Cambridge Healthtech Institute’s Kent Simmons recently spoke with Dr. Kathryn Hastie, a Staff Scientist in the Immunology and Microbiology department at The Scripps Research Institute, about her upcoming PEGS Young Scientist Keynote presentation entitled “The Lassa Virus Glycoprotein: Stopping a Moving Target”
Kathryn Hastie, PhD, Staff Scientist, Immunology and Microbiology, The Scripps Research Institute
To what extent is the shape of the molecule displayed in a vaccine important in eliciting the right immune response?
Viruses often express a single, glycosylated protein on their surface. These proteins are metastable machines that facilitate viral entry into the host cell by rearranging from a mature viral-surface state to a postfusion state, with receptor bound intermediate conformations presented along the way. The viral glycoprotein is often the only antigen on the viral surface and is therefore a major target for neutralizing and protective antibodies. Many neutralizing antibodies function by directly blocking the steps required for fusion, whether it be receptor binding or conformational changes that allow release of the fusion peptide. Antibodies that target epitopes that are only accessible after receptor engagement and/or fusion initiation or completion are often not effective in preventing viral infection. Hence, the particular conformation of the glycoprotein presented to the immune system can have a critical effect on the types of antibodies elicited and therefore protection against infection as a whole.
How can 3D structures inform rational design of vaccines and immunotherapeutics?
Determination of a 3D structure is key to understanding the epitope landscape of an antigen, particularly those from pathogens such as viruses, which express proteins on their surface that are glycosylated and adopt more than one conformational state. For Lassa, we found that the majority of the human antibody response targeted epitopes either poorly presented or not presented at all on the trimeric, prefusion viral form of the GP. These antibodies instead recognize portions of the GP only exposed after entry and fusion. In contrast, more than 80% of the neutralizing response is targeted to quaternary epitopes displayed only the context of the prefusion GP. Biochemical data was important in our initial understanding of the types of antibodies elicited during Lassa infection, but we still didn’t understand where and how neutralizing antibodies interact with Lassa GP and why neutralizing antibodies are poorly elicited during a natural infection. The crystal structure of Lassa GP illuminated the very few sites on the trimer available for antibody binding and demonstrated that of those antibodies that neutralize, the most protective are those that span across multiple GP protomers rather than just a single monomer or individual subunit. This structure and our analysis of the humoral response against Lassa has provided the blueprints for an immunogen most likely to elicit the most effective neutralizing antibodies.
Can we engineer our way to better immune responses?
Viruses are in a constant arms race with the immune system, balancing fitness of the virus as a whole with immune escape. Often, this is achieved by cloaking the more immunogenetic proteinaceous portions with host-cell derived glycans or by presenting more than one target to the immune system. Different conformations of surface attachment proteins are often requisites for the virus to enter the cell. However, these conformations, although expressed transiently, can also act as effective decoys for the immune response as they often expose epitopes that are not hidden by glycosylation and thus are more immunogenic. Efforts spanning multiple viruses have focused on engineering the glycoprotein in particular in order to lock the protein in the conformation most vulnerable to immune surveillance. In addition to providing the immune system with a single, stable target, researchers are also investigating the benefits of site-selective removal of glycosylation sites that may conceal otherwise protective epitopes.
What was the major technical hurdle you had to overcome to determine the Lassa virus glycoprotein structure?
The Lassa virus glycoprotein, GP, is produced as a polyprotein that is post-translationally processed by cellular proteases in the receptor-binding GP1 and fusion-mediating GP2 subunits. These subunits assemble into a trimer of non-covalently associated GP1 and GP2 heterodimeric units. Previous work demonstrated that only processed GP is incorporated into the virion, suggesting that processing was a requirement for proper trimerization. However, in the absence of the transmembrane domains, the GP ectodomain is unstable as a GP1-GP2 protomer. Attempts to purify the wild-type prefusion GP were unsuccessful due to the propensity of GP1 and GP2 to separate, and GP2 to spring into its post-fusion conformation. Through significant protein engineering efforts, we were able to identify point mutations that stabilized the GP1-GP2 interaction, provided an increased energetic barrier for the pre-to-post fusion transition of GP2 and allowed for efficient processing of the GP protomer.
Kathryn Hastie, PhD, Staff Scientist, Immunology and Microbiology, The Scripps Research Institute
Dr. Hastie studied Ecology and Environmental Biology, Molecular Biology and Biochemistry at the University of Colorado, Boulder. She then joined Erica Ollmann Saphire’s group at The Scripps Research Institute and completed her graduate studies in October, 2011. As a Staff Scientist in the Ollmann Saphire lab, Dr. Hastie conducts an independently NIH-funded research project aimed at expanding structural knowledge of glycoproteins of Lassa and other arenaviruses. In addition, she serves on international task forces to steer thought about how to better elicit and detect the right responses and to deliver a much-needed vaccine for Lassa virus, which infects hundreds of thousands of under-served in West Africa every year.
The PEGS Boston Young Scientist Keynote was launched at the 2017 PEGS to recognize a rising star in the field of protein science who is currently in a postdoc program or who has completed a postdoc in the last five years. Nominations of candidates for this role were solicited from leading industry and academic research labs in the fall of 2018, and Dr. Hastie was selected for this presentation on the basis of votes from a 15-person group of scientific advisors. Please visit the PEGS website following the 2019 meeting for details on how you can nominate a candidate for the 2020 event.