Finding the answers hidden in our antibodies

Jason Ladner, an assistant professor in the Department of Biological Sciences and the Pathogen & Microbiome Institute (PMI), was a leading innovator on PepSeq, a technology that allows scientists to test antibody binding against hundreds of thousands of protein targets at one time, instead of testing one at a time. When we're trying to track a contagious virus like coronavirus and figure out how to respond to it, getting answers quickly can be the answer between life and death.

This protocol is laid out in detail in an article published earlier in November in Nature Protocols.

"PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries" describes the novel approach for conducting highly multiplexed serology assays and details the team's approach for designing and synthesizing custom PepSeq libraries, as well as how to use these libraries to conduct assays and interpret the data. The hope, Ladner said, is to provide a roadmap for scientists everywhere to use the protocol in their own research, moving us closer to answers on some major infectious diseases.

It's an important step forward as concerns about bioterrorism, zoonotic diseases and the next pandemic are never far away and understanding these pathogens will help enable scientists to develop vaccines as well as tracking their movement and evolution.

"Serology assays are important for diagnosing many human and animal infections," he said. However, traditional assays often lack sensitivity and/or specificity. Our approach can help to identify which proteins most commonly stimulate an antibody response during infection and which epitopes are specific for the pathogen of interest, rather than being cross-reactive across related pathogens."

What is PepSeq?

At its most basic, the protocol allows scientists to test how protein targets, or antigens, bind to antibodies hundreds of thousands at a time. Antigens, which are pieces of a pathogen -- like a virus or a bacteria -- are the targets of antibodies in the host's blood. New antibodies are produced in response to each new infection, these antibodies are highly specific for particular antigens and they can persist for months or even years. Therefore, antibodies help to protect from future infections with similar pathogens, but they also provide a record of past exposures.

One of the big questions on the medical side, then, is which antibodies provide protection against infection. This is where humanity found itself in 2020 -- not knowing which were the right antibodies that the body needed to develop to respond to COVID-19. That information was needed to create an effective vaccine and, in the future, could help develop vaccination approaches that would broadly protect against coronaviruses like SARS-CoV-2, including those that we have yet to identify.

The big question on the medical side, then, is identifying which antibodies are needed to attack a pathogen's antigens. This is where humanity found itself in 2020 -- not knowing which were the right antibodies that the body needed to develop to respond to COVID-19. That information was needed to create an effective vaccine.

PepSeq, which was initially developed by a team that included John Altin, now a researcher at TGen, Ladner's collaborator and a co-author on the study, contributes to this process in two key ways. First, it allows scientists to simultaneously assess antibody binding across a large number of different antigens. Because antibodies are evidence of a host's past infections, knowing what antibodies a person has can offer clues of what pathogens they have been exposed to in the past. This assay allows researchers to broadly explore exposure history by testing for evidence of past infections by all viruses known to infect humans.

"This can help us to better understand the epidemiology of infectious diseases, and it is also empowering us in our search for potential viral triggers for non-infectious diseases like diabetes and celiac disease," Ladner said.

Second, the antigens used in PepSeq are short peptides, which means they are typically no more than 64 amino acids in length. That means that each time an antibody binds to a PepSeq antigen, the signal is specific to a particular region of a protein, which then allows Ladner and his team to dissect the antibody response in minute detail. It helps suss out which portions of a pathogen are most commonly targeted by antibodies and which antibodies are specific to particular pathogens, like the novel coronavirus that causes COVID-19, and which cross-recognize -- and potentially cross-protect against -- closely related pathogens, like the coronaviruses that can cause the common cold.

"In particular, we've been very interested in a couple of epitopes that are highly conserved between SARS-CoV-2 and 'endemic' human coronaviruses that have been infecting humans for decades and generally just cause a common cold," he said. "We've found that the dynamics of the antibody response to these epitopes are distinct, and responses directed against these regions could provide broad protection against coronaviruses."

The third key factor overarching every assay: PepSeq allows these tests to be run quickly and with a much smaller sample volume than other assays -- important because often these samples are precious and in short supply.

What happens now?

This study provides the information needed for any researcher to use PepSeq. It also provides their rationale for the decisions they made in the process so others can build on the technology. As innovative as this protocol is, the researchers' goal is for others to innovate even further. Ladner's team is leading the development of bioinformatics protocols and custom software packages that will create the PepSeq libraries and allow for interpretation of data collected through the protocol.

From a big-picture standpoint, having these kinds of conversations helps the field as a whole progress, Ladner said. It helps develop trust in the process and the results.

"Even if no one outside of PMI and TGen end up using PepSeq, we want the broader scientific community to trust that the results are reliable, and the best way to do that is through transparency about the process," he said. "In this type of protocol manuscript, we can go into much greater detail about the methods than we can in a manuscript focused on the results of a particular research project that utilizes PepSeq."

Ladner also is refining a protocol for new sample types, including human saliva and urine and blood-fed mosquitoes. He's also using PepSeq to better understand the distribution and ecology of viruses that affect other animals but not humans (yet). This will allow his team to explore antibody reactivity in other animals, like bats, which will demonstrate what types of viruses are commonly infecting these reservoir species.

Although most of this research is aimed at questions in the fact-finding part of the process, it should lay a foundation that leads to a more effective response during future outbreaks as well as vaccines and therapeutic treatments for viral illnesses. Ladner's team has funding from the U.S. Department of Defense's Defense Threat Reduction Agency to use PepSeq to better understand the advantages and disadvantages of different vaccine delivery platforms.

"One of the main goals for vaccination is to stimulate the production of protective antibody responses, and PepSeq can help us to understand the antibody response to vaccination," Ladner said. "The results from PepSeq assays could certainly help to inform strategies and approaches that are more directly linked to outbreak preparation and response."