a series of HPV16/6 hybrid VLPs with surface loops swapped between two types to alter the epitope structure. This report is the first on the elicitation and epitope identification of a potent neutralizing antibody response against HPV16 VLPs in humans. The precise interpretation of neutralizing determinants provides a basis for prophylactic vaccine design
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