NEW YORK (GenomeWeb) – A new mass spectrometry-based protein quantitation method developed by scientists at the US Army Medical Research Institute of Infectious Diseases could greatly improve quality control in manufacturing vaccines based on virus-like particles (VLPs).
Led by Lisa Cazares, researchers from USAMRIID and the US Army's Edgewood Chemical Biological Center described a quantitative method of detecting the glycoprotein GP1, the main antigenic target of an Ebola virus VLP vaccine. GP1 is a viral transmembrane protein that, along with extracellular matrix protein VP40, forms the empty shell of a VLP, which can induce an immune response.
The method uses high-performance liquid chromatography and isotope dilution standards along with high-resolution mass spec. For the study, the researchers used a Thermo Fisher Scientific LTQ-Orbitrap Elite mass spectrometer in tandem-MS mode. They published their results last week in Clinical Proteomics.
"GP1 concentration is a critical attribute of [Ebola virus] vaccines and accurate measurement of the amount of GP1 present in [Ebola VLP] lots is crucial to understanding variability in vaccine efficacy," the authors wrote.
Using purified standards, the method was able to account for GP1 with an average accuracy of 83 percent. Compared with Western blot, the mass spec assay was designed to account for truncated products in the recombinant GP1 quantitation standard materials.
While Western blot has been the go-to assay for assessing how much of the target protein is produced in VLP vaccine lots, Cazares told GenomeWeb that the field has always been searching for a better way. "We knew in the beginning that Western blot would not be accurate enough," she said. Despite the considerable difficulty of working with GP1, she and her co-authors have found a more precise way to quantify it. The mass spec method even seems to have a direct link to the biology of the Ebola VLP vaccine.
"Compared to a Western blot, mass spec quantitation seemed to better predict the amount of GP1 needed to confer protection against Ebola in an animal model," she said.
It could be an important step forward in expanding the list of VLP-based vaccines and provides some interesting considerations for scientists working with glycoprotein quantitation.
VLPs have already been successfully deployed in Gardasil, the vaccine for human papillomavirus.
"They're sort of little empty viral shells, which have the same glycoprotein expression on the surface but not the genetic material inside," Cazares said. "They mimic an infectious virion but they're not infectious." USAMRIID is developing a VLP-based vaccine for Ebola virus, which has been in the works for several years and Cazares has been working on characterizing the protein content.
GP1 is the primary antigen the body uses to develop Ebola immunity and VLPs are a great way to introduce the antigen because they mimic the wild-type structure but are non-infectious. "It's nice because you're presenting the protein to the immune system in its native state," Cazares said.
While commercial production of Ebola VLPs is routine, achieved by transfecting human cells, the amount of GP1 in each VLP lot can be highly variable.
For the kinds of studies that go into developing a vaccine, it's important to know how much antigen is required for a protective dose. "In the past, people had used things like total protein concentration and recombinant glycoprotein as a standard in Western blotting to estimate the amount [of GP1]," Cazares said, adding that Western blotting inherently relies on antibodies, which can be poorly characterized and lead to overestimations.
Mass spec presents its own challenges and a method for GP1 quantitation had remained elusive. "There are many reagents and a lot of other variables," Cazares said. "But if you control for all of that, the quantitation is going to be so much better than just straight Western blotting."
One of the problems Cazares' team ran into was that GP1 is " a very tricky protein,' she said. The glycosylation made trypsin digestion complicated and the researchers had to add a trypsin enhancer, Protease Max.
Perhaps an even bigger hurdle was creating a reference standard for GP1. "Viruses are very thrifty, their mRNA sequences often express multiple proteins by using overlapping reading frames," Cazares said. "They may have truncated or overlapping protein products because of an mRNA editing site." Degradation in the lengthy VLP production process can also truncate proteins. Because they were using recombinant GP1 for their reference standards, the scientists realized that they would need a highly purified, full-length protein standard to get the true quantity of GP1 in the VLPs.
Using HPLC, they removed all truncated versions and other contaminating proteins. "We had the best standard that we could get for quality control for the method," Cazares said. "It was very accurate, without that standard we never would have been as confident in our results."
She suggested that getting that high-quality reference standard is an important takeaway from the study for general protein quantitation. Additionally, the team experienced some issues with missed trypsin cleavage sites, so they simply created standards for missed cleavages and quantitated them.
These were problems that Cazares said might crop up in VLP vaccines being developed for other viruses, "Membrane-bound viruses usually have some glycoproteins on their surface," she said. "Any VLP vaccine preparation that relies on an envelope protein as its primary antigen could benefit from an assay like this."
The method could also help determine the amount of antigen needed to confer protection from Ebola. In a small mouse study, the USAMRIID scientists took vaccines prepared from five different Ebola VLP lots, gave them to mice, and challenged them with Ebola.
The results showed a strong correlation between the amount of GP1 in the vaccine, as determined by their mass spec method, and survival following exposure to Ebola.
Moreover, Cazares said that by going back to the initial VLP lots, she could put a number on how much antigen she'd need to confer protective immunity in future studies.
"Being able to standardize the vaccine by giving a number, providing the concentration of the primary antigentic protein, that helps determine the protective dose," she said. "And will help down the line in developing a human vaccine."