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[Preprint]. 2025 Jul 24:2025.07.23.25332064.
doi: 10.1101/2025.07.23.25332064.

Effective Aerosol Inoculation of Dose-Escalated Seasonal Influenza H3N2 Virus in Controlled Human Infection Model

Nadine Rouphael  1 , Ralph Tanios  1 , Jessica Traenkner  1 , Matthew D Pauly  2 , Nishit Shetty  3 , Meredith J Shephard  2 , A J Campbell  2 , Christelle Radi  1 , Shamika Danzy  2 , Jin Pan  4 ; Flu CHIM Study Group; Andrew P Catchpole  5 , Alex Mann  5 , Joshua F Detelich  6 , Anice C Lowen  2 , Linsey C Marr  7 , Seema S Lakdawala  2
Affiliations

Effective Aerosol Inoculation of Dose-Escalated Seasonal Influenza H3N2 Virus in Controlled Human Infection Model

Nadine Rouphael et al. medRxiv. .

Abstract

Human challenge models (CHIMs) are instrumental in advancing influenza research but have traditionally relied on intranasal inoculation, which does not mimic the natural aerosol transmission of the virus. We conducted a dose-escalation influenza CHIM study to evaluate the safety and feasibility of two modern aerosol delivery systems: a flow-focusing monodisperse aerosol generator (FMAG) and a medical nebulizer. Fourteen healthy adults aged 18-49 years were exposed to influenza A/Perth/16/2009 (H3N2) in a controlled inpatient setting. Infection rates were 75% (3/4) with FMAG and 50% (2/4) with the nebulizer at the higher dose. Infections were self-limited, with sinus congestion, rhinorrhea, and cough being the most common symptoms. No serious adverse events occurred. Viral shedding was reproducible across respiratory sites, and seroconversion occurred in 33% of infected participants. Symptom timing and viral kinetics were comparable to those observed in prior intranasal CHIMs. Participants receiving nebulizer-delivered virus showed earlier viral detection in the oral cavity, suggesting broader airway deposition. These findings demonstrate that aerosol influenza challenge is both safe and effective and can simulate natural infection more accurately than intranasal delivery. This reintroduction of aerosolized influenza challenge provides a robust platform for studying transmission dynamics, tissue-specific immune responses, and for evaluating next-generation vaccines and therapeutics under conditions that better approximate real-world exposure.

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Conflict of interest statement

NR: Emory University receives grants and contracts supporting NR’s research from Merck, Sanofi, Pfizer, Vaccine Company, Immorna, and the NIH; NR has received honoraria from Virology Education and Medscape and travel support from Sanofi and Moderna; she serves on advisory boards for Moderna, Sanofi, Seqirus, and Pfizer and on safety committees for EMMES, ICON, BARDA, CyanVac, Imunon, and Micron; NR holds leadership roles on the ARLG and CDC Pertussis Challenge advisory boards, the UAB Immunology Institute advisory board, and the DMID Safety Monitoring Committee for a malaria challenge model; she has also received equipment and other support from the Georgia Research Alliance. AM and AC are employees of hVIVO and hold stock in the company. ME serves as a paid consultant to Medtronic and Boston Scientific and has received advisory-board payments for a Medtronic patent committee. CK receives CTSA, CFAR, and industry trial support to her institution; she consults for Rebiotix/Ferring (payments to her personally) and has served as an expert witness for Womble Dickinson (payments to her); she has received travel support to ASM meetings; holds a pending patent on a fecal-microbiota processor; serves unpaid on the boards of Project Mercy and the National MPS Society; and has received equipment from the Georgia Research Alliance. SL receives core laboratory support from the Flu Lab; has received honoraria from NIH, Indiana University, Yale, University of Colorado, Cleveland Clinic, Boston University, NYU, and the University of Wisconsin–Madison; and serves on the APPA Medical Advisory Board. LM: Flu Lab support to her institution; grants from NSF and Wellcome Leap (to institution); consulting fees from MITRE (paid to her); honoraria from The New York Times, University of Hong Kong, Queensland University, and Washington University (paid to her); travel support from MITRE and ISIRV; and a pending patent on a surface-enhanced Raman spectroscopy biosensor. ALow: Flu Lab support to Emory University; multiple NIAID grants (U01 AI144673, R01 AI146260, R01 AI154894, R01 AI165644, R01 AI127799, P01 AI186819, 75N93021C00017) paid to Emory; and equipment from the Georgia Research Alliance.. AJC, AL, AKM, SDB, CR, DG, KP, EF, GQ, JN, KB, KBush, JT, JD, JP, MP, MV, NS, RT, MS, and NVM report no relationships, activities, or interests to disclose.

Figures

Fig. 1.
Fig. 1.. Symptom scores and nasopharyngeal swab titers
The mean daily symptom scores for nine stacked, color-coded symptom categories are plotted on the left axis. Scores for each symptom range from 0–4 and the number of symptoms included in each category is indicated in parentheses in the legend. Participants were inoculated on Day 1. Baseline symptoms were collected on Day 1 prior to inoculation. The cycle threshold (Ct) value obtained from daily testing of nasopharyngeal swab samples is plotted as a black line on the right axis. Samples in which influenza was not detected were given a Ct value of 45. Participants are grouped by aerosol generation device with the FMAG (flow-focusing monodisperse aerosol generator) on the left and the medical nebulizer on the right, and by virus dilution: 1:100 at the top and 1:10 at the bottom. Red participant identifiers indicate participants who met the MMID case definition. Asterisks denote the first day that daily oseltamivir phosphate doses were administered to a participant.
Fig. 2.
Fig. 2.. Quantification of influenza viral RNA at different anatomical sites over time
The amount of influenza viral RNA was determined by quantitative reverse-transcription PCR (qRT-PCR) of daily nasopharyngeal swab (blue), anterior nasal swab (red), buccal swab (yellow), oropharyngeal swab (green), and saliva (purple) samples for all participants. The limit of detection is indicated by a dotted line. Participants are grouped by aerosol generation device with the FMAG (flow-focusing monodisperse aerosol generator) on the left and the medical nebulizer on the right, and by virus dilution: 1:100 at the top and 1:10 at the bottom. Inoculations occurred on Day 1. Red participant identifiers indicate those who met the MMID case definition. Asterisks denote the first day that daily oseltamivir phosphate doses were administered to a participant.
Fig. 3.
Fig. 3.. Infectious virus titers at different anatomical sites.
The amount of infectious virus was determined by plaque assay of daily nasopharyngeal swab (blue), anterior nasal swab (red), buccal swab (yellow), oropharyngeal swab (green), and saliva (purple) samples for the six participants with MMID. The limit of detection was 5 PFU/mL and is indicated by a dotted line. Participants are grouped by aerosol generation device with the FMAG (flow-focusing monodisperse aerosol generator) on the top and the medical nebulizer on the bottom. Inoculations occurred on Day 1. Asterisks denote the first day that daily oseltamivir phosphate doses were administered to a participant.
Fig. 4.
Fig. 4.. Comparison of viral replication kinetics and symptoms between intranasal and aerosol inoculation routes.
(A) The influenza genome copies measured in nasopharyngeal swab samples tested by quantitative reverse-transcription PCR (qRT-PCR) assay. Participants were inoculated via either the intranasal route15 (black, n=14, including six from reference 15 and nine unpublished) or the aerosol route using an FMAG (flow-focusing monodisperse aerosol generator) (blue) or medical nebulizer (red) on study Day 1. Only participants with MMID are shown. The assay’s limit of detection is indicated by the dashed line. (B) The sum of the mean FLU-PRO symptom responses for the nine symptom categories listed in Figure 1, each scored on a scale from 0 to 4, for the same participants as in panel A. Responses for study Day 1 were collected in the evening, after participants were inoculated in the morning.
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