Unmasking Lassa virus glycoprotein: Mutations that enable antibody escape

This month’s story comes from the Bloom lab using their signature deep mutational scanning technique to identify mutations in the surface protein of Lassa virus -called glycoprotein- that escape antibody neutralization. 

Lassa virus is a potentially deadly virus causing hemorrhagic fever - similar to Ebola virus. It is endemic in parts of West Africa, where it is primarily spread by the common African rat. The virus has also caused cases in the United States and Europe. In October 2024, the first known death from Lassa virus in the United States was reported, involving a middle-aged individual in Iowa who had recently traveled to West Africa.

Humans typically become infected with Lassa virus through exposure to food or household items contaminated with the urine or feces of infected rats.

Although the Lassa virus was first identified in 1969 in Lassa, Nigeria, - hence its name- no vaccine has been developed. Due to its potential to cause a global public health emergency, Lassa virus is part of the World Health Organization’s R&D Blueprint for priority pathogens, emphasizing the urgent need for research into vaccines and treatments.

In a recent study led by Caleb Carr, a graduate student in Dr. Jesse Bloom, the team identified viral mutations in the glycoprotein of Lassa virus that can mediate antibody escape. Identifying mutations that enable antibody evasion is crucial.  The glycoprotien’s high variability poses a significant challenge, enabling the virus to evade antibodies and escape vaccine-induced immunity—a challenge seen in other viruses like SARS-CoV-2- a challenge when designing vaccines. 

To identify mutations that enable escape, researchers in the Bloom lab turned to deep mutational scanning, a high-throughput technique that examines the effects of single mutations in the glycoprotein on viral entry and antibody neutralization. Carr explained that this work uses a novel pseudovirus platform -deep mutational scanning- to safely measure the functional and antigenic effects of nearly all amino acid mutations in the Lassa virus glycoprotein. The pseudovirus strategy uses the core of a replication-defective virus to display surface proteins of the virus being studied without risking actual infection.  This approach provides critical data on the natural diversity of the virus and its implications for vaccine and therapeutic development.

Using the deep mutational scanning platform, researchers created libraries of Lassa glycoprotein-pseudotyped lentiviruses containing nearly 10,000 possible single amino acid mutations. Each viral genome was tagged with a unique barcode for sequencing and identification.

The team first examined the effects of mutations on the glycoprotein's ability to mediate viral entry into human cells. They found that unmutated glycoprotein variants and those with synonymous mutations—changes in DNA sequence that do not alter the protein’s amino acid sequence—had no effect on viral entry compared to the wild-type strain. Variants with premature codons showed impaired entry, while those with nonsynonymous mutations -changes that do alter the protein sequence- exhibited a wide range of effects, from deleterious to enhanced viral entry. 

Next, the researchers mapped mutations that allowed the glycoprotein to escape neutralization by monoclonal antibodies. By incubating the glycoprotein libraries with neutralizing monoclonal antibodies and conducting neutralization assays, they identified several mutations that conferred escape from the antibodies tested. “This finding underscores the necessity of developing a robust cocktail of antibodies to counteract viral escape,” the team explained. 

To determine if these escape mutations occur naturally, the team analyzed existing Lassa glycoprotein sequences and found instances of these mutations in samples collected from both human and rodent hosts. They validated their findings by testing glycoprotein sequences from natural Lassa virus strains containing one or more of these escape mutations. Consistent with the deep mutational scanning results, these natural variants were more resistant to the predicted antibodies.

These findings demonstrate the ability of deep mutational scanning to identify mutations in the strain of Lassa virus tested that also occur in natural virus sequences, providing valuable insight into how the virus evades immune responses.

“This highlights how the development of antibodies for use against Lassa virus should explicitly consider the potential for viral escape in order to avoid the problems that have plagued antibody-based countermeasures against other viruses like SARS-CoV-19,” the team explained. 

Looking ahead, the Bloom lab aims to extend this work to study polyclonal antibody responses generated by natural infections or vaccine candidates. Carr emphasized that understanding polyclonal responses could inform the design of therapeutics and vaccines capable of protecting against all current and future Lassa virus variants.

The spotlighted research was supported by grants from the National Cancer Institute, the Genomics & Bioinformatics Shared Resource of the Fred Hutch/University of Washington Cancer, he  Flow Cytometry Shared Resource of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium and the Fred Hutch Scientific Computing

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Jesse Bloom and Neil King contributed to this work. 

Carr CR, Crawford KHD, Murphy M, Galloway JG, Haddox HK, Matsen FA 4th, Andersen KG, King NP, Bloom JD. Deep mutational scanning reveals functional constraints and antigenic variability of Lassa virus glycoprotein complex. Immunity. 2024.