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Abbott Uses Pegivirus Discovery to Increase Human Virome Knowledge, Update Research Assay


NEW YORK (GenomeWeb) – Despite the discoveries, technological advances, and sheer amount of data researchers have generated on human biology in the past decades, the virome remains somewhat shrouded in mystery. New viruses are discovered regularly, but we have yet to understand what purpose many of them serve in the human body or whether they are pathogenic.

That's where the University of California, San Francisco-Abbott Viral Diagnostics and Discovery Center comes in.

In December, a team from the VDDC identified a new human pegivirus. As they reported in PLOS Pathogens at the time, the researchers did metagenomic sequencing on blood samples from more than 150 individuals with chronic liver disease, identifying a handful of reads corresponding to human pegivirus 2, or HPgV-2, in an individual who had been infected with hepatitis C virus and died from sepsis. They then used new diagnostic assays aimed at the virus to screen thousands of additional blood samples, turning up 11 more HPgV-2-postive samples, all from individuals with HCV.

Now, thanks to what the researchers uncovered about the existence of this new pegivirus and how to find it in human samples, Abbott has been able to redesign its assay to better detect HPgV-2, in the hopes that learning more about the virus will lead to a greater understanding of its pathogenic potential in humans.

"We recognized that only a subset of the HPgV-2 RNA positive samples were also antibody positive," John Hackett Jr., divisional vice president for applied research and technology in diagnostics at Abbott, told GenomeWeb in email. "Data published in our studies and [others] suggested that HPgV-2 was capable of establishing a chronic infection. It would be surprising if HPgV-2 remained in the blood stream for months but did not trigger an immune response. We wondered if we weren't detecting everyone with an antibody response, and if better tools (tests) could do this."

Abbott presented a poster at the European Association for the Study of the Liver's 2016 International Liver Congress in April showing that some people infected with HPgV-2 were making antibodies to peptides derived from the virus's amino acid sequences. The researchers thought they could find a biomarker that could be used to detect antibodies to the virus, so they made some recombinant proteins that they believed would be more useful in detecting antibodies. The recombinant proteins were also derived from the pegivirus amino acid sequence but included many more of the sequences than the small peptides, the poster noted. Whereas the peptides most likely represented antibody binding sites to small linear amino acid stretches, the larger recombinants represented more complex structures that can bind antibodies.

One recombinant found to have value extended the region covered by one of the peptides in the original study. The second recombinant covered an envelope glycoprotein, E2, which is found on the surface of the virus and is mostly likely exposed to the infected individual's immune system, according to Abbott.

"The original antibody screening assay utilized peptides as the antigen to detect antibodies in the blood. We tested many different peptides spanning the HPgV-2 genome and identified three having value as a diagnostic for HPgV-2 infection," Hackett said. "These three peptides gave us an indicator of areas of the HPgV-2 polyprotein which had potential to be antigenic. In the new assay we used recombinant proteins which span a much larger area of the polyprotein. We extended one of the regions of interest from the peptide study."

This discovery also highlights some of the challenges underlying serology, he added. The VDDC is constantly trying to identify new viruses in order to ascertain whether and how they may threaten human health. Part of that mission involves the constant development and refining of tools to aid in this detection.

"Although our original peptide-based tests could identify some individuals with HPgV-2 infection, we reasoned that longer polypeptides would enhance sensitivity of detection as they contain more potential antigenic regions and may display theses epitopes in a manner more similar to the native virus," Hackett said.

People can make antibodies to both linear or conformational epitopes, he added. Peptides are most likely representative of linear epitopes while recombinant proteins represent both linear and complex structures. Those complex structures contain conformational epitopes.  And while the original assay detected antibodies directed against linear epitopes, it likely missed antibodies to conformational epitopes.

In the second assay, the researchers expressed an envelope glycoprotein from HPgV-2 in a human cell line. "This protein will be expressed on the surface of the virus during infection and most likely plays a role in entry of HPgV-2 into a target cell where the virus can replicate," Hackett noted. "Based on experience with other flaviviruses, envelope proteins are usually antigenic. Expression of this protein in human cells should mimic as closely as possible the folding this protein would undergo during infection. We wanted to have an antigen that was as close to the state it would be in during infection, when antibodies would be made."

And though some of the discoveries and improvements made here are specific to this pegivirus, Hackett said the general knowledge gained from this experience could inform other discovery efforts as well. More importantly, it shows the depth of knowledge there is left to plumb about the human virome.

It starts with identifying and characterizing a novel virus, and then using that information to develop tracking tools. "Design of tests to screen for the virus is an iterative process," Hackett said. "As we increase our understanding of the biology of the virus, the nature of the immune response, and epitopes that trigger an antibody response we can improve the tools to detect infection. With better tools we can more accurately determine how many people HPgV-2 has infected by looking at the serologic fingerprint of the virus."

Both iterations of the VDDC assay, the company added, are prototypes that are intended for early-phase research studies. The decision of whether to develop a formal assay and launch it commercially would be based on its clinical utility and need.