Early Innate Immune Signatures Imprint Clinical Outcomes of Bordetella pertussis Challenge in a Controlled Human Infection Model

Background: Despite widespread vaccination, Bordetella pertussis (B. pertussis) continues to re-emerge globally, highlighting critical gaps in our understanding of vaccine-induced protective immunity. Controlled human infection models (CHIMs) offer a powerful platform to interrogate host-pathogen interactions in vivo, define correlates and mechanisms of protection, and inform next-generation pertussis vaccine design. Methods: This open label, phase 1, dose escalation CHIM trial (NCT05136599) was conducted at the Canadian Center for Vaccinology (Nova Scotia, Canada). Healthy adults aged 18-40 years with distinct infant vaccination histories (whole cell [wP] vs acellular [aP]) and low pre-existing antibody levels (anti-pertussis toxin [≤] 20 U/mL) were intranasally inoculated with B. pertussis isolate D420. Blood, serum, plasma, peripheral blood mononuclear cells (PBMCs), nasopharyngeal aspirates (NPA), and nasal washes (NW) were collected at baseline and multiple time points post-challenge. Multicolor flow cytometry assay was used to profile innate cellular immune responses, while Luminex-based assays were employed to quantify cytokines, chemokines and cytolytic molecules in NW samples and complement proteins in plasma samples. PBMC samples collected at selected timepoints were stimulated with heat-killed (HK) B. pertussis in vitro for assessing natural killer (NK) cell activation, effector function and maturation. Data from 59 participants receiving 106 - 108 colony-forming units (CFU) were analysed according to clinical outcome (non-infected, asymptomatic, symptomatic), sex, and vaccination history. Findings: Although infection and symptom development followed a dose-dependent pattern, 20.34% (12/59) of participants remained non-infected and had no evidence of seroconversion across all challenge doses, suggesting the existence of intrinsic resistance enabling spontaneous clearance of infection. Notably, 91.7% (11/12) of non-infected participants were wP-immunized whereas 69.5% (16/23) of aP-immunized participants developed clinical symptoms. Innate immune profiling revealed distinct immune signatures emerging within 1-3 days after challenge among CHIM participants with different clinical outcomes. Non-infected participants exhibited sustained expansion of circulating NK cells together with early mucosal production of granzyme A, granzyme B, IL-29 (type III interferon lambda 1), and MCP-2, connecting rapid cytotoxic and antiviral-like effector programming with spontaneous clearance. In contrast, symptomatic participants displayed robust early complement activation and mucosal production of eotaxin-2 and MIP-1, accompanied by broad expansion of monocytes, eosinophils, and NK cells as well as depletion of circulating neutrophils in peripheral blood. Asymptomatic individuals exhibited an intermediate phenotype characterized by early I-TAC and TRAIL production with concurrent depletion of circulating neutrophils. In vitro assays further demonstrated that B. pertussis directly induced NK cell activation and degranulation, promoting production of granzymes, perforin, and IFN-gamma; together with CD16 upregulation. Importantly, NK-cell subset mapping revealed a hierarchical pattern linking NK-cell maturation states to clinical outcomes. Non-infected participants were enriched for adaptive/memory-like NK cells and highly cytotoxic NK1C subsets, whereas symptomatic participants exhibited marked attenuation of NK-cell maturation and expansion of immature NK2 subset. Interpretation: Our results demonstrate that distinct early innate immune programs are associated with divergent clinical trajectories following B. pertussis challenge. Robust type 1 memory-like NK-cell responses likely serve as an effective first line of defense, promoting spontaneous bacterial clearance through direct cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) in non-infected participants. In contrast, increased production of type 2 inflammatory mediators, together with expansion of immature NK2 subsets, is associated with a less favorable immune environment for bacterial control. This state is accompanied by pronounced complement activation and broader engagement of innate and adaptive immune responses. Collectively, these findings reveal a previously underappreciated role for memory-like cytotoxic NK-cell responses in mediating sterilizing immunity and identify NK cell-mediated protective mechanisms as promising targets for the rational design of next-generation pertussis vaccines. Our results further highlight adaptive/memory-like cytotoxic NK cells as promising candidates for vaccine-induced immunological correlates of protection. Funding: Centers for Disease Control and Prevention (CDC), National Institutes of Health (NIH), Canadian Institute of Health Research (CIHR), IWK Health Center