Preparation and properties of a papillomavirus infectious intermediate and its utility for neutralization studies - PubMed (original) (raw)
Preparation and properties of a papillomavirus infectious intermediate and its utility for neutralization studies
Joshua W Wang et al. Virology. 2014.
Abstract
We show that minor capsid protein L2 is full length in clinical virion isolates and prepare furin-cleaved pseudovirus (fcPsV) as a model of the infectious intermediate for multiple human papillomavirus (HPV) types. These fcPsV do not require furin for in vitro infection, and are fully infectious in vivo. Both the γ-secretase inhibitor XXI and carrageenan block fcPsV infection in vitro and in vivo implying that they act after furin-cleavage of L2. Despite their enhanced exposure of L2 epitopes, vaccination with fcPsV particles fails to induce L2 antibody, although L1-specific responses are similar to PsV with intact L2. FcPsV can be applied in a simple, high-throughput neutralization assay that detects L2-specific neutralizing antibodies with >10-fold enhanced sensitivity compared with the PsV-based assay. The PsV and fcPsV-based assays exhibit similar sensitivity for type-specific antibodies elicited by L1 virus-like particles (VLP), but the latter improves detection of L1-specific cross-type neutralizing antibodies.
Keywords: Carrageenan; Furin; Gamma secretase; Heparin; Human papillomavirus (HPV); Infectious intermediate; L2; Minor capsid protein; Neutralization; Papillomavirus.
© 2013 Published by Elsevier Inc.
Figures
Figure 1. 293TTF cell line overexpresses furin and facilitates production of furin-cleaved PsV
Expression of endogenous furin in parental 293TT cells (lane 1), 293TT transiently transfected with furin gene (lane 2), 293TTF cells stably transfected with furin in the presence (lane 3) and absence (lane 4) of puromycin (2μg/ml). Equivalent amounts of conditioned media supernatant of parental 293TT (lane 5) or 293TTF in the presence (lane 6) and absence (lane 7) of puromycin (2μg/ml) were used to assess furin secretion. Image analyses and relative densitometry using ImageJ indicate that for 293TTF cells endogenous furin expression and secretion was at least 150-fold higher compared to 293TT control cells. Band seen at ~60kDa was a furin splice variant as indicated in the material data sheet of the antibody used (A). Equivalent amounts of HPV16 PsV (solid squares) or HPV16 fcPsV (open squares) based on L1 content, were added onto pre-plated furin deficient cells FD11 (B) or FD11F (FD11 cells re-complemented with furin gene) (C), 293TT cells (D) and 293TT cells with 20μM of furin inhibitor (E). All experiments were performed in triplicate
Figure 2. Furin-cleaved pseudovirus (fcPsV) exhibits 80-90% L2 cleavage without compromise of morphology or infectivity in vivo
Transmission electron microscopy of HPV16 PsV (180,000x) (A) and fcPsV (135,000x) (B), black bar indicates 100nm. Infection time course of HPV16 PsV versus fcPsV encapsidating a GFP reporter was added to 293TT cells (60,000 cells/well) (significance was calculated using paired t-test: 24 hours P=0.0349, 48 hours P=0.0282, 72 hours P=0.0654) (C). In vivo vaginal challenge of mice for 72 hours with HPV16 PsV or fcPsV encapsidating a luciferase reporter (unpaired t-test, P=0.0901), or PBS as negative control (D). Commassie gel showing L2 cleavage status of HPV16 PsV using the standard PsV protocol or in 293TTF (fcPsV) matured in various calcium concentrations (0, 2.5, 5.0 and 7.5 mM) for 24 hours (E) Western blot analysis of L2 cleavage on HPV PsV genotypes 16, 18, 45, 58 made in either in 293TT with the standard HPV PsV protocol or in 293TTF with modified maturation timing of 48 hours and 5mM of calcium chloride (F)
Figure 3. Absence of furin-cleaved L2 from papillomaviruses of natural isolates
Western blot of papillomavirus pseudovirions (PsV) and PV virions L2 extracted from clinical isolates of warts of the same papillomavirus genotype; HPV26 (A) , BPV-1 (B), HPV6 (C), HPV57 (D), MmuPV1 (E) and table showing classification and clinical source of respective papillomavirus.
Figure 4. Heparin, Carrageenan and γ-secretase inhibitor (XXI) inhibit both HPV16 PsV and fcPsV
Inhibition assays were performed using equivilant amounts of HPV16 PsV (solid circles) or fcPsV (open circles) based on L1 content with varying concentrations of heparin (A), carrageenan (B) in 293TT cells. The same assay was performed using fcPsV16 infection of HSPG-deficient PGSA-745 cell line in the presence of either heparin (open triangles) or carrageenan (closed triangles) (C). In vivo mouse (n=5) vaginal challenge of HPV16 fcPsV or PsV in the presence or absence of heparin or carrageenan (D). Inhibition assay using HPV16 fcPsV in the presence or absence of XXI (500nM) in FD11 cells (furin-deficient CHO cells) (E). In vivo mouse (n=10) challenge with HPV16 fcPsV or PsV in the presence or absence of XXI. * indicates significance (P value= 0.0089 for HPV16 PsV comparison, P value= 0.0101 for HPV16 fcPsV comparison).
Figure 5. Vaccination with HPV16 fcPsV particles which have enhanced RG-1 epitope exposure does not elicit an enhanced L2 immune response compared to PsV
RG-1 Immunoprecipitation of equivalent amount of HPV16 fcPsV and PsV (based on L1 content) and immunoblot with antibody to L1. The arrow indicates amount of L1 pulled-down (A). ELISA results for mouse sera vaccinated with HPV16 PsV or fcPsV (plus alum-MPL adjuvant) using HPV16 PsV or full length HPV16 L2 peptide as antigens. (B)
Figure 6. Generation of LoVoT cell line
Stable expression of the SV40 T-antigen in LoVoT (lane 2) with parental LoVo cells (lane 1) and 293TT (lane3) as controls (A). Validation of LoVoT cell line demonstrating superior infectivity (luminescence signal) for the same input of HPV16 fcPsV using virus dilution of 1:8000 (B).
References
- Bourne GL, Grainger DJ. Development and characterisation of an assay for furin activity. Journal of immunological methods. 2011;364:101–108. - PubMed
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