A new study from scientists at La Jolla Institute for Immunology (LJI) has given researchers a guide to neutralizing the deadly Lassa virus (LSV) using a trio of rare antibodies isolated from survivors of Lassa virus infection. The LJI scientists showed exactly how a cocktail of three human antibodies can block viral infection, which they say may prove valuable in upcoming clinical trials for Lassa therapies. “We now know where these three therapeutic antibodies act and how exactly they act,” said Kathryn Hastie, PhD, an LJI instructor and director of the Antibody Discovery Center at LJI.

The team plans to use their new map of the Lassa virus surface glycoprotein to design a much-needed vaccine. Hastie and colleagues reported on their work in Science Translational Medicine, in a paper titled, “A cocktail of protective antibodies subverts the dense glycan shield of Lassa virus,” in which they conclude: “ … these results explain protective mechanisms of rare, broad, and potent antibodies and identify a strategy for the rational design of therapeutic modalities against LF and related infectious diseases.” The research was led by the Saphire Lab at LJI, including instructor Haoyang Li, PhD, Hastie, and professor Erica Ollmann Saphire, PhD, in collaboration with Luis Branco, PhD, of Zalgen Labs.

Lassa virus is a deadly virus endemic to West Africa, where it is primarily spread by rodents. The virus causes Lassa fever (LF), a disease that strikes up to 300,000 individuals each year. The infection typically presents with flu-like symptoms at the start, but can lead to severe illness, long-lasting symptoms, such as deafness, and even death. Lassa virus is particularly dangerous for pregnant women: 90% of infections during pregnancy are fatal.

In 2017, Hastie and her colleagues in the Saphire Lab (then at Scripps Research) published the first-ever structural images of the Lassa virus glycoprotein. Lassa uses glycoproteins to enter host cells and initiate infection. Hastie’s glycoprotein structure gave researchers an idea of what they were up against.

Hastie’s breakthrough came as researchers hunted for the rare human antibodies that could break through Lassa’s defenses. The hope was for researchers to harness these neutralizing antibodies to develop Lassa fever therapeutics or vaccines. This was achieved when research partners at Tulane University and Zalgen Labs isolated a promising group of Lassa-fighting antibodies from the blood of Lassa fever survivors. Collaborators from the University of Texas Medical Branch went on to test a cocktail of three neutralizing antibodies in nonhuman primates. This antibody therapy, called Arevirumab-3, proved 100% effective in treating Lassa fever, even in animals with advanced disease. “The antibody cocktail comprising these three antibodies, termed Arevirumab-3, provides complete protection in nonhuman primates, even when delivered in late-stage LF (8 days after infection with death typically occurring on day 11),” they stated. “This was a groundbreaking finding,” added Saphire. “The dogma had been that antibodies would not be protective against Lassa virus.”

However, the FDA was not prepared to launch clinical trials until the researchers could uncover the mechanism that made the therapy so effective. Exactly how did these neutralizing antibodies target Lassa virus glycoprotein and prevent infection? As the authors acknowledged, “Although the protection efficacy of Arevirumab-3 has been confirmed in nonhuman primates, details of epitopes and protective mechanisms of these antibodies had remained unknown, which has hindered clinical application.”

To answer this question, researchers needed a more detailed map of Lassa glycoprotein. Hastie’s original structure of the glycoprotein required complicated molecular engineering to provide sufficient stability for imaging. Her structure gave scientists a critical glimpse of Lassa glycoprotein, but not the full picture. Plus, some of the promising therapeutic antibodies could not recognize this or any version of engineered Lassa glycoprotein. Researchers needed to isolate a natural glycoprotein target for further investigation.

Fortunately, the Saphire Lab had the tools and expertise to reveal these molecular details. Li led the effort to produce a “native” Lassa glycoprotein. Thanks to advances in protein production and three years of perseverance, Li’s version of the glycoprotein was a copy of the real thing and could be recognized by all three antibodies used in Arevirumab-3. Li then used a technique called cryo-electron microscopy single-particle analysis to image the native glycoprotein together with the three antibodies. “A thorough understanding of the structures, GPC recognition, and neutralizing mechanisms of these protective mAbs will explain the action of this first-in-class antibody-based therapeutic candidate and may provide additional insights into vaccine design,” the scientists pointed out. “Here, we describe biochemical analyses and high-resolution cryo-electron microscopy structures of a therapeutic cocktail of three broadly protective antibodies that target the LASV glycoprotein complex (GPC), previously identified from survivors of multiple LASV infections.”

Hastie noted, “Haoyang’s ingenuity and hard work enabled us to see the structures we couldn’t see before. Based on the high-resolution structures and several functional assays, the team revealed exactly how the three antibodies used in Arevirumab-3 neutralize Lassa virus.

Hastie was amazed to see how antibody 8.9F binds to the very top of the glycoprotein structure. This area of the glycoprotein is where three molecules (called protomers) come together to form a “trimer,” a kind of twisted trefoil, as Hastie describes it. Lassa would normally use this region of the glycoprotein to bind with receptors on host cells, but Li’s structure shows how a single 8.9F jumps in and binds to all three protomers simultaneously to block infection.

“The structure is really a beautiful illumination of how this antibody essentially mimics the host receptor to block the glycoprotein receptor from binding,” commented Hastie. “It’s an absolutely gorgeous structure to see.”

Meanwhile, the neutralizing antibody called 12.1F binds to just one protomer in the three-sided trimer. Fortunately, any therapeutic would have many copies of 12.1F. Moving as a team of three, each 12.1F antibody can bind to a protomer to aid in neutralizing the virus.

At the same time, copies of antibody 37.2D target Lassa virus by binding in a way that anchors adjacent protomers together. This antibody activity is a huge problem for Lassa, since the virus needs to open up its trimer (where the protomers come together) to infect host cells. With 37.2D on the scene, its entry machinery is locked up, unable to function. “Lassa has another trick,” Saphire commented “It shields itself using a thick coat of human carbohydrate molecules—like a wolf in sheep’s clothing. Haoyang’s structures clearly show how these potent, protective antibodies breach or even utilize the carbohydrates to target and neutralize the virus.”

“The structures reveal how the antibodies breach or selectively use GPC glycans and collaboratively inhibit virus entry,” the investigators noted. “Structural and mechanistic analyses reveal compatible neutralizing epitopes and complementary neutralization mechanisms that offer high potency, broad range, and resistance to escape,” the investigators explained.

“The findings fill a critical gap in Lassa virus research and may pave the way for Arevirumab-3 to move into clinical trials,” said Branco, who will lead the Zalgen team to perform future clinical trials. “Furthermore, results from complementary biochemical assays, monoclonal antibody-resistant mutant (MARM) analysis, and protein engineering studies elucidate the protective mechanisms of this preclinical therapeutic cocktail and provide guidelines for LASV antigen design,” the team noted in the published paper. “This study, together with our previous LASV challenge studies in animal models, demonstrates an opportunity for therapeutic treatment of LF, a WHO and CEPI priority infectious disease.”

 The study results give the researchers a guide to better targeting three Lassa epitopes, or weak spots. Two of these critical epitopes had never been mapped before. In fact, just this year, the Saphire Lab published three articles on anti-Lassa neutralizing antibodies (including this paper). The other two investigations were published in Cell Reports and mBio.

“This body of work now offers the first-ever full-epitope map, revealing every vulnerable target of the Lassa glycoprotein,” said Saphire. “We now have a very clear idea about the neutralizing epitope surface and the requirements needed on the glycoprotein for anybody binding and recognition,” added Hastie.

Li and Hastie are using the new glycoprotein epitope map to guide vaccine design. They hope a future vaccine could prompt those at risk to make neutralizing antibodies on their own.