Design and antiviral assessment of a panel of fusion proteins targeting human papillomavirus type 16

Abstract

Cervical cancer ranks as the third most prevalent malignancy in women worldwide. The persistence of Human papillomavirus (HPV) infection stands out as the foremost risk factor for cervical cancer development. Among the numerous HPV subtypes, HPV16 infection emerges as the primary pathogenic determinant of cervical cancer. To date, no specific drugs have been approved. In this study, we engineered two high-affinity fusion protein targeting HPV16 L1 protein based on the alpaca-derived single-domain antibody 2C12 previously obtained in our laboratory. These two fusion proteins exhibited potent neutralizing activity against HPV16 pseudovirus with IC50 values of 7.8 nM and 6.5 nM, respectively. Molecular docking analysis revealed that 2C12 formed ten pairs of hydrogen bonds with HPV16 L1, among which Arg39 and Thr100 established multiple pairs of hydrogen bonds with HPV16 L1, indicating their crucial roles in antigen-antibody binding process. These structural and biological findings underscore the effective binding capacity of these fusion proteins to HPV16, leading to reduced viral load and providing valuable insights into therapeutic antibody and vaccine development against HPV 16 infection.

Fig 1. Purification and identification of AHPVD and AHPVT protein.

(A) Results of AHPVD protein HiLoad 16/600 Superdex 75 prep grade gel filtration chromatography. (B) Results of AHPVT protein HiLoad 16/600 Superdex 75 prep grade gel filtration chromatography. (C) SDS-PAGE identification of AHPVD and AHPVT protein.

Fig 2. 2C12, AHPVD and AHPVT binding affinities to HPV 16 L1.

Z6 was used as a negative control. The red color represents the fitted curve. The binding profiles are shown with time (s) on the x-axis and response units (RU) on the y-axis. KD represents the equilibrium dissociation constant, KD values shown are the mean ± standard deviation (SD) of three independent experiments.

Fig 3. The neutralizing capacity of 2C12, AHPVD and AHPVT for HPV 16 pseudoviruses: Z6 was used as a negative control.

(A) Z6; (B) 2C12; (C) AHPVD; (D) AHPVT; (E) The green fluorescencewas detected by CQ1 microscopic reading method. Three independent experiments were performed with two replicates. The curves and IC50 values are one representative data, in which the error bar for each concentration is presented as mean ± SD.

Fig 4. Structure analysis of 2C12 complex with the HPV16 L1.

(A) Ribbon representations of the complex of 2C12 (pink) with the HPV16 L1 (green), the hydrogen region is highlighted by black circle in the side view; (B-D) Zoomed-in view of 2C12 with HPV16 L1 polar interaction; (E) Interaction surface is displayed in surface representation model and colored in red and blue, key residues are labeled.