Quantitative and epitope-specific antigenicity analysis of the human papillomavirus 6 capsid protein in aqueous solution or when adsorbed on particulate adjuvants

Abstract

Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as "a black box" due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples.

Neutralization sites of human papillomavirus-6 relate to virus attachment and entry phase in viral infection

“…BALB/c mice for hybridoma preparation were immunized subcutaneously three times with HPV6 VLPs absorbed with aluminum adjuvant as described in our previous study [29]. A set of anti-HPV6 murine mAbs were generated by ascites fluid in a standard hybridoma assay and screened by enzyme-linked immunosorbent assay and pseudovirus-based neutralization assay.…”

“…HPV6 PsVs were produced as described in our previous study [29]. The L1/L2 expression vector p6sheLLr (No.…”

“…We replaced each of the surface loops of the HPV6 L1 protein with the homologous loops of the HPV16 L1 protein separately, hereafter referred to as HPV6-16 chimeric VLPs. Genes encoding the HPV16 loop (BC, DEa, DEb, DEc, EF, FGa, FGb, FGc, HIa, HIb) were cloned into pTO-T7-HPV6 L1 to replace HPV6 loops by Gibson Assembly [29]. The recombinant plasmids were transformed into E.coli ER2566 strain for the expression of the HPV6-16 loop mutant VLPs.…”

Escherichia coli-derived virus-like particles in vaccine development

“… 79 The functional epitopes can be quantitated by their ability to bind to a panel of specific monoclonal antibodies. 92 – 94 Monoclonal antibodies have been developed as specific probes to identify and characterize virion-like epitopes. 34 , 95 , 96 Binding activity to a certain neutralizing epitope can serve as an excellent surrogate marker for in vivo immunogenicity or vaccine efficacy.…”

Concepts in Light Microscopy of Viruses

“…In recent years, manufacturers of confocal microscopes have developed systems that combine high-content image-based screening with spinning disk confocality. Examples of such systems include the IXM-C series from Molecular Devices [ 181 ], the Yokogawa CSU-X1 [ 182 ], Opera Phenix from Perkin Elmer [ 183 ], the IN Cell Analyzer 6000 from GE Healthcare [ 184 ], or further instruments from other suppliers [ 185 ]. The commercial systems differ in image quality and practicability, for example, the possibility of phase-contrast acquisition, light sources, image file formats, the feasibility to exchange the optical filters, or price.…”