Influenza virus is transmitted via respiratory expulsions, but detecting infectious virus in expulsions is challenging. Here, we describe quantification and genotyping of infectious virus in respiratory particles using a modular influenza sampling tunnel (MIST). The particles deposit on cell monolayers, enabling culture, quantification, and sequencing of viruses. Concomitantly, water-sensitive paper and fine particle samplers yield respiratory particle counts over a broad size range. Using the MIST, we captured infectious virus from humans experimentally infected with the influenza virus on multiple days post-inoculation. The recovered respiratory particles varied in quantity over three orders of magnitude and contained viral variants also detected in samples from infected individuals. Expulsion of infectious virus was associated with infectious viral load in saliva and nasopharyngeal swabs and with clinical symptoms. These data reveal maintenance of viral diversity in expelled aerosols and suggest heterogeneity among individuals in the magnitude of infectious expulsions, impacting forward transmission potential.

BACKGROUND:S-337395 is a novel inhibitor of the respiratory syncytial virus (RSV) L-protein, and a potential oral antiviral against RSV. This phase 2a, randomized, double-blind, placebo-controlled, single-center, proof-of-concept trial conducted in the United Kingdom (April-October 2024) evaluated the efficacy, safety, and dose-response relationship of S-337395 against RSV infection in a human challenge model. METHODS:Healthy adults aged 18-55 years meeting predefined eligibility criteria were inoculated with RSV-A Memphis 37b on Day 0. Nasal washes were collected for quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) assessment twice daily on Days 2-12 and once on Day 13. Participants were randomized 5:13:13:13:13 to receive S-337395 (1, 10, 30, or 300 mg) or placebo, once daily for 5 days upon evidence of infection. Efficacy analyses were conducted in participants with confirmed RSV infection in intent-to-treat infected (ITT-I) set. Safety was assessed in all randomized participants. Dose-response was evaluated across a wide dose range (1-300 mg). RESULTS:Of 114 randomized participants, 60 comprised the ITT-I set. Covariate-adjusted mean viral load area under the curve (VL-AUC) was significantly lower in the 30-mg and 300-mg groups by qRT-PCR (64.87% and 88.94% reductions, respectively, both p<0.05) and by viral culture assay (72.32% and 86.17% reductions, respectively, both p<0.05). Total Symptom Score AUC was 78.15% lower in the 300-mg group (p<0.05). S-337395 was well-tolerated without any safety concerns. CONCLUSIONS:S-337395 significantly reduced viral load, viral titer, and symptoms, and was well-tolerated in this human challenge study.

BACKGROUND:Controlled human infection models are widely used to study infectious diseases. Since the 1960s, these studies have primarily relied on intranasal inoculation, as earlier aerosol-based methods raised safety concerns and were largely abandoned despite better mimicking the natural route of infection. METHODS:Utilizing modern advances in aerosol delivery, we conducted a dose-escalation study to evaluate the safety and feasibility of aerosol inoculation for human influenza challenge. Healthy adults aged 18-49 years were inoculated with influenza A/Perth/16/2009 (H3N2) using either a flow-focusing monodisperse aerosol generator (FMAG) delivering coarse particles or a medical nebulizer delivering fine particles. Participants were monitored in a controlled inpatient setting with symptom tracking, virologic sampling, and serology. Doses were escalated according to predefined safety and attack rate thresholds. RESULTS:Fourteen participants were enrolled. Flow-focusing monodisperse aerosol generator delivery at higher dose levels resulted in infection in 75% of participants (3/4), while nebulizer delivery produced infection in 50% (2/4). Illnesses were mild and self-limited. Viral shedding was detected at multiple respiratory sites, and adverse events were infrequent and generally mild. No serious adverse events occurred. Antibody responses were observed in a subset of infected participants. CONCLUSIONS:In this small pilot, aerosolized challenge using modern delivery systems was feasible under controlled conditions; no safety concerns were identified, and MMID was induced in a subset of participants. These data establish a methodological framework for future studies evaluating pathogenesis and mucosal immune responses to a variety of respiratory pathogens.

Selection of advantageous mutations drives the emergence of dominant variants during seasonal influenza epidemics. However, within-host detection of such variants remains rare, limiting our understanding of how selection operates at the scale of individual hosts. In this study, we used a controlled human infection model to examine the within-host evolutionary dynamics in thirteen participants intranasally infected with a seasonal H3N2 influenza A virus. Although this clinical trial is ongoing, our work represents a pre-planned, interim, exploratory analysis. Results in this system were contrasted with those observed in a ferret model of infection. The inoculum, used in both humans and ferrets, carried standing diversity that enabled evaluation of variant trajectories during infection. Although the dynamics were variable among participants, in humans, the minor variants in the PA and NP gene segments tended to increase in frequency as infection progressed. Variant dynamics were more consistent among ferrets but showed differences from humans in the fate of the minor NP allele. Based on these observations, we fit a population genetic model to longitudinal measurements of variant frequencies. Estimates of variant selection coefficients and effective viral population sizes indicated that in humans the two minor variants had a selective advantage over the major variants, but genetic drift was strong, limiting the efficiency of selection. In ferrets, the PA minor variant also was estimated to have a selective advantage, while the NP minor variant was estimated to have a selective disadvantage. Moreover, effective viral population sizes were estimated to be considerably higher in ferrets than in humans, indicating that genetic drift was weaker in ferrets. Our analyses reveal differing selective environments acting on influenza viruses in human and ferret hosts and indicate that selection at the within-host level is weakened by genetic drift.

Quantifying T cell response during primary infection in humans is crucial for understanding adaptive immunity. Leveraging a controlled human challenge to SARS-CoV-2, we characterized antigen-specific T cell response within and across individuals. Notably, individual clones reached similar maximum frequencies despite differences in the timing of their peak expansion. Mathematical modeling showed that this observation is consistent with precursor frequency, but not TCR signal strength, as the source of inter-clonal variability. Single-cell profiling revealed distinct temporal programs for CD4+ and CD8+ T cells, with CD4+ cells expanding earlier but contracting to a lower frequency. Clones with similar receptors, likely recognizing the same antigen, expanded at similar times. Together, these findings highlight how clone-intrinsic properties such as precursor frequency and lineage shape T cell clonal kinetics. These insights provide a quantitative framework for understanding T cell response in humans, with implications for vaccine design. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Human challenge trials evaluate protection from influenza exposure following vaccination but have not been reported for nucleoside-modified messenger ribonucleic acid (modRNA) influenza vaccines. METHODS:This phase 2a, double-blind trial randomly assigned healthy adults 18-55 years of age in a 1:1 ratio to receive either a modRNA or quadrivalent influenza vaccine (QIV) 30 days before an influenza A/H1N1 challenge. Control group participants from a separate trial were exposed to the same virus but were not vaccinated. Four primary efficacy end points were used to evaluate events from day 1 to 8 postchallenge. Two end points were the percentage of participants with laboratory-confirmed symptomatic influenza (grade 2 or higher) and the percentage with febrile influenza (with a temperature of at least 37.9°C). Influenza end points were compared between vaccine and control groups to calculate vaccine efficacy (VE [1 minus relative risk]). Two end points were the median differences between the vaccine and control groups for the area under the viral load (VL)-time curve (VL-AUC) and for peak VL. Vaccine safety end points were examined. RESULTS:The per-protocol cohort included 55, 48, and 52 participants in the modRNA, QIV, and control groups, respectively. Symptomatic influenza occurred in 0%, 4.2%, and 26.9% of participants in the modRNA, QIV, and control groups, respectively, with a VE of 100.0% (95% confidence interval [CI], 75.2% to 100.0%) for the modRNA vaccine and 84.5% (95% CI, 43.4% to 96.0%) for the QIV. Febrile influenza occurred in 0%, 0%, and 17.3% of participants in the modRNA, QIV, and control groups, respectively, with a VE of 100.0% (95% CI, 61.2% to 100.0%) for the modRNA vaccine and 100.0% (95% CI, 55.9% to 100.0%) for the QIV. The median differences in the VL-AUC between the vaccine and control groups were -88.66 for the modRNA (95% CI, -261.95 to -1.35) and -67.01 for the QIV (95% CI, -254.13 to -0.97). The median differences in peak VL between the vaccine and control groups were -4.52 for the modRNA vaccine (95% CI, -5.29 to 0.00) and -1.49 for the QIV (95% CI, -5.25 to 0.00). No serious adverse events were reported. CONCLUSIONS:Following an influenza challenge, the modRNA vaccine was associated with greater VE and reduced VL compared with the control, without any serious adverse events. (Funded by hVIVO and Pfizer; clinical trial registration number, ISRCTN13789612.).

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.

Identifying host factors that mediate protection against newly-emergent viruses is needed for improved pandemic preparedness. Here, we analysed pre- and early post-exposure immune factors associated with resisting SARS-CoV-2 infection after human challenge in seronegative individuals, using multiplex protein, cytometric and RNA sequencing approaches in the nasopharynx and circulation. Pre-existing cross-reactive antibodies correlate poorly with clinical outcome. Instead, protection is associated with heightened nasopharyngeal CCL13 levels locally produced by conventional dendritic cells and monocytes, along with cross-reactive T cells and less differentiated NK cells. Conditional independence network analysis implicates nasal CCL13 as the central node connected to pre-existing non-structural protein-specific T cells by CD1c+ DCs. In those who became infected, baseline cross-reactive T cell and less differentiated NK cell frequencies also correlate with shorter infection duration. Thus, pre-existing mucosal chemokine levels may promote rapid innate and innate-like responses that effectively block infection. ClinicalTrials.gov identifier NCT04865237.

Abstract Background Influenza is a significant cause of morbidity and mortality globally. CD388 is a multivalent conjugate of a dimeric zanamivir stably linked to a proprietary human IgG1 Fc fragment engineered for extended half-life. In preclinical and two Phase 1 studies CD388 has been shown to be safe. Here we describe a post-hoc analysis of prophylactic activity of CD388 against influenza disease in a Phase 2a human challenge study. Methods Healthy participants (18-55 yrs) were randomized to receive subcutaneous (SQ) CD388 or placebo. CD388 was administered 5 days before intranasal challenge with A/Perth/16/2009 (H3N2). A hemagglutinin inhibition assay (HAI) was performed at the onset of quarantine, on Day 0 (prior to inoculation) and on Day 28. A 4-fold increase in Day 28 HAI titer with respect to Day 0 titer was considered as confirmation of influenza infection. Participants completed a graded symptom scoring system 3 x daily. Symptomatic infection was defined as RT-PCR-confirmed (2 quantifiable measurements on ≥ 2 independent samples over 2 days) or culture confirmed (1 quantifiable TCID50 measurement) influenza infection from Day 1 (pm) to Day 8 (am) AND fever or ≥ 2 symptoms at a single time point or any symptom of grade ≥ 2 at a single time point. Prophylactic efficacy was assessed among participants who experienced a 4-fold increase in HAI titer. Results 28 participants received 150 mg of CD388 and 28 received placebo. 17 participants in the CD388 arm (60.7%) and 15 participants in the placebo arm (53.6%) had a 4-fold increase in the HAI titer on Day 28 (Table 1). Although these end-points were not powered, of those with the 4-fold change in HAI, the number of symptomatic participants was significantly lower in the CD388 arm (3/17, 17.6%) compared to placebo (9/15, 60%; p= 0.017), and among these symptomatic participants the median AUC TCID50 (log TCID50/mL x number of days) was significantly lower in the CD388 arm (6.1, min 3.3, max 9.7) compared to placebo (10.8, min 5.7, max 17; p=0.008, Table 1). Conclusion A single SQ dose of CD388 administered 5 days prior to influenza challenge was effective in preventing symptomatic disease. A Phase 2b clinical influenza prevention study for CD388 will begin in the Fall of 2024. Disclosures Ozlem Equils, MD, Cidara Therapeutics: Employee|Cidara Therapeutics: Stocks/Bonds (Public Company) Shawn Flanagan, PhD, Cidara Therapeutics: Salaried Employee|Cidara Therapeutics: Employee|Cidara Therapeutics: Stocks/Bonds (Public Company)|Cidara Therapeutics: Stocks/Bonds (Public Company) Sy-Shi Wang, Ph.D., Janssen Research & Development LLC: Employee|Janssen Research & Development LLC: Stocks/Bonds (Public Company) Johan Vingerhoets, PhD, Janssen Pharmaceutica NV: Stocks/Bonds (Public Company) Wilbert van Duijnhoven, n/a, Janssen Pharmaceutica NV: Employee|Janssen Pharmaceutica NV: Stocks/Bonds (Public Company) Alex James Mann, MSc, hVIVO: Employee Roxana E. Rojas, M.D., Ph.D., Janssen Research & Development LLC: Employee|Janssen Research & Development LLC: Stocks/Bonds (Public Company)|Johnson and Johnson: Stocks/Bonds (Public Company) Taylor Sandison, MD, MPH, Cidara Therapeutics Inc: Employee|Cidara Therapeutics Inc: Stocks/Bonds (Public Company)

BACKGROUND:Influenza is a significant public health concern, especially in immunocompromised patients. CD388 is a novel multivalent zanamivir conjugate that is stably linked to a proprietary human immunoglobulin G1 Fc with a long half-life for prevention of influenza. Here we report a proof-of-concept study on the prophylactic activity of subcutaneously administered CD388 against influenza challenge. METHODS:In a randomized, double-blind, placebo-controlled, phase 2a human challenge study, a single dose of CD388 (50 or 150 mg) was subcutaneously administered in healthy participants 5 days before intranasal challenge with influenza A (H3N2). Safety, pharmacokinetics, infection rate (by reverse transcription-quantitative polymerase chain reaction methods [RT-qPCR]), intranasal viral load (VL), and symptoms were compared between the CD388 and placebo treatment groups. RESULTS:The area under the VL-time curve (VL-AUC), primary endpoint, was lower in the CD388 150 mg group (n = 28) compared with the placebo group (n = 28; mean 10.70 log10 [copies/mL] × days vs mean 16.09 log10 [copies/mL] × days; P = .0390). Peak VL and the rate of RT-qPCR-confirmed influenza infection were lower in the CD388 group versus the placebo group (P = .0185 and P = .0248, respectively). Clinical symptom scores were numerically lower among participants treated with CD388 compared with participants treated with placebo. There were a limited number of treatment-emergent adverse events. Anti-drug antibody development was rare and not clinically relevant. CONCLUSIONS:CD388 was well-tolerated and demonstrated prophylactic activity against RT-qPCR-confirmed influenza infection in a human challenge study. The efficacy of CD388 in preventing influenza will be confirmed in larger studies. Clinical Trials Registration.  ClinicalTrials.gov identifier: NCT05523089.

Background Patient-reported outcomes and cross-sectional evidence show an association between COVID-19 and persistent cognitive problems. The causal basis, longevity and domain specificity fi city of this association is unclear due to population variability in baseline cognitive abilities, vulnerabilities, virus variants, vaccination status and treatment. Methods Thirty-four young, healthy, seronegative volunteers were inoculated with Wildtype SARS-CoV-2 under prospectively controlled conditions. Volunteers completed daily physiological measurements and computerised cognitive tasks during quarantine and follow-up at 30, 90, 180, 270, and 360 days. Linear modelling examined differences between ' infected ' and ' inoculated but uninfected' ' individuals. The main cognitive endpoint was the baseline corrected global cognitive composite score across the battery of tasks administered to the volunteers. Exploratory cognitive endpoints included baseline corrected scores from individual tasks. The study was registered on ClinicalTrials.gov with the identifier fi er NCT04865237 and took place between March 2021 and July 2022. Findings Eighteen volunteers developed infection by qPCR criteria of sustained viral load, one without symptoms and the remainder with mild illness. Infected volunteers showed statistically lower baseline-corrected global composite cognitive scores than uninfected volunteers, both acutely and during follow up (mean difference over all time points = - 0.8631, 95% CI = - 1.3613, - 0.3766) with significant fi cant main effect of group in repeated measures ANOVA (F (1,34) = 7.58, p = 0.009). Sensitivity analysis replicated this cross-group difference after controlling for community upper respiratory tract infection, task-learning, remdesivir treatment, baseline reference and model structure. Memory and executive function tasks showed the largest between-group differences. No volunteers reported persistent subjective cognitive symptoms. Interpretation These results support larger cross sectional fi ndings indicating that mild Wildtype SARS-CoV-2 infection can be followed by small changes in cognition and memory that persist for at least a year. The mechanistic basis and clinical implications of these small changes remain unclear.

Human infection challenge permits in-depth, early, and pre-symptomatic characterization of the immune response, enabling the identification of factors that are important for viral clearance. Here, we performed intranasal inoculation of 34 young adult, seronegative volunteers with a pre-Alpha severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. Of these participants, 18 (53%) became infected and showed an interferon-dominated mediator response with divergent kinetics between nasal and systemic sites. Peripheral CD4 + and CD8 + T cell activation and proliferation were early and robust but showed distinct kinetic and phenotypic profiles; antigen-specific T cells were largely CD38 + Ki67 + and displayed central and effector memory phenotypes. Both mucosal and systemic antibodies became detectable around day 10, but nasal antibodies plateaued after day 14 while circulating antibodies continued to rise. Intensively granular measurements in nasal mucosa and blood allowed modeling of immune responses to primary SARS-CoV-2 infection that revealed CD8 + T cell responses and early mucosal IgA responses strongly associated with viral control, indicating that these mechanisms should be targeted for transmission-reducing intervention.

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited 1 . Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection. A human SARS-CoV-2 challenge study in individuals without previous exposure to the virus or vaccines provides detailed profiles of local and systemic epithelial and immune cell response dynamics over time and infection status.

Blood transcriptional biomarkers of acute viral infections typically reflect type 1 interferon (IFN) signalling, but it is not known whether there are biological differences in their regulation that can be leveraged for distinct translational applications. We use high frequency sampling in the SARS-CoV-2 human challenge model to show induction of IFN-stimulated gene (ISG) expression with different temporal and cellular profiles. MX1 gene expression correlates with a rapid and transient wave of ISG expression across all cell types, which may precede PCR detection of replicative infection. Another ISG, IFI27, shows a delayed but sustained response restricted to myeloid cells, attributable to gene and cell-specific epigenetic regulation. These findings are reproducible in experimental and naturally acquired infections with influenza, respiratory syncytial virus and rhinovirus. Blood MX1 expression is superior to IFI27 expression for diagnosis of early infection, as a correlate of viral load and for discrimination of virus culture positivity. Therefore, MX1 expression offers potential to stratify patients for antiviral therapy or infection control interventions. Blood IFI27 expression is superior to MX1 expression for diagnostic accuracy across the time course of symptomatic infection and thereby, offers higher diagnostic yield for respiratory virus infections that incur a delay between transmission and testing. It's not always clear whether blood biomarkers are differentially expressed in the time course of viral infections. In this SARS-CoV-2 human challenge study, the authors identify distinct single-gene blood transcriptional biomarkers for early stages of infection or for symptomatic infection.

Background Effectively implementing strategies to curb SARS-CoV-2 transmission requires understanding who is contagious and when. Although viral load on upper respiratory swabs has commonly been used to infer contagiousness, measuring viral emissions might be more accurate to indicate the chance of onward transmission and identify likely routes. We aimed to correlate viral emissions, viral load in the upper respiratory tract, and symptoms, longitudinally, in participants who were experimentally infected with SARS-CoV-2. Methods In this phase 1, open label, first-in-human SARS-CoV-2 experimental infection study at quarantine unit at the Royal Free London NHS Foundation Trust, London, UK, healthy adults aged 18-30 years who were unvaccinated for SARS-CoV-2, not previously known to have been infected with SARS-CoV-2, and seronegative at screening were recruited. Participants were inoculated with 10 50% tissue culture infectious dose of pre-alpha wild-type SARS-CoV-2 (Asp614Gly) by intranasal drops and remained in individual negative pressure rooms for a minimum of 14 days. Nose and throat swabs were collected daily. Emissions were collected daily from the air (using a Coriolis mu air sampler and directly into facemasks) and the surrounding environment (via surface and hand swabs). All samples were collected by researchers, and tested by using PCR, plaque assay, or lateral flow antigen test. Symptom scores were collected using self-reported symptom diaries three times daily. The study is registered with ClinicalTrials.gov, NCT04865237.Findings Between March 6 and July 8, 2021, 36 participants (ten female and 26 male) were recruited and 18 (53%) of 34 participants became infected, resulting in protracted high viral loads in the nose and throat following a short incubation period, with mild-to-moderate symptoms. Two participants were excluded from the per-protocol analysis owing to seroconversion between screening and inoculation, identified post hoc. Viral RNA was detected in 63 (25%) of 252 Coriolis air samples from 16 participants, 109 (43%) of 252 mask samples from 17 participants, 67 (27%) of 252 hand swabs from 16 participants, and 371 (29%) of 1260 surface swabs from 18 participants. Viable SARS-CoV-2 was collected from breath captured in 16 masks and from 13 surfaces, including four small frequently touched surfaces and nine larger surfaces where airborne virus could deposit. Viral emissions correlated more strongly with viral load in nasal swabs than throat swabs. Two individuals emitted 86% of airborne virus, and the majority of airborne virus collected was released on 3 days. Individuals who reported the highest total symptom scores were not those who emitted most virus. Very few emissions occurred before the first reported symptom (7%) and hardly any before the first positive lateral flow antigen test (2%). Interpretation After controlled experimental inoculation, the timing, extent, and routes of viral emissions was heterogeneous. We observed that a minority of participants were high airborne virus emitters, giving support to the notion of superspreading individuals or events. Our data implicates the nose as the most important source of emissions. Frequent self-testing coupled with isolation upon awareness of first symptoms could reduce onward transmissions.