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Hybrid Tech Uses Electron Microscopy and Nextgen Sequencing to Speed Antibody Discovery

Scripps Research scientists devised an approach to help shortcut a key step in modern vaccine development. The researchers married data from high-resolution…



Scientists at Scripps Research have devised a method that may be able to shortcut one of the big steps in modern vaccine development. The researchers showed that they could apply high-resolution, low-temperature electron microscopy (cryo-EM) data to rapidly characterize antibodies – elicited by a vaccine or infection – that bind to a desired target on a virus at an atomic level. The method utilises a “structure-to-sequence” computer algorithm that can relate monoclonal antibody (mAb) structure determined by cryo-EM, to the DNA sequence that would produce that structure.

“The COVID-19 pandemic has highlighted the need for robust and rapid vaccine and antiviral technologies,” said study senior author Andrew Ward, PhD, a professor in the Department of Integrative Structural and Computational Biology at Scripps Research. “We are optimistic that our new approach will help fill that need by greatly streamlining antibody discovery.”

Ward and colleagues described their technology in Science Advances, in a paper titled “From structure to sequence: Antibody discovery using cryoEM.”

A key rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies, the authors noted. “One of the rate-limiting steps with traditional methods for antibody isolation is screening mAb libraries to identify the clones with desired epitope specificity … Comprehensive analyses of immune responses to infection or vaccination are laborious and expensive.” As study co-first author, Aleksandar Antanasijevic, PhD, pointed out, “Traditionally, identifying antibodies that are useful against a virus involves the laborious sorting and testing of antibody-producing B cells to find the right ones—a process that takes months.”

The researchers’ feat was enabled in part by recent improvements in cryo-EM, a technology that uses a beam of electrons to illuminate and image targets far below the scale of ordinary light microscopy. In a study published in Nature Communications in August, for example, the researchers used high-resolution cryo-EM to rapidly and precisely map where antibodies in rhesus macaque monkeys bind to synthetic versions of the HIV envelope protein that are being developed for potential HIV vaccines. “Recently, we developed an approach that uses cryo–electron microscopy (cryoEM) for characterization of polyclonal antibody (pAb) responses elicited by vaccination or infection (cryoEMPEM) on the level of immune sera,” they wrote.

For their new study, the investigators took this line of research a step further. They employed a “structure-to-sequence” computer algorithm that can relate antibody structure determined by cryo-EM, to the DNA sequence that would produce that structure. “In this study, we expanded the applicability of cryoEMPEM data by introducing a method for identification of functional antibody sequences from structural observations,” they wrote.

To enable this, the team assembled a library of all the antibody-encoding DNA sequences from the rhesus macaque monkeys, which could obtained by quickly bulk sequencing the genetic material from antibody-producing B-cells from the animals’ lymph nodes. Applying the algorithm to the cryo-EM data and the antibody sequence library, the scientists could reliably match an antibody of interest in their cryo-EM images to a unique antibody defined in the sequence database.

A new method developed at Scripps Research uses cryoEM technology to more quickly identify antibodies for use in vaccine development. In this example, the algorithm screened between approximately 100,000 to 1,000,000 possible antibody sequences from the database to identify the sequence (left) that best matches the antibody observed in their cryoEM images (transparent gray surface). [Scripps Research]
The researchers showed that they could confirm the accuracy of the result by making copies of a monoclonal antibody using the sequence data, and verifying with cryo-EM that the antibody bound in an identical way to the antibody that was originally imaged. “ .. we developed a method to determine mAb sequences directly from cryoEMPEM maps,” the team stated. “This hybrid approach, consisting of electron microscopy (EM) and next-generation sequencing (NGS), enabled sequence assignment of variable regions of polyclonal Fabs (Fv) including the complementarity-determining regions (CDRs) … This approach provides an alternative to traditional mAb discovery methods based on single B cell sorting, hybridoma, and phage display technologies.”


“With this new method we can go from blood sample collection from infected or immunized patients to identifying all the elicited antibodies of interest in about ten days,” stated staff scientist and study co-first author Charles Bowman, PhD.

The authors also noted that their reported proof of concept study used lymph node B cells with specificity for a particular antigen. However, they anticipate that their approach will work with B cells obtained from other sources, such as peripheral blood, spleen, bone marrow, or plasma cells, and without the presorting for antigen binding. “By directly imaging the serum antibodies using cryoEM, we have a proxy for abundance, affinity, and clonality,” they commented.

The scientists are now refining their technique to optimize its speed and usability, and are applying it to several areas: to rapidly evaluate human antibody responses to experimental HIV vaccines; to develop antibody-blocking treatments for autoimmune diseases; and to discover antibodies that could therapeutically hit other protein targets on cells.

They expect that future improvements in cryo-EM technology and structure-to-sequence algorithms will allow the even more rapid identification of antibodies using high-resolution structural images alone, with no need for DNA sequencing of B cells.

“This structure-to-sequence approach has a lot of potential in immunology and beyond,” Antanasijevic said. “We envision being able to use it someday to study protein-to-protein interactions generally, for example, to discover a given protein’s binding partners.” As the authors concluded, “ … our approach will open up new doors for both the discovery of mAbs and analyzing antibody responses to infection and vaccination. The ongoing COVID-19 pandemic has highlighted the need for such robust and rapid technologies.”

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