Hepatitis C or HCV, causes a chronic liver infection that can lead to permanent liver scarring and, in severe cases, cancer. It affects approximately 71 million people worldwide and causes approximately 400,000 deaths each year.
Although treatments are available for HCV-related infections, they are expensive, difficult to access, and do not protect against reinfection. A vaccine that can help prevent HCV infection is a major unmet medical and public health need.
One of the main reasons there hasn’t been an HCV vaccine yet is that scientists have yet to identify the appropriate antigen or part of the virus that would trigger a protective immune response in the body.
Decades of research have identified HCV E1E2, the virus’s only surface protein, as the most promising vaccine candidate. However, the development of an HCV vaccine based on this protein is limited by uncertainty about its appearance. Knowing the structure of the protein is necessary to understand how the immune system reacts to the virus.
So how do researchers capture the structure of a single protein on a shape-changing virus?
We are researchers specializing in microscopy and vaccine design. Using new technology, we were able to visualize the molecular details of this elusive protein, unlocking key information about how this virus works and offering a potential blueprint for a future vaccine.
That’s how we did it.
The challenges of catching a shape-shifting virus
One of the reasons it has been so difficult to grasp the structure of the HCV E1E2 protein is that it is both flexible and fragile. It changes shape so often and breaks so easily that it is difficult to purify it.
By analogy, imagine a bowl of spaghetti dipped in tomato sauce. Now imagine trying to take a picture of each piece of spaghetti in the same position over time while the bowl is shaking. Hard to do, right? That’s how to imagine the complete E1E2 protein.
There were also technological barriers. Until recently, available imaging techniques were limited in their ability to visualize microscopic proteins. X-ray crystallography, for example, is unable to capture molecules that change and distort frequently, such as HCV. Additionally, other options, such as nuclear magnetic resonance spectroscopy, required cutting large parts of the protein or chemically manipulating it in a way that would transform its physiological state and potentially alter its function.
So, to look at the structure of E1E2, we needed a way to extract and purify, stabilize and trap the entire shape-changing protein in one configuration.
How to take a picture of a protein
Cryo-EM, or cryo-electron microscopy, is a type of imaging technique that visualizes specimens at cryogenic temperatures, in this case the boiling point of nitrogen: minus 320.8 degrees Fahrenheit (minus 196 Celsius). With such cold temperatures, the ice freezes so quickly that it does not have time to crystallize. This creates a beautiful glass-like frame around the protein of interest, allowing an unobstructed view of every structural detail. Cryo-EM also requires very little protein to operate, which reduces the amount of material to be purified.
Winner of the 2017 Nobel Prize in Chemistry and Nature The magazine’s 2015 “Method of the Year” award, cryo-EM is superb for imaging biological macromolecules in their native or natural state in the aqueous environment of human blood. Cryo-EM has also played a pivotal role in characterizing the structure of the COVID-19 virus and its variants.
So how do you take a picture of a protein?
First, we integrated the genetic code to make E1E2 into human cells in a Petri dish in order to have sufficient amounts of proteins to study. After purification of the protein, we immersed it in liquid ethane followed by liquid nitrogen. Liquid ethane is used to freeze protein because it has a higher boiling point than liquid nitrogen. This means it is able to capture more heat before turning into gas, allowing the protein to freeze much faster than it would in liquid nitrogen and avoid structural damage.
Once the protein was vitrified or in a glass-like state of ice, we were able to not only see its overall structure, but also capture several individual configurations of the protein that it takes on when it changes shape, including its shapes the least stable.
At this point, our protein was ready for its close-up. We used a microscope that uses a high-energy focused beam of electrons and a very sophisticated camera that detects how the electrons bounce off the surface of the protein. This created a 2D image which we then mathematically transformed into a 3D model. And that’s how we got the coveted “close-up” of the HCV surface protein.
Our next step then was to assess the location of each amino acid, or building block of the protein, in 3D space. Because each amino acid has a unique shape, we used a computer program that could identify each in our 3D map. This allowed us to manually reconstruct a high-resolution model of the protein, one building block at a time.
A new tool for designing an HCV vaccine
Our 3D map and model of the HCV E1E2 protein supports previous research describing its structure while providing new insights into the characteristics that will help pave the way for a long-sought vaccine design against this virus.
For example, our structure reveals that the interface between the two main parts of the protein is stabilized by sugars and hydrophobic patches, or areas that repel water molecules. This creates sticky binding centers along the protein and prevents it from collapsing – a potential site for protective antibodies and new drugs to target.
Researchers now have the tools to design antiviral drugs and vaccines against HCV infection.
This article was originally published on The conversation by Lisa Eshun-Wilson and Alba Torrents from the Pena at the Scripps Research Institute. Read the original article here.