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A novel immunity-based test for early diagnosis of Lyme disease

Periodic Reporting for period 4 - ID-Lyme (A novel immunity-based test for early diagnosis of Lyme disease)

Reporting period: 2019-07-01 to 2020-12-31

The ID-Lyme project was initiated in 2016 to develop a novel diagnostic test for the early detection of Lyme disease (Lyme borreliosis, LB). LB is an infectious disease caused by Borrelia bacteria that are transmitted by ticks. LB can be treated using cost-effective antibiotics, at least when discovered at an early stage of disease. Left untreated, patients may suffer from serious long-term effects, including paralysis, damage to joints, heart, and nervous system. Due to the difficulty to diagnose LB, in many cases damage has already occurred before the underlying cause is identified.
Standard laboratory testing is often unable to give a clear answer to whether a patient has been infected or not, and whether the bacterium is still alive. The result is that a true infection may remain untreated, which is a significant health concern. In later stages, diagnosis becomes more difficult and treatment more expensive and less effective. Inadequately treated LB is a significant health and economic burden for society.
With ID-Lyme, the EU funded a project that aimed to deliver to the market a diagnostic test based on cell-mediated immunity (CMI) that can identify LB infections in the early stage prior to the onset of symptoms. This would enable focussed LB management, improve patient health outcomes and reduce LB-related healthcare costs. A better LB test would have significant impact on EU health care and society, as currently over 2.6 million tests per year are performed in the EU on people suspected of a Borrelia infection.
CMI-tests consist of two steps: A stimulation step where immune cells are brought in contact with pathogen proteins (antigens), and a read-out step where small proteins released by immune cells (called cytokines) are measured. The challenge of a CMI-test is that it has to be performed within 12 hr from blood drawing to ensure immune cells remain viable. This is a logistic challenge and makes such tests expensive and unreliable if timelines and transport conditions are not met. To overcome this challenge, the ID-Lyme test, and any other CMI-based test, should be performed close to the point of blood drawing. For the new test, we envisaged a Point of Care (PoC) device that can be sent out to the end-user.
During the first year, the team discussed pros and cons of PoC Lyme tests with potential end-users. The conclusion was that a true PoC test would not be needed, but that at least the first step should be close to point of blood drawing. We performed a Health Technology Assessment to quantify the economic burden and benefits for patients and society and looked at reimbursement schemes in The Netherlands, Sweden, Germany and Austria.
Technically, a vast amount of work was done to design and develop a device (named WOLF) that would enable performing the stimulation step of the test at the point of blood drawing and thus help overcome the logistic hurdles for CMI tests. We further investigated the optimal Borrelia antigen mixture to be used for stimulation step and assessed stability and the storage conditions. Antigen production at DIARECT was optimized to enable kit manufacturing once all components of the kit are ready. For read-out of the relevant CMI biomarkers (cytokines), the consortium compared sensitivity and cost of three methods, in order to make a sensitive test affordable to many people.
In the second year, the team came to the conclusion that despite extensive troubleshooting efforts at Innatoss, the Lyme CMI-test was not fit to distinguish between infected and non-infected individuals. The same was found upon testing other marketed LB CMI-tests for LB . Therefore, efforts to develop a novel Lyme-CMI test were abandoned. This was important information for the Lyme community since CMI-tests for Lyme are often claimed to outperform the classical serological tests.
The WOLF device developed in period 1, however, was considered to be an immensely useful tool to open up CMI-based diagnostics in any disease context. Therefore, the team continued this effort. The devices were manufactured in China, directed by npk design. A patent application was filed in 2017 and is under review.
In the 3rd period, Innatoss completed development of two sensitive, enzyme-linked immunosorbent assays (ELISA) for measuring cytokines to be used in the second, read-out step of CMI-tests. All relevant parameters of assay performance, including stability, precision, interference, and sensitivity were validated. Various redesigns of the WOLF devices were produced and tested. Risk analyses were performed and release procedures written. The team also explored other applications of both the ELISAs and the WOLF device.
During the final sprint towards the end of the project, COVID-19 changed the world around us. The COVID-19 pandemic thwarted any planning, first in China, and later in Europe. Production of devices in China was delayed, clinical studies became virtually impossible, due to the fact that hospitals closed and health care professionals were hard to find. As a team, we dealt in a positive manner with the continuing changes in planning. The Commission facilitated it by extending the program.
Our manufacturer in China re-opened its facilities quickly, continuing with required changes. The validation lots of the ELISAs were produced just before the world came to a halt, and testing performance continued during the pandemic. User reviews and stakeholder meetings could not be organized, but the team creatively got the feedback it needed: A group of medical students joined Innatoss for COVID-19 testing. Being future users, they were asked to provide feedback on the use of the devices.
In Vienna, the application of WOLF for Chlamydia was slowed down due to closure of the out-patient clinic. Nevertheless, the team made substantial progress in generating materials for a future test.
In the process of designing a CMI-test for Lyme borreliosis, the team developed the WOLF device that will be key in overcoming the logistic hurdles of CMI-tests in any disease setting. The device can be applied both in human and veterinary diagnostics, including tuberculosis, Q-fever and Chlamydia. In parallel, the team developed sensitive cytokine ELISAs to be used as the read-out of such CMI-tests.
Even though the COVID-19 pandemic created serious delays to the project, it also brought a new CMI-test application: Assessing cellular immunity towards the new coronavirus SARS-CoV-2 could make a difference in understanding protection from the virus following either infection or vaccination.

The ID-Lyme consortium designed and developed an innovative device, WOLF, that will make CMI testing accessible to the health care community.

WOLF is an easy to use ‘split and prime’ test kit for point-of-care blood stimulation that will:
• Limit logistic hurdles
• Reduce time and labour costs
• Facilitate testing at remote locations
• Avoid high local investments

Most importantly, the WOLF device will stimulate development of novel CMI-based tests.

Besides scientific output, the program forged close collaborations between partners that were previously not aware of each other. DIARECT (now BBI solutions) and the Medical University of Vienna worked together on tests for B. miyamotoi, and Innatoss and DIARECT started to investigate the use of tick-borne disease arrays for serological diagnosis. All partners envision future collaborations for new products.
The WOLF device: Key to implementation of cell-based immunity diagnostics