Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in Ebola virus-like particle vaccine preparations

Abstract

Background Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from ‘host’ cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy.MethodsAfter production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP1 concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP1 protein. Purified recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm.ResultsConditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP1 with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6 %. Unlike western blot values, the LC-HRMS quantitation of GP1 in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice.ConclusionsThis method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1. By monitoring variability in GP1 content, the eVLP production process can be optimized, and the total amount of GP1 needed to confer protection accurately determined.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9119-8) contains supplementary material, which is available to authorized users.

T-cell-dependent mechanisms promote Ebola VLP-induced antibody responses, but are dispensable for vaccine-mediated protection

“…The particular lot of VLP within this study was previously used for EBOV vaccine studies and has been extensively characterized. 19 , 32 The GP content for these studies was determined by Western blot and fixed at a 10 μg GP dose for the vaccinations. The VLPs were maintained at −80 °C and diluted in sterile saline and/or combined with poly-ICLC prior to vaccination.…”

Advances in Designing and Developing Vaccines, Drugs, and Therapies to Counter Ebola Virus

“…Thus, an EBOV vaccine capable of effectively inducing a long-lasting neutralizing antibody response is desirable for developing appropriate prevention strategies in combating the infection. In this line, the mucin-like domain of EBOV envelope glycoprotein GP1 has been identified to be critical in induction of protective humoral immune response ( 46 , 47 ). Filorab 1 vaccine revealed desirable immunogenicity without the side effects.…”

“…Ebola VLPs (EBOV-VLPs or eVLPs) are generated from the expression of viral transmembrane glycoprotein (GP) and structural matrix protein (VP40) in mammalian cells, which undergo self-assembling and budding from host cells and display morphological similarity to infectious EBOV particles ( 47 ). Baculovirus-derived eVLPs comprising GP, VP40, and NP of EBOV have been found to induce human myeloid DC maturation, suggesting their immunogenicity.…”

“…Quantitation of EBOV antigenic particles using proteomic assays like liquid chromatography high resolution mass spectrometry method can be employed for determining the batch quality of vaccine constructs as well as in optimizing the dosages by assessing the amount of GP1 needed to confer effective protection ( 47 ).…”

The expanding role of mass spectrometry in the field of vaccine development

“…To improve on the conventional approaches for absolute quantitation of GP1 in Ebola virus‐like particles (eVLPs), an isotope dilution full‐scan liquid chromatography‐high‐resolution mass spectrometry method was developed using an UltiMate 3000 HPLC and an Orbitrap Elite Hybrid Ion Trap‐Orbitrap mass spectrometer . The reported MS quantitation method provided not only a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1 but also ensured adequate preclinical/clinical dosing.…”