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Tuberculosis Assay in Lophius' Pipeline as Immune-Response CMV Test Gains Traction

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NEW YORK (360Dx) – When a virus or bacteria lies dormant in the body, diagnostic tests to detect pathogens in patient samples will falter. Lophius Biosciences, a Regensburg, Germany-based diagnostics maker, has now developed a technology to interrogate the host immune system in vitro using biomarkers produced when a pathogen antigen is used to stimulate immune cells in a patient's blood sample, indicating a past active infection.

With its latent cytomegalovirus test for the transplant market gaining traction, the firm is now applying similar technology to a tuberculosis assay in the works.

"Our core competence is interrogating the immune system," Lophius' CEO Bernd Merkl said in an interview.

Lophius' CMV product, a kit called T-Track CMV, has been CE marked and marketed for about two years, Merkl said. It measures cell-mediated immunity and is being used to stratify the risk of rejection among recipients of solid organ and bone marrow transplants.

The CMV virus is present in anywhere between 30 percent and 90 percent of adults in populations worldwide, but "as long as you're healthy nothing happens, because the immune system keeps the virus under control," Merkl said. However, in immune-compromised or immunosuppressed people, such as patients with HIV or who have received transplants, CMV can develop into a serious, and occasionally life-threatening, infection, and this can be related to CMV present in the patient but also in the donor tissue.

As described in a 2017 BMC Immunology report, the Lophius CMV test involves in vitro stimulation of mononuclear cells in peripheral blood samples using two CMV-specific immunogenic proteins, called T-activated IE-1 and T-activated pp65.

Following stimulation, the biomarkers indicating activation of T cells in the patient sample are detected with ELISpot technology.

Essentially, the method tells physicians about how active the immune system is vis a vis the virus. Since the memory T cells of the immune system would only be able to be activated if the patient had previously been infected, the test tells physicians that a patient has a latent infection lurking somewhere that could be reactivated upon immunosuppression. In the case where a donor had a CMV infection and the recipient did not, the test can also tell physicians whether the recipient is mounting an immune response to the donor CMV.

In combination with determining viral load of CMV itself using standard qPCR to see how active the virus is, the method can help physicians make better decisions when to start, resume, or stop antiviral therapy, Merkl said.

The test has been incorporated into international guidelines for solid organ transplantation and is currently used in Europe to manage therapy or prophylaxis for special patient cases, Merkl said.

The test has been validated in many areas, such as for hemodialysis patients hoping to get kidney transplants, for monitoring cell-mediated immunity in kidney transplant patients, and for informing clinicians if a kidney transplant recipient might be protected from CMV reactivation. A study in South Korea, which has a CMV seropositivity rate among adults of greater than 95 percent, found that a pre-transplant test of a kidney donor could accurately predict if a recipient was at moderate risk of developing a post-transplant CMV infection.

The test has also been used for hematopoetic stem cell transplant patients, as described in the Journal of Clinical Virology, as well in pneumonia patients.

Furthermore, the firm recently announced preliminary results of a prospective, longitudinal, observational, multicenter CMV study, called AlloProtect. In the study, hematopoetic stem cell transplant recipients were followed post-transplantation for CMV reactivation. Patients with a positive T-Track CMV test after resolution of the first CMV reactivation, as well as at day 100 post-transplantation, remained free from future recurrent CMV reactivation. The firm said the study demonstrated that T-Track CMV allows an improved risk stratification of CMV-related clinical complications.

"If you look at bone marrow transplant patients, the immune system is basically transplanted from the donor to the recipient, so the immune system has to develop," Merkl explained. And there are also high-risk groups, such as donors who are CMV seronegative, and for whom the CMV immunity needs to build up. "For doctors, it is important to understand if the CMV immunity is already there, or not, and this is what our test in a bone marrow transplant setting would tell you," Merkl said.

The test could also potentially be used as a sort of companion diagnostic. There is a new drug from Merck called Prevymis that has market approval to help prevent CMV infection in bone marrow transplant patients for the first 100 days, for example. "There is interest from certain clinicians to also use our test in their investigator-initiated trials, and it could be something like a companion diagnostic, meaning it would help the doctor, together with viral load measurements, to make better decisions when to start, resume, or stop antiviral therapy," Merkl said.

Martina Koch, who heads the visceral organ transplantation and transplantation immunology divisions at the University Medical Center of the Johannes Gutenberg University Mainz is using the T-Track CMV with her solid organ transplant (SOT) patients.

Koch explained that the Lophius T-Track assay shows not only the patient's immune status against CMV but also the general immune reactivity if the patient carries a CMV infection. For this purpose, a donor-specific test would theoretically be more reliable, "but donor material is not always available, and tests with donor material are more complicated," Koch said in an email.

On the other hand, the T-Track test is "easy to use," and Koch said she also uses it as a measurement for over- or under-immunosuppression in SOT patients.

"As long as an adequate reactivity against CMV is detectable, the patient might be able to cope with other infections, too," she noted, adding that "tests like this might help to tailor immunosuppressive therapies in SOT."

Other tests that measure CMV-specific cell-mediated immunity include ones from Qiagen and Oxford Immunotec, Merkl said.

TB testing

In addition to the ELISpot-based CMV test, the firm is also working on other projects, some of which will use PCR-based detection methods.

For latent tuberculosis, "You can't detect the bacteria because it is hiding somewhere in the body, but you can detect the immune cells by giving the blood a stimulus with an antigen, and then the immune cells produce biomarkers and you can measure these biomarkers with RT-qPCR," Merkl said. In this case, the biomarkers would be transcripts induced by T-cell activation.

The company is now developing a next-generation infection screening test for TB, and is working on a differential diagnosis test that will be able to distinguish a latent infection and a treatment-requiring active infection, he said. 

Importantly, these TB tests are blood-based, not sputum-based, and they are multimarker tests.

Other latent TB tests on the market include one from Qiagen, which recently received approval in Canada. Qiagen is also developing a handheld reader for its QuantiFeron-TB Gold Plus assay for latent TB detection, and recently began a collaboration with DiaSorin to port its latent TB test to that firm's automated Liaison instrument that has resulted in CE marking of the workflow.

To date, Lophius has no funding from organizations like the Foundation for Innovative New Diagnostics or the Gates Foundation, but Merkl said the company has had conversations with FIND, and that the company is represented in tech development roadmaps that FIND has presented.

In addition to Lophius' technology, according to a recent FIND presentation, non-sputum-based tests in development for latent or incipient TB include the QIA-Predict and QIA-TB Signature assays from Qiagen, the Incipient TB Assay from Abbott, and the T-cell Immune Profiling test from Becton Dickinson. And researchers at Stanford University and the Center for Infectious Disease Research in Seattle are also developing assays for mRNA Signatures of latent TB. 

For its next-generation infection detection screening test, Lophius is currently in the optimization phase, Merkl said, and the company plans to start the clinical trial for market approval during the next year. For the TB differential diagnosis test, Lophius is still in the marker evaluation phase, but expects to bring an assay to clinical trials in 2020.