Infections occurring at mucosal tissues are one of the major causes of global mortality and morbidity. For example, respiratory viral infections account for approximately 4 million deaths annually, primarily in immunocompromised individuals, children, and elderly patients (Khales et al., 2025). Severe outbreaks of influenza, respiratory syncytial virus (RSV) and SARS-CoV-2 occurred simultaneously several times in the last six years, with increased healthcare burden on all continents. Influenza virus and RSV are responsible for respectively 100, 000 and 290, 000-650, 000 deaths every year (WHO.int) (B27, WHO, n.d.), while SARS-CoV-2 has led to 600 million reported cases and close to 7 million deaths from its initial outbreak (Healthdata.org). In addition to directly compromising the respiratory health, these viruses also aggravate diseases including chronic obstructive pulmonary disease and asthma (Traves and Proud, 2007). The recent introduction (RSV and SARS-CoV-2) or increasingly widespread use (influenza virus) of vaccines against these pathogens led respectively to 35 to 64% (RSV) (Du et al., 2025), 90 to 95% (SARS-CoV-2) (Sheikh et al., 2021), and 42% (influenza virus) (Yegorov et al., 2025) decreases in hospitalizations among immunized adults, illustrating the potency of vaccines. However, despite their instrumental role in limiting the mortality rate, these intramuscularly administered vaccines proved insufficient to halt the transmission of pandemic pathogens and to provide long-lasting protection. This highlights the necessity to develop new effective vaccination strategies, with a particular focus on the protection of mucosal surfaces that constitute both the entry route and a major site of replication for most human pathogens (Tobias et al., 2024). To this end, it is essential to understand the distinctive features of mucosal immunity.

The mucosa is the first line of defense against infections, allowing immunological monitoring. It stretches across an area approximately 200 times greater than the skin (Brandtzaeg, 2009; Mowat and Agace, 2014). The mucosa consists of an epithelial structure embedded with mucosa-associated lymphoid tissues (MALTs) such as the tonsils in the upper respiratory tract (Talks et al., 2024).

Mucosal immunity presents an important role in human defense by reducing the transmission of pathogens at their entry points (Miquel-Clopes et al., 2019; Tscherne and Krammer, 2025). Although promising, mucosal vaccines are rare, with only few of them having received human approval (Figure 1) (Shakya et al., 2016; Fraser et al., 2023). A number of persisting problems such as the enzymatic and structural barriers that hinder stability and antigen absorption, pre-existing immune tolerance, and the limited availability of potent licensed mucosal adjuvants (Rhee et al., 2012) hampers their widespread use. However, significant advantages such as the localized protection at infection sites and needle-free delivery (Song et al., 2024; Boyaka, 2017; Pilapitiya et al., 2023) might improve vaccine acceptance and coverage. Certain studies have also suggested that these vaccines are more effective when compared to systemic vaccines in preventing respiratory infections (Paris et al., 2021; Matsuda et al., 2021; Li et al., 2025), which has led to a spark of interest in mucosal vaccines, especially in the context of COVID-19 and influenza virus (Singh et al., 2023; Kastenschmidt et al., 2023).

Recent advances in regulatory frameworks, manufacturing methods, and delivery platforms designed specifically for mucosal immunization such as the inhalable or oral tablet forms are accelerating progress in this field (Zafar et al., 2023; Zhang et al., 2025).aApproximately 34 mucosal vaccine candidates reached clinical trials to date (Knisely et al., 2023; AbsolutelyMaybe.plos.org) indicating an emerging trend in diversifying mucosal vaccines despite the limited number of approved candidates.

One of the factors hampering the translation to the clinic of innovative mucosal vaccines is that animal models are poorly predictive of their immunogenicity in humans (Kraan et al., 2014; Jameson and Masopust, 2018; Kayesh et al., 2021; Gibbons and Spencer, 2011; Tscherne et al., 2025). This limitation has been attributed to interspecies variations in mucosal structure, innate immunity, IgA class switching patterns, and receptor expression. For example, rodents lack tonsils, which are a major site of mucosal immunization in humans, and contrary to human mucosal and lymphoid tissues cannot be infected by CD46-tropic adenovirus vaccine vectors (Sakurai et al., 2006). Consequently, unlocking the potential of mucosal vaccines requires experimental models that are more representative of human mucosal immunity may unlock the potential of mucosal vaccines (Kessie and Rudel, 2021), which is an area where human-derived ex vivo cultures are rapidly gaining relevance.

Ex vivo models derived from human primary tissues are recognized as critical tools to bridge the gap between in vitro and animal models. Building upon early 2D primary cultures or multicellular spheroids, the field of ex vivo models diversified exponentially in the 21^st century. Prominent systems include tumoral self-organizing organoids used for precision oncology and drug screening (Davies, 2018), and organs-on-chips relying on microfluidics systems (Ingber, 2022). Contrary to simple 2D cultures of immortalized cell lines, these platforms offer a more biologically relevant context to investigate immune activation, host-pathogen interactions etc (Fergusson et al., 2025). Unlike animal models, primary ex vivo models originate from human donors, and thus retain crucial immune cell composition, stromal components, and other species-specific characteristics (Figure 2) (Hoffmann et al., 1995). Here, we will present an overview of current ex vivo mucosal models and discuss how they have been applied or could be applied in the future to address critical challenges in mucosal vaccine development.

Experimental models should be predictive of mucosal vaccines’ immunogenicity in humans. Consequently, highly valued readouts from ex vivo models include physiologically relevant antigen penetration and presentation, but also predictiveness of adaptive immune response features such as the clonality and neutralizing titers of humoral responses. In particular, primary adaptive responses have been more challenging to reproduce experimentally due to difficulties in maintaining physiological function of diverse interacting cell types and in reaching sufficient numbers of antigen-specific naïve lymphocytes. Models able to reliably mount such responses could be instrumental in the development of potent single-dose vaccines, a major goal of pandemic preparedness.

Although these successes highlight the multiple advantages of human ex vivo primary cultures compared to traditional animal models or established cell lines, certain technical and biological challenges have not been fully addressed to date. A number of experimental systems are still limited to a low throughput by low tissue availability, ethical requirements or technical complexity. However, certain models including tonsil lymphoid organoids or commercial ALI models are more accessible and benefit from a higher experimental throughput, while the relentless progress seen in the last decade already helped democratizing organ-on-chips technologies.

Most mucosal COVID vaccines that reached clinical trials were repurposed systemic vaccines with little to no prior experience in mucosal delivery. For example, the ChAdOx1 and SAd36 adenovirus platforms were initially developed and tested for intramuscular vaccination (Antrobus et al., 2014; Fonseca et al., 2017). The former proved insufficiently immunogenic as a mucosal vaccine (Madhavan et al., 2022), while the latter was licensed as the iNCOVACC vaccine. However, it can be expected that the success rate of clinical translation would have been higher if suitable mucosal ex vivo models had been available instead of or complementarily with animal experiments. Likewise, the Flumist intranasal live influenza vaccine was reported to insufficiently protect against H1N1 viruses, because the H1N1 strain present in this multivalent vaccine tends to be outcompeted by other strains in vivo (Dibben et al., 2021). We thus hypothesize that using airway organoids during vaccine development may have helped to identify and address this limitation earlier.

The technical progress in ex vivo models align with regulatory innovations aimed at reducing the reliance of biomedical research on animal experiments, following the 3R principles. Notably, the recent FDA Modernization Act 2.0 authorizes clinical trials to be undertaken on the basis of ex vivo studies. This further increases the scientific and medical relevance of advanced culture systems. Nevertheless, mucosal vaccines still face regulatory hurdles as compared with the better-known systemic vaccines, in particular due to the scarcity of robustly validated mucosal correlates of protection (King et al., 2024). However, certain high-throughput ex vivo models may facilitate systematic screening of vaccinees’ serum or mucosal secretions for protection from pathogen infection in challenge studies. These experiments could help identify biomarkers of vaccination success based on multi-OMICs phenotyping of vaccinees’ samples (Kessie and Rudel, 2021).

Though the use of complex organotypic and/or immunocompetent cultures is still limited by technical skills requirements of lack of throughput or availability of tissues of origin, it is becoming increasingly accessible due to technical developments in culture longevity, readout sensitivity and more. For example, ex vivo models mounting primary immune responses, once considered unfeasible, are described on an increasingly frequent basis (Wagar et al., 2021; Zhai et al., 2025; Suhito et al., 2025)(We expect that increased cross-talk between scientific fields will help lower entry barrier and facilitate the adaptation of existing models, such as systemic lymphoid follicle-on-chips, to mucosal tissues. To fully realize the potential of mucosal ex vivo cultures, clinical validation of the immune response of each model would be required. Such experiments are still very rare both for mucosal and systemic cultures, going so far little further than recapitulating known immune activation mechanisms (Kastenschmidt et al., 2023) ex vivo, or showing that organoids grown from PBMCs of older donors secrete less antibodies than those of younger donors (Dauner et al., 2017). Therefore, ambitious studies comparing the immune response of ex vivo cultures to well-known vaccines with responses of tissue donors vaccinated after sample donation are warranted. Though not feasible for all models due to ethical requirements, these studies may prove critical in identifying best-in-class models and demonstrating clinical relevance. Together, these trends emphasize that while the field is advancing, bridging experimental progress with real-world impact will require coordinated innovation in immunology, engineering, and regulation.

The increasing burden of respiratory infections, worsened by concurrent outbreaks of SARS-CoV-2, influenza virus, and RSV, continues to represent a significant challenge on global public health systems, especially among vulnerable populations. While conventional systemic vaccines have considerably decreased hospitalizations and severity of the disease, their inadequate efficacy to prevent pathogen transmission highlights the need for novel mucosal vaccine platforms.

Mucosal vaccines provide localized immunity at the site of infection and therefore hold significant potential for infection control despite persisting challenges. However, biological, technical, and regulatory barriers such as mucosal tolerance, restricted antigen absorption, and the lack of approved mucosal adjuvants hinder their advancement (Sallard and Aydin, 2024). To overcome these, advances in vaccine administration methods such as licensed intranasal sprays and oral tablets or experimental microneedle patches (Paris et al., 2021) are paving the way toward the next generation of vaccines. Nevertheless, the inability of existing animal models and in vitro systems to precisely mimic human mucosal responses remain a challenge (Kayesh et al., 2021). Under these physiological constraints, ex vivo primary human tissue models, such as tonsil organoids or airway ALI cultures offer a more biologically relevant platform to investigate antigen penetration of target tissue or epithelial–immune interactions. Current models may be further refined to provide a holistic and accurate overview of the mucosal immunogenicity of candidate vaccines.

Furthermore, advanced immunophenotyping methods and standardized donor stratification might improve interpretability and reproducibility of existing models. Moreover, considering the significant influence of microbiome on mucosal immune regulation, including them into ex vivo cultures might also be beneficial.

In conclusion, mucosal vaccines are highly promising but remain an underdeveloped approach in respiratory disease control. To unlock their full potential, concurrent advances in tissue engineering, bioanalytical modelling and immunology are required. Ex vivo human-derived platforms may drive the design, evaluation, and translation of next-generation mucosal vaccines by merging experimental standardization and biological complexity, ultimately advancing effective vaccination strategies.