This section contains data from the Surveillance of Respiratory Viruses in Healthcare and Animal Workers (SENTINEL) research project in the Netherlands.
Outbreaks with respiratory viruses occur continuously. This project is designed to detect early circulation of (novel) respiratory viruses in both symptomatic and asymptomatic individuals, either by direct viral detection or changes in local or systemic immunity. Additionally, SENTINEL will provide information on infectivity of viruses during outbreaks, and (protective) immune responses after infection and/or vaccination.
Highly pathogenic avian influenza A(H5) viruses pose a pandemic threat, with a history of mammalian adaptation and zoonotic spillovers into humans. We aimed to determine whether pre-existing cross-reactive immune responses to recent A(H5) clade 2.3.4.4b influenza viruses are present in the general population. To that end, we conducted an observational cross-sectional study within the prospective ‘Surveillance of rEspiratory viruses iN healThcare and anImal workers in the NethErLands’ (SENTINEL) cohort, in which we analyzed a subset of 107 healthcare workers (HCW) who completed their periodic study visit in August or September 2024. Blood samples were analyzed for influenza A(H5)-specific antibody binding, hemagglutination inhibition, Fc-effector functions, neuraminidase (NA) inhibition, and T-cell responses. Median age of included HCW was 50 years (IQR 40–58); 77/107 (72%) were female, 29/107 (27%) male, and 1/107 (1%) did not report. Low-level binding antibodies directed against the A(H5) hemagglutinin (HA) head were detected in up to 28 individuals (depending on the antigen), but without hemagglutination inhibition activity. Nevertheless, we detected A(H5)-reactive antibodies with Fc-effector functions in all participants, likely targeting the conserved HA stalk. Additionally, we observed high levels of NA inhibiting antibodies (GMT: 208 (95% CI: 153-284)) in up to 97% of the HCW against avian N1, and T-cell responses against HA and NA from A(H5) influenza viruses in the majority of HCW. A(H5)-specific responses correlated with immune responses targeting A(H1N1), indicating they were likely induced by prior exposures to seasonal influenza viruses. Together, our findings suggest that partial cross-reactive immunity to A(H5) influenza viruses exists in humans, which may play an important role during future outbreaks, potentially by blunting disease severity. Characterizing such baseline immunity is crucial for accurate pandemic risk assessment and preparedness planning.
Recent human infections with highly pathogenic avian influenza viruses (HPAIV) of the A(H5) hemagglutinin (HA) subtype underscore their pandemic potential, highlighting the need for stockpiled vaccines as a pandemic preparedness measure. The Netherlands obtained the MF59-adjuvanted inactivated A(H5N8) vaccine (manufactured by CSL Seqirus) based on A(H5) clade 2.3.4.4b A/Astrakhan/3212/2020, through a joint European procurement, and offered it to healthcare workers (HCW) at risk of occupational exposure. Given the limited data on the breadth of immune responses induced by this vaccine, this study evaluated antibody and T-cell responses upon vaccination of naïve HCW and HCW previously vaccinated with A(H5) influenza vaccines. As a sub-study of an ongoing observational cohort study, immune responses at baseline and on days 7 and 28 after each of the two vaccine doses were evaluated in 39 HCW. Of those 39 HCW, 6 had been previously vaccinated with A(H5) influenza vaccines containing antigenically distinct HA. HA-binding antibodies were measured via protein microarray (PMA), and functional antibodies were quantified by hemagglutinin inhibition (HI), neuraminidase inhibition (NI), and antibody-dependent cellular cytotoxicity (ADCC) assays. T-cell responses were detected by interferon-gamma release assays (IGRA) and activation-induced marker (AIM) assays. HI antibodies targeting clade 2.3.4.4b A(H5) influenza viruses, including A/Astrakhan/3212/2020 (vaccine antigen) and A/Texas/37/2024 (isolated during the cattle outbreak in the United States), were induced in all naïve HCW. When post-boost sera were tested against 80 antigenically diverse A(H5) influenza viruses from our A(H5) antigenic map (PMID: 41094140) , clade 2.3.4.4b-restricted HI responses were primarily detected in naïve HCW, in contrast to a broad response against almost all tested viruses in previously vaccinated HCW. Additionally, de novo induction of N8-specific antibodies was detected. Most HCW had detectable A(H5)-reactive ADCC antibodies and T-cells at baseline, which were boosted by vaccination. Our findings show that the zoonotic influenza vaccine induced HI antibodies targeting clade 2.3.4.4b A(H5) viruses in naïve HCW. In addition to HI antibodies, the induction of NI antibodies, and boosting of ADCC antibodies and T-cells targeting the vaccine antigen was detected. While naïve individuals developed HI responses restricted to clade 2.3.4.4b, previously vaccinated individuals had broad HI reactivity across all A(H5) clades. This highlights the potential of heterologous prime-boost vaccination strategies to induce broad A(H5) immunity.
The upper respiratory tract is a key entry point for pathogens, yet local tissue-resident memory T cells (Trm) remain understudied compared to memory T cells in peripheral blood. Here, we systematically compared nasal curettes and flocked swabs for immune cell sampling from the nose, assessing yield, operator variability, and T cell phenotypes across the nasal turbinates and nasopharynx. The use of flocked swabs yielded higher immune cell numbers while being similarly tolerable. Nasal turbinate-derived Trm subsets were consistent, whereas nasopharyngeal Trm displayed a more recently recruited phenotype. Multiple cryopreservation media were compared, all of which demonstrated high viability after thawing. Antigen-specificity was assessed using activation-induced marker (AIM) assay, cytokine release assays and peptide–HLA tetramer staining. Notably, influenza virus-specific T cells were reliably detected in more than half of the cryopreserved nasal samples. These findings establish a robust approach for nasal Trm sampling and profiling, while demonstrating that cryopreservation preserves antigen-specific T cells. This work enables standardized, minimally invasive nasal immune monitoring for multi-center studies, including mucosal vaccination trials and controlled human infection models, providing a critical framework for understanding local immunity at the site of pathogen entry.