Respiratory viral infections caused by influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 remain major causes of morbidity, hospitalization, and mortality among older adults worldwide. Current antiviral therapies have limited effectiveness and generally require administration within the first 48 hours after symptom onset. Increasing evidence suggests that the programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) immune checkpoint pathway plays an important role in the host immune response during acute viral respiratory infections. Upregulation of PD-L1 has been associated with impaired antiviral T-cell activity, immune exhaustion, and disease progression in influenza, RSV, and SARS-CoV-2 infections.

The AIRE-INT study is a prospective, observational, multicenter, non-interventional study designed to characterize the temporal kinetics of PD-L1 expression and inflammatory biomarkers in adults aged 60 years or older presenting with acute respiratory viral infection.

The study will be conducted in primary care centers and urgent care facilities within the Barcelonès Nord and Maresme healthcare regions in Catalonia, Spain. A total of 150 participants will be enrolled, including 75 with confirmed viral respiratory infection and 75 controls with negative rapid antigen tests for influenza, RSV, and SARS-CoV-2.

Eligible participants must be aged ≥60 years and present within 24 to 72 hours after the onset of respiratory symptoms compatible with acute viral infection, including fever, cough, nasal congestion, or dyspnea. Participants in the infected group must have a positive rapid antigen test for influenza, RSV, or SARS-CoV-2. Control participants must test negative for all three viruses. Written informed consent will be obtained before enrollment.

Participants with severe immunosuppression, chronic immunosuppressive therapy, active oncologic disease, terminal illness, or autoimmune diseases associated with PD-L1 dysregulation will be excluded.

Each participant will be followed for up to 60 days and will complete two in-person study visits and one follow-up assessment.

Visit 1 will occur between 24 and 72 hours after symptom onset and will include informed consent, collection of demographic and clinical data, assessment of symptoms and medical history, rapid antigen testing, and blood sample collection for PD-L1 and inflammatory biomarker analyses.

Visit 2 will occur between 5 and 9 days after symptom onset and will include repeat clinical assessment and blood sample collection to evaluate temporal changes in PD-L1 expression and inflammatory responses during the acute phase of infection.

Visit 3 will occur between 30 and 60 days after symptom onset and will consist of clinical follow-up through telephone contact and electronic medical record review to assess symptom resolution, complications, hospitalization, intensive care admission, and mortality.

Laboratory analyses will include flow cytometry quantification of PD-L1 expression and evaluation of inflammatory biomarkers, including C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), complete blood count parameters, renal and hepatic function markers, and virus-specific IgG antibodies. Blood samples will be processed according to standardized laboratory procedures at the Hospital Germans Trias i Pujol Microbiology Department.

The primary objective is to characterize the temporal profile of PD-L1 expression from symptom onset to infection resolution in older adults with influenza, RSV, or SARS-CoV-2 infection.

Secondary objectives include:

Describing the evolution of inflammatory serum biomarkers and their association with disease severity.

Identifying biomarkers useful for screening, prognosis, and clinical monitoring.

Establishing a biological reference framework for future evaluation of PD-L1 inhibitors in respiratory viral infections.

The primary outcome measure is the level and temporal evolution of PD-L1 expression and inflammatory biomarkers between study visits. Secondary outcomes include hospitalization, intensive care admission, mortality at 60 days, symptom duration, and clinical progression.

Statistical analyses will include descriptive and univariate analyses, longitudinal modeling of biomarker kinetics using locally estimated scatterplot smoothing (LOESS) and nonlinear mixed-effects regression models, and predictive logistic regression models evaluating associations between biomarkers and clinical outcomes.

The study is expected to provide important information regarding the kinetics of PD-L1 expression and inflammatory responses during acute respiratory viral infections in older adults. The findings may support the development of prognostic biomarkers and future host-directed therapeutic strategies targeting the PD-1/PD-L1 pathway across multiple respiratory viruses.

Respiratory viral infections remain a major global public health challenge, causing substantial morbidity, hospitalization, and mortality, particularly among older adults and individuals with chronic medical conditions. Influenza viruses, respiratory syncytial virus (RSV), and SARS-CoV-2 are among the most clinically relevant respiratory pathogens worldwide and continue to impose a considerable burden on healthcare systems. Seasonal influenza epidemics affect up to 20% of the population annually, depending on circulating strains, and are responsible for an estimated 290,000 to 650,000 deaths globally each year. Although most infected individuals recover spontaneously within a few days, severe disease and complications are more common in adults aged 60 years or older, pregnant women, healthcare workers, young children, and individuals with underlying comorbidities. Current preventive strategies rely mainly on annual vaccination; however, vaccine effectiveness has remained limited in recent years due to viral antigenic drift and the continuous emergence of new strains.

Although antiviral therapies are available for some respiratory viruses, their effectiveness is limited and treatment generally must be initiated within a narrow time window after symptom onset. Furthermore, currently available antiviral agents target virus-specific mechanisms, limiting their applicability across different respiratory pathogens. These limitations have stimulated interest in host-directed therapeutic strategies capable of modulating common immune pathways involved in viral pathogenesis.

The programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint pathway has emerged as a potential regulator of antiviral immunity. PD-L1 is an immunoregulatory molecule that limits excessive immune activation and tissue damage by suppressing T-cell responses. While this mechanism is essential for maintaining immune homeostasis, excessive activation of the PD-1/PD-L1 pathway may contribute to impaired antiviral immunity, immune exhaustion, delayed viral clearance, and prolonged inflammation.

Experimental and translational studies have demonstrated increased PD-L1 expression during acute respiratory viral infections, including influenza, RSV, and SARS-CoV-2. In influenza infection, PD-L1 upregulation has been associated with reduced CD8+ T-cell activity and impaired antiviral responses. Similar observations have been reported in SARS-CoV-2 and RSV infections, suggesting that modulation of this pathway may represent a common biological mechanism across multiple respiratory viruses.

Preclinical studies have further suggested that PD-L1 expression follows a dynamic temporal pattern during infection, with expression levels increasing during the acute phase and subsequently declining during recovery. These findings raise the possibility that characterization of PD-L1 kinetics in humans may identify biologically relevant periods of immune dysregulation and help define potential therapeutic windows for future host-directed interventions.

Despite increasing interest in the PD-1/PD-L1 pathway, its temporal behavior during naturally acquired respiratory viral infections in humans remains poorly understood. In particular, limited information is available regarding the relationship between PD-L1 expression, systemic inflammatory responses, and clinical outcomes in populations at highest risk for severe disease and complications.

The AIRE-INT study seeks to generate clinical and biological data on PD-L1 expression and associated inflammatory responses during acute respiratory viral infections caused by influenza, RSV, and SARS-CoV-2. By improving understanding of immune checkpoint dynamics in naturally occurring infection, the study aims to contribute to the identification of biomarkers with potential prognostic and monitoring value and to provide foundational evidence for future research evaluating host-directed therapeutic approaches targeting the PD-1/PD-L1 pathway.

The expected impact of this study is to provide a detailed characterization of PD-L1 kinetics and inflammatory responses during acute respiratory viral infections in older adults. The results may contribute to the identification of prognostic and monitoring biomarkers and establish the biological basis for future proof-of-concept clinical trials evaluating PD-L1-targeted therapies in respiratory viral infections. This study may ultimately support the development of innovative host-directed therapeutic strategies capable of acting across multiple respiratory viruses and extending the therapeutic window beyond currently available antiviral approaches.

Inclusion Criteria:

GROUP A (Infected-Positive)

• Age ≥60 years.
• Symptoms compatible with viral respiratory infection (fever, cough, nasal congestion, dyspnea, etc.) between 24h and 72h from symptom onset.
• Confirmed diagnosis, positive for influenza, RSV, or SARS-CoV-2 by rapid test.
• Signed informed consent at visit 1.

GROUP B (Non-Infected-Negative. Controls)

• Age ≥60 years.
• Negative diagnosis for influenza, RSV, or SARS-CoV-2 by rapid test.
• Signed informed consent at visit 1.

Exclusion Criteria:

• Known severe immunosuppression (e.g., transplant recipient, uncontrolled HIV).
• Chronic immunosuppressive treatment (except low-dose corticosteroids (≤10 mg/3 months)).
• Participation in another clinical trial within the last 30 days.
• Inability to comply with the visit schedule.
• Immunosuppressive drugs.
• Drugs indicated for treatment.
• Oncology patients.
• Terminal patients.
• Autoimmune diseases associated with alterations in PD-L1.
• Systemic lupus erythematosus (SLE).
• Rheumatoid arthritis (RA).
• Multiple sclerosis (MS).
• Type 1 diabetes mellitus (T1DM).
• Sjögren's syndrome.
• Myasthenia gravis.
• Autoimmune thyroiditis (Hashimoto's, Graves' disease).

People aged 60 years or older who attended the healthcare service with flu-like symptoms.