This was a phase II, randomised, double-blind, placebo-controlled study. The study was conducted at a single site in the United Kingdom (hVIVO Services Limited, Queen Mary BioEnterprises Innovation Centre, London, UK). The study was initiated on August 12, 2022, and was completed on May 03, 2023. This study was approved by the South Central-Berkshire B Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki, the principles of the International Council for Harmonisation Good Clinical Practice, and applicable local regulatory requirements.

The inclusion criteria were adults aged between 18 and 55 years (inclusive) with a body weight ≥ 50 kg, body mass index ≥ 18 kg/m2; no medical history of clinically significant medical conditions; a negative pregnancy test (female participants) and use of an effective contraceptive method; and who were serosuitable (haemagglutination inhibition titre ≤ 1:10) for infection with the influenza A/Perth/16/2009 (H3N2) challenge virus at generic screening. The exclusion criteria were symptoms or signs of upper or lower RTI within 4 weeks prior to the first study visit; any history or evidence of any clinically significant or currently active condition that may have interfered with completion of the study; females who were breast-feeding or had been pregnant within 6 months prior to the study; history of anaphylaxis or history of severe allergic reactions; and nasal surgery or any significant abnormality affecting the nose or nasopharynx that may have interfered with the study within 3 months of the study. At the generic screening, participants were asked what biological sex they were assigned at birth (male or female). All participants provided informed written consent.

Participants were randomised to one of three study arms (daily HEX17 doses for 3 days, single-dose HEX17 on day − 3 and placebo on day − 2 and day − 1, or daily placebo on day − 3 to day − 1) in a 3:3:4 ratio. An independent statistician provided a computer-generated randomisation code to determine which study medication regimen participants received. Randomisation was by block, with a block size of 10. Participants were dispensed blinded investigational medicinal product (HEX17/placebo) labelled with their randomisation number. The principal investigator/investigator, all clinical and nonclinical staff (excluding the unblinded pharmacist, unblinded statistician and quality assurance auditors where necessary) and participants were blinded until after the database was locked and unblinding was approved.

Participants were resident within the quarantine unit for the inpatient phase of 13 days (day − 4 to day 8). Baseline assessments and randomisation were performed up to day − 3, ahead of the first study dose.

HEX17 and placebo were provided by Pneumagen (Fife, Scotland, UK) and formulated as liquid sprays for intranasal administration. HEX17 was administered as a 10 mg/mL solution, at a dose of 2.8 mg in 0.28 mL (1.4 mg [0.14 mL] per nostril). The HEX17 dose in each treatment arm was determined based on the tolerability profile observed in the first-in-human study. HEX17 was delivered intranasally, from an Aptar Cartridge Pump System (CPS) Spray Pump. Matching placebo was administered at a volume of 0.28 mL (0.14 mL per nostril). Participants in the daily HEX17 arm received HEX17 once daily for 3 days (day − 3 to day − 1). Participants in the single-dose HEX17 arm received HEX17 on day − 3, and placebo once daily on day − 2 and day − 1. Participants in the placebo arm received placebo once daily for 3 days (day − 3 to day − 1). Influenza A/Perth/16/2009 (H3N2) was provided by hVIVO (London, UK) and formulated as a liquid for intranasal drop administration in a capped vial. The virus was administered at an approximate dose of 105.5 TCID50 (50% tissue culture infectious dose). Participants were closely monitored for 24 h following challenge virus inoculation.

Nasal samples were collected by nasopharyngeal swabs on day 1 (once; PM), twice daily on day 2–7 (taken approximately 12 h apart), and on day 8 (once; AM). These samples were used to determine the incidence of influenza infection and viral load by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and viral culture (for detailed methodology, see Supplementary Information).

Symptomatic influenza infection was defined as two detectable RT-qPCR measurements on two or more independent nasal samples over 2 days, or one quantifiable cell culture measurement, from day 1 to 8, and any symptoms of grade ≥ 2 at a single time point.

The severity of influenza symptoms was assessed using the total symptoms score (TSS). The TSS was based on diary cards that were completed by participants three times a day, from day 1 (AM) to day 8 (AM). Participants provided a grade from 0 to 3 for the following 11 symptoms: runny nose, stuffy nose, sneezing, sore throat, earache, malaise/tiredness, headache, muscle and/or joint ache, chilliness/feverishness, cough, and shortness of breath. The grading scale was as follows: grade 0—no symptoms; grade 1—just noticeable; grade 2—clearly bothersome from time to time but does not interfere with me doing my normal daily activities; grade 3—quite bothersome most or all of the time, and it stops me participating in activities. Shortness of breath had an additional grade, grade 4—symptoms at rest. An individual TSS was derived for each assessment as a sum of the symptom scores (with possible values ranging from 0 to 33). The peak TSS was defined as the maximum observed value for TSS from day 1 to 8. Area under the curve (AUC) of viral load and TSS was calculated using the trapezium rule:

With n + 1 measurements yi at times ti, (i = 0, n), the AUC is calculated as:

$$\text=\frac \sum_^\left(_- _\right) \left(_+ _\right)$$

The actual time of each assessment will be used in the calculation.

For viral load–area under the curve (VL-AUC), 14 measurements are used for the computation (1 on day 1, 2 on each day from day 2 to day 7 and 1 on day 8). For TSS-AUC, 24 measurements are used for the computation (3 on each day from day 1 to day 8).

Participants were discharged from the quarantine unit on day 8, having tested negative for the influenza virus by rapid viral antigen test and had no clinically significant symptoms. The final follow-up visit was conducted on day 28 (± 3 days).

The co-primary outcomes for this study were the effect of HEX17 in reducing the incidence of symptomatic influenza infection and/or the severity of symptoms after influenza viral challenge, compared with placebo. The co-primary endpoint was chosen to allow determination of both symptomology as well as viral load, rather than viral load alone. Secondary outcomes included the effect of HEX17 compared with placebo on the following: reducing the incidence of influenza infection; the number of participants with grade 2 or higher symptoms; and reducing the influenza viral shedding/load. The safety of HEX17 and the challenge virus were also assessed. Adverse events (AEs; including serious AEs [SAEs]) and AEs of special interest (for HEX17, namely a clinically significant reduction in forced expiratory volume in 1 s and forced vital capacity) were recorded up to the follow-up visit. For the challenge virus, AEs and SAEs were recorded from day 0 to the planned discharge from quarantine (day 8). AEs were classified as solicited or unsolicited, and reported using descriptive statistics. Solicited AEs included bleeding, burning sensation, pain or irritation of the nose, loss of taste or smell, sensation of needing to sneeze, sneezing and unpleasant taste. Unsolicited AEs were reported by patients, assessed for relatedness to HEX17 prophylaxis and graded 1–4 for severity. Exploratory pharmacokinetic assessment of HEX17 plasma concentrations was also performed. Blood samples for this analysis were collected on days − 3, − 2 and − 1; these were collected pre-dose (within 2 h before study medication administration), and 1, 2 and 3 h post-dose. Immunogenic response to HEX17 was not analysed in this study.

The sample size was based on the primary comparison between the placebo arm and the two HEX17 dose arms pooled together. The number of participants in the placebo arm (40) and in the pooled HEX17 arm (60; 30 in each dose arm) had a power of at least 80% to detect a significant reduction in the following primary outcomes:

A 70% reduction in symptomatic influenza infection rate compared with placebo assuming a symptomatic infection rate of 27.8% in the placebo arm

A 65% reduction in peak TSS compared with the placebo arm, assuming a coefficient of variation of 125%, using a one-sided 0.05 type I error rate without adjustment for multiple testing

All analyses were prespecified. For the co-primary outcomes, pairwise comparisons of qRT-PCR confirmed symptomatic influenza infection and mean peak TSS were performed between the pooled HEX17 and placebo arms. Exploratory endpoints included comparisons between each HEX17 dose arm and the placebo arm. The incidence of symptomatic influenza infection was summarised for each arm using counts and percentages with comparisons between arms using the Pearson chi-square test. Peak TSS was summarised for each arm using means and medians, while the differences between arms were summarised using Hodges–Lehmann estimation and tested using the Wilcoxon rank-sum test. Tests for both primary outcomes used a nominal one-sided error of 0.05 without adjustment for multiplicity, because this was not assumed to be a requisite for a phase 2 aiming to provide proof of concept for the efficacy of HEX17. The primary efficacy analysis was conducted on the per protocol population data, defined as participants who had received all doses of the study medication and the challenge virus, completed the quarantine period, and presented with no major protocol deviation likely to impact data evaluation. Safety analysis was conducted on the safety analysis dataset, defined as participants who had received at least one dose of HEX17 or placebo. Statistical analysis conducted for the secondary outcomes was performed on the per protocol dataset as follows: incidence of RT-qPCR confirmed influenza infection was assessed by Pearson chi-square test; and viral shedding (as determined by viral load and peak viral load from RT-qPCR and viral culture) was assessed by Wilcoxon rank-sum test.

Statistical analysis was performed using SAS version 9.04.01M6P11072018. No data monitoring committee was used. This study was registered with EudraCT, 2022-001853-22 (and on ClinicalTrials.gov, NCT05507567).