Characterization of gH/gL/pUL128-131 pentameric complex, gH/gL/gO trimeric complex, gB and gM/gN glycoproteins in a human cytomegalovirus using automated capillary western blots

Human cytomegalovirus (HCMV) is currently a major cause of congenital disease in newborns and organ failure in transplant recipients. Despite decades of efforts, an effective vaccine against HCMV has yet to be developed. However, the discovery of pentameric gH complex on viral surface which contains potent neutralizing epitopes may help enable development of an effective vaccine. In our company ongoing Phase II clinical trial of whole-live virus HCMV vaccine (V160), the pentameric gH complex has been restored on the surface of live attenuated AD169 virus strain. The reconstructed HCMV virus contains a variety of surface glycoproteins including pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, gB glycoprotein, and gM/gN heterodimer complex. To further characterize this virus and enable the monitoring of multiple viral antigens during vaccine process development an effective and efficient analytical strategy was required to detect and quantify several viral surface proteins. In this paper, we present an innovative approach based on capillary western blot technology that allows fast and accurate quantitation of pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, and gB glycoprotein. This method is suitable for analyzing target proteins in multiple sample types including supernatants from infected cell culture, purification intermediates, concentration bulk, and the final vaccine product. In addition, the capillary western blot-based technology identified a previously unknown biochemical profile present in some HCMV viruses: triplet gH peaks of viral surface proteins in non-reducing environ-ment, which could potentially present a new strategy for specificity and identity testing. (c) 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Automated summary

This paper presents an innovative approach using capillary western blot technology for rapid and accurate quantification of key viral surface proteins of HCMV. The method is versatile in analyzing multiple sample types and can aid in vaccine process development and virus characterization.