New Tools for Diagnosing Post-Acute Infection Syndromes

Millions of people worldwide continue to experience lingering health problems long after recovering from an initial infection—a group of conditions now known as post-acute infection syndromes (PAISs), which include long COVID, post-treatment Lyme disease syndrome, and post-dengue fatigue syndrome, among many others. These syndromes can affect various parts of the body and cause persistent, often disabling symptoms that disrupt daily life. Despite their growing prevalence, PAISs are frequently misunderstood and underdiagnosed, leaving many patients without clear answers or effective treatment options.

In this Innovation Spotlight, Nigel McCracken, chief operating officer at Virax Biolabs, explores the science behind PAISs, focusing on immune system dysfunction. He also introduces the emerging ViraxImmune™ platform, which aims to bring much-needed clarity to this complex medical landscape by measuring T cell exhaustion markers in response to infection.

What are PAISs and what do individuals with these conditions typically experience?

PAISs are a range of complex conditions that manifest after an acute infection. They may involve clinical signs and symptoms that vary in severity and may persist for weeks, months, or years and encompass a number of organ systems. Common general symptoms include fatigue, pain, and sleep disturbance; however, symptoms may affect multiple systems including neurological, musculoskeletal, gastrointestinal, and respiratory systems.

Various bacterial and viral infections are possible triggers for post-acute infection syndrome, including those caused by SARS-CoV-2, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV), enteroviruses, human papillomavirus (HPV), Borrelia burgdorferi, and Zika virus among others.

PAIS diagnosis can be very difficult, with affected individuals often neglected by healthcare systems when a definitive diagnosis is not made and a management plan is not implemented. An estimated 100 million individuals are currently affected worldwide, with an annual incidence projected at 1–2 percent of the population due to ongoing exposures to infections and vaccines. Despite their prevalence, no early diagnostic tools exist.

What are some common biological hallmarks for these disorders?

PAISs are often associated with immune system dysfunction, notably progressive T cell exhaustion marked by diminished effector activity, reduced antigen killing, upregulation of exhaustion markers, metabolic reprogramming, mitochondrial dysfunction, and increased oxidative stress. Without proper intervention, T cell dysfunction may progress to terminal exhaustion.

Chronic antigenic stimulation plays a key role in T cell exhaustion. Persistent exposure to viral antigens can drive the upregulation of inhibitory receptors and metabolic dysfunction in T cells, contributing to the immune dysregulation observed in PAIS.

In your research, how have you assessed T cell exhaustion and what have you found?

We analyzed the expression of T cell exhaustion markers following prolonged antigenic stimulation. PBMCs from healthy donors were stimulated for four days with peptide pools derived from SARS-CoV-2, EBV, or CMV. Expression of exhaustion markers PD-1, LAG-3, TIM-3, TIGIT, and CD39 was assessed to evaluate the effect of stimulation on T cell functionality. Our analysis demonstrated a significant upregulation of exhaustion markers on CD4+ and CD8+ T cell subsets following non-specific stimulation of PBMCs induced by anti-CD3, anti-CD28, and anti-CD2 antibodies. Additionally, stimulation with pathogen-specific peptide pools (SARS-CoV-2, EBV, or CMV) resulted in detectable, but significantly less pronounced increases in exhaustion marker expression, consistent with the selective activation of antigen-specific memory T cells.

T cell exhaustion is also well documented to correlate with reduced cytokine production. We see significant changes in the levels of cytokines involved in the immune response and inflammation between PAIS patients compared to healthy controls using ViraxImmune™, our FluoroSpot T cell assay.

How does the ViraxImmune™ platform work?

The ViraxImmune™ PAIS assay measures the specific T cell response when stimulated with an array of infection-derived peptide pools. Detection of a range of crucial cytokines, such as IL-2, IL-6, IL-1β, IFN-γ, and TNF-α, provides insight into both T cell dysfunction and chronic inflammation. Upon activation, T cells secrete cytokines, which are immediately captured on the assay surface below the cell. The detection is done by a cocktail of antibodies with unique fluorescent labels for each cytokine. Those labels are read using a fluorescent scanner such as a microscope or a specific reader.

Then, a composite score is calculated for each patient based on the individual cytokine analyses undertaken, providing a single, easy-to-interpret value. This provides a more comprehensive assessment than individual cytokine responses alone, offering clinicians a better tool for interpreting patient outcomes and helping them assess the severity of the patient’s condition and guide management decisions.

What studies are currently underway for your PAIS diagnostic platform?

Virax Biolabs has initiated a number of clinical studies in the United Kingdom aimed at assessing the performance of the ViraxImmune™ assay in detecting T cell dysfunction in confirmed PAIS patients.

The planned clinical studies, including our multi-center clinical validation study (NCT06731179), are evaluating the role of T cell dysfunction in PAIS participants experiencing persistent symptoms of chronic fatigue and cognitive impairment. This will be undertaken as part of evaluation of the clinical performance of the ViraxImmune™ PAIS assay. Validated symptom questionnaires for chronic fatigue and cognitive impairment are being assessed along with the participant’s composite score for T cell dysfunction to establish a link between the participant’s T cell dysfunction and their symptoms of chronic fatigue and cognitive impairment.

We are also in discussions with US-based clinical research centers to run additional clinical studies to support US regulatory approval and pricing acceptance.

How could clinical implementation of the platform help improve patient outcomes?

There are currently no reliable diagnostics available for PAISs. By utilizing infection-derived peptide pools to activate memory T cells and then measuring the cytokine profiles related to specific infections, ViraxImmune™ PAIS is designed to give a measure of T cell function and to address the diagnostic needs of multiple post-acute infection syndromes and help simplify the patient journey.

ViraxImmune™ aims to provide information to clinicians about an individual’s T cell dysfunction, linked to their symptoms of PAIS, and reduce time to diagnosis for PAIS patients and improve early treatment management. This approach is expected to offer significant diagnostic potential for evaluating and monitoring immune status in chronic conditions.

Is there anything else on the horizon that you’d like to share?

Virax Biolabs is looking to bring to the market a comprehensive set of T cell diagnostics and immune profiling solutions utilizing its novel ViraxImmune™ platform. We have talked about the utility in PAIS; however, we also have a focus on developing solutions in the area of protective immunity, in particular in post-transplant infection and vaccine efficacy.

Having worked for many years in developing drugs in oncology, we believe there is a real potential opportunity in the area of CAR T cell therapy. Because CAR T cell therapy is moving towards a more allogenic “off the shelf” production, we believe there is an opportunity to better identify individuals who may benefit from it as well as provide additional information on how to optimize dosing for better efficacy and less risk of cytokine release syndrome.