Mucosal and Systemic Immunity Correlates of Protection Against Influenza in Young Children, Aged 2-5 Years (MUSIC-Flu) (MUSIC-Flu)

Annual influenza epidemics cause an estimated 290 000-650 000 deaths globally, with a disproportionately high burden in sub-Saharan Africa. In South Africa, approximately 20% of the population develops influenza-associated illness annually, with the highest rates of severe illness and mortality among infants and the elderly. Children under five years of age show particularly high infection rates (up to 67 per 100 person-seasons) and play a key role in transmission. Pediatric vaccination programmed in high-income countries have shown indirect protection to vulnerable populations, but such strategies are not implemented in sub-Saharan Africa in part because the mechanism for the indirect effects are not well understood and because influenza vaccination is only moderately effective. To elucidate the mechanism through which the indirect effects are acting, such as through reducing transmission, and to develop more effective vaccines, further studies and trials are needed.

Correlates of protection are immune markers that predict if an individual will be protected from infection or illness. For vaccine trials, it is a useful tool to allow for the estimation of immune responses without doing large efficacy trials. For influenza, the commonly accepted correlate of protection, especially for inactivated vaccines, is antibodies against the haemagglutinin surface protein, measured as HAI titers. However, limitations exist with HAI titer as a correlate of protection, particularly in children where higher titers are needed for protection compared to adults. More recent research has suggested promise that mucosal immune markers may better correlate with influenza infection and illness, as this is where the infection begins, but these markers have not yet been fully identified or described. In this study we aim to identify additional correlates of protection/infection, including mucosal correlates, to provide essential evidence to inform vaccine development, regulatory pathways, and implementation strategies aimed at reducing influenza transmission and disease burden in South Africa and other similar settings.

Aim:

To describe and compare humoral and cellular immune responses to natural influenza virus infection in the mucosa and blood to identify possible correlates of protection against influenza infection, illness and duration of virus shedding.

Primary objectives:

In children aged 2-5 years:

1. Characterize mucosal and systemic immune markers profiles among children with and without PCR- and/or serologically-confirmed naturally acquired influenza infection
2. Characterize mucosal and systemic immune marker profiles among children with and without PCR-confirmed naturally acquired influenza-associated illness
3. Characterize how natural infection-induced mucosal and systemic immune markers may correlate with reduction in viral load and duration of shedding during influenza infection Secondary objectives

1. Determine whether immune markers that correlate with protection against infection also correlate with protection against illness and with reduction in viral load/duration of shedding 2. Assess the impact of respiratory viral co-infection between influenza virus and RSV or SARS-CoV-2 on influenza presentation, viral load, duration of shedding, and on the magnitude/direction of mucosal/systemic immune responses 3. Quantify the waning of naturally-induced mucosal and systemic immunity over time and estimate the half-life of immune markers 4. Evaluate seroconversion and boosting rates following PCR-confirmed infection and correlate these with mucosal markers 5. Utilize mathematical modelling to define the optimal target product profiles for pediatric transmission-reducing vaccines capable of significantly lowering influenza burden in all ages 6. In a subset of participants: 6.1. Describe how cellular immunity markers (CD4+, CD8+ T cells) from mucosa and periphery correlate with 6.1.1. Protection against PCR- and/or serologically-confirmed influenza infection 6.1.2. Protection against PCR-confirmed influenza illness 6.1.3. Duration of shedding and viral load during PCR-confirmed influenza infection 6.2. Describe the kinetics of cellular immunity (CD4+ and CD8+ T cells)

Methods:

Study design: Prospective, non-interventional, cohort study. Study site and population: The study will be conducted in Jouberton in the North West Province of South Africa, which was the site of previous studies of community influenza and SARS-CoV-2 transmission (PHIRST and PHIRST-C) and an intensive cohort study among toddlers that estimated the incidence of influenza infection (ISiT). Enrolled participants will be healthy children aged 2-5 years, resident in Jouberton and available for follow up for the duration of the study.

Sample size: We will enrol a maximum of 150 children, which even if loss to follow-up reaches close to 30% and the final sample size is 100, will provide 80% power to detect a 50% protection with an influenza attack rate of 45%.

Enrolment and follow up: We will enrol healthy children at clinics in Jouberton, attending for Expanded Progamme of Immunization (EPI) vaccination or other well-child visits. Children aged 2-5 years will be pre-screened and those meeting enrolment criteria will be formally enrolled at the clinic following written informed consent from a parent.

Potential participants will be evaluated for study eligibility and eligible children with caregiver consent will be enrolled and baseline data collected from March through April 2026.

Children will be followed from April through October 2026 during the influenza season. Follow-up includes collection of thrice weekly mid-turbinate nasal swabs to be tested by reverse transcription polymerase chain reaction (RT-PCR) for influenza and other respiratory viruses. One swab per week will be collected by study nurses during a household visit, and two additional swabs per week will be collected by the caregiver. Data on symptoms and tympanic temperature will be collected by nurses at weekly visits and daily by caregivers using an electronic symptom diary. Serum will be collected before and after the season, and at two additional time points during the follow-up period. Peripheral blood mononuclear cells (PBMCs) will be collected before and after the season, and nasal mucosal fluid, oral fluid and nasopharyngeal swabs will be collected monthly to test for immunological markers, including antibodies, cytokines and T- and B-cells.

Analysis: To explore how mucosal and systemic immune markers correlate with protection against influenza infection, illness and virus shedding, we will summaries and compare marker levels between groups with and without influenza infection using geometric means and regression modelling.