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Institute for Systems Biology Using NIH Grant to Develop Peptide Dx for Lyme Disease

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NEW YORK (GenomeWeb) – Researchers at the Institute for Systems Biology are the latest group taking a proteomics approach to the early detection of Lyme disease.

The work is being led by the ISB's Robert Moritz, whose lab this year received $293,521 for the first year of a two-year, approximately $525,000 grant from the National Institutes of Allergy and Infectious Diseases to investigate peptide biomarkers for Lyme disease. Specifically, Moritz's team is using mass spectrometry to look in patient blood samples for peptides shed by Borrelia burgdorferi, the bacteria that causes the disease.

Collaborators on the project include Klemen Strle, an assistant professor at Harvard Medical School, Melissa Caimano, an associate professor at UConn Health, and Andrea Varela-Stokes, an associate professor at Mississippi State University.

Lyme disease can be challenging to diagnose. Serological assays that measure a patient's antibody response to infection are most commonly used, but these tests can often give false negatives or inconclusive results, particularly early in the disease when a patient's immune response may not be large enough to be detected.

There are also PCR-based tests that detect B. burgdorferi nucleic acids directly, but, due to the small concentration of B. burgdorferi in patient blood, these tests are often not sensitive enough to detect the disease. Moritz also noted that false positive results have been a problem for these assays.

The ISB researchers and their collaborators are approaching the challenge from the protein angle, looking for peptides shed by B. burgdorferi during the acute phase of infection that they can use to diagnose the disease.

This approach comes with challenges of its own, however, Moritz said. Perhaps most notable is the high level of genetic variability across different B. burgdorferi strains. This means the researchers must be careful to choose peptides that are conserved across these different strains.

Moritz offered the example of the protein OspC, which has been a molecule of interest for researchers looking to develop Lyme tests and vaccines.

"The problem is the whole protein is highly variable across strains," he said. "So that means that while you could make an assay [based on detecting OspC] to one strain, it wouldn't pick up many other strains."

Another challenge is developing assays sensitive enough to detect peptides shed by B. burgdorferi, though Moritz said that previous work his group has done in tuberculosis gives him confidence that this will be possible.

"We believe we have the sensitivity to do it because we did it with TB," he said, noting a project in which ISB researchers used mass spec to detect TB peptides shed into plasma and urine.

The researchers are using selected reaction monitoring (SRM) mass spec to develop a peptide panel that can be used for diagnosing Lyme disease. Assuming that effort is successful, they would then develop immunoassays to the peptides of interest, which would enable for easier, more widespread clinical implementation.

Moritz was one of the leaders of the ISB's Human SRMAtlas project, which developed SRM mass spec assays to the human proteome, allowing for quantitation of the 20,123 of the 20,203 annotated human proteins in the UniProtKB/Swiss-Prot database as well as several thousand protein isoforms and post-translational modifications.

He and his colleagues are taking a similar approach to the Lyme disease project, developing an SRMAtlas to B. burgdorferi that will contain targeted mass spec assays to the full set of the bacteria's proteins. Like the Human SRMAtlas, the B. burgdorferi atlas will be publicly available, allowing other research groups to use the assays in their work, Moritz said.

To build the atlas, the researchers are analyzing B. burgdorferi taken from infected patients, which Moritz said is necessary to capture proteins key to the organism's infectious qualities.

"We need to understand the variation not only within the chromosome but also the plasmids [carried by B. burgdorferi]," he said. "Because the plasmids harbor some of the proteins that maintain [the bacteria] in an infectious state."

"When you grow them in the lab, the bacteria tend to toss off some of those plasmids, and then they are no longer as infectious," he added. "So there is always a question about which bacteria are really working [as they would in an infected host]."

Moritz said his lab is also working with Varela-Stokes to look at how the interaction between B. burgdorferi and its vector, ticks, affect its proteome.

He said he and his colleagues have identified an initial list of high priority B. burgdorferi peptides that they are now testing whether they can detect in patient blood samples.

Given the poor performance of existing Lyme disease diagnostics, a number of research groups and commercial firms are exploring tests for the condition.

One such outfit is Manassas, Virginia-based Ceres Nanosciences, which, like the ISB team, is focused on detection of B. burgdorferi proteins. Ceres' diagnostic, which the company offers as a CLIA test, uses its nanotrap technology to enrich the B. burgdorferi protein OspA in patient urine, which is then measured using an immunoassay.

In a 2015 paper in the Journal of Translational Medicine, the company and affiliated researchers looked at 24 patients with a clinical diagnosis of Lyme disease, 12 of which had positive serology, five of which had negative serology, three of which had indeterminate  serology, and four of which did not have serology tested. All 24 of these patients tested positive for Lyme disease using the Ceres test.

They also looked at 120 patients without Lyme disease, three of whom were symptomatic and 117 of whom were asymptomatic. All 120 tested negative using the Ceres test.

The test this summer received Breakthrough Device designation from the US Food and Drug Administration, which could speed its progress through the regulatory process.

In April, Finnish diagnostics firm Reagena launched a point-of-care test for Lyme disease that detects the chemokine CXCL13, which is linked to Lyme infection, in cerebrospinal fluid.

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