Air-liquid interface (Ali) systems allow the establishment of a culture environment more representative of that in vivo than other culture systems. They are useful for performing mechanistic studies of respiratory epithelial cells as drug permeation barriers and can be used to study the interactions between hosts and respiratory pathogens. However, there have been few studies concerning Ali cultures of primary swine tracheal epithelial cells (STECs) and an immortalized STEC line, and the differences between these two systems remain poorly defined. In this study, we established Ali culture systems for primary STECs and for immortalized STEC line, and we systematically compared the differentiation capacities and immunological functions of these systems for the first time. Under Ali culture conditions, immortalized STEC line and primary STECs could survive for at least forty days, formed tight junctions and differentiated into stratified cells. They both possessed complete abilities to produce mucin and inflammatory cytokines and develop cilia. However, in contrast to primary STECs, which had a heterogeneous morphology, Ali-cultured immortalized STEC line appeared to be a homogenous population. The formation of tight junctions in Ali-cultured primary STECs was superior to that in immortalized STEC line. In addition, cilia in Ali-cultured immortalized STEC line were more pronounced, but their duration of expression was shorter than in primary STECs. Ali-cultured primary STECs and immortalized STEC line systems possessing complete abilities to undergo ciliary differentiation and inflammatory cytokine production were established for the first time in this study, and several differences in morphology and the formation of tight junctions and cilia were observed between these two systems. These two systems will be important tools for drug screening studies, as well as for detailed analyses of the interactions between hosts and respiratory pathogens.

The respiratory mucosa serves as the primary site for Pseudorabies Virus (PRV) invasion, which can elicit both local mucosal and systemic immune responses. However, due to the presence of mucosal barrier, nasal immunization with inactivated vaccines typically results in a limited immune response; thus, a mucosal adjuvant is necessary to enhance this response. Cyclic GMP-AMP (cGAMP) acts as a potent agonist of STING, exhibiting significant mucosal adjuvant properties. It plays a vital role in the regulation of innate immune responses to DNA viruses. In this study, we developed a nasal vaccine for PRV by integrating cGAMP with PRV antigen. Both mucosal and systemic antibody responses were enhanced in a dose-dependent manner by PRV antigen or cGAMP adjuvant administration. cGAMP adjuvanted PRV vaccine induced a persistent IgG but resulted in a transient IgA mucosal antibody response. Intranasal immunization with cGAMP effectively enhanced CD3⁺CD4⁺T cell responses in vivo. cGAMP adjuvanted vaccine conferred effective protection against PRV challenge. Furthermore, we also established a well differentiated air-liquid cultured mice bronchial epithelial cell (Ali-MBECs). Utilizing this model, it was demonstrated that cGAMP activates IRF3 signaling, which subsequently induced the expression of MX1, ISG56, and IFN-β. This study found that cGAMP significantly enhanced the nasal immunological efficacy of the PRV inactivated vaccine by activating the STING signaling pathway within respiratory mucosa. Therefore, cGAMP demonstrated significant potential as an effective mucosal immune adjuvant to be employed in inactivated PRV vaccine. This approach offered novel technical support for the immunization and prevention against PRV infection.

A recently identified mesenchymal cell population, telocytes (TCs), has been found in many tissues of different animal species. Jet needle-free injection (JNFI) is a promising non-invasive drug-delivery method that can trigger effective immune responses in the skin. In this preliminary morphological study, an inactivated porcine circovirus vaccine was delivered into pig neck skin by JNFI, and untreated normal neck skin served as the control. Histopathology, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to evaluate TC distribution, ultrastructure, and selected quantitative TEM parameters 24 h after injection. TCs were widely distributed in porcine skin, located between collagen fibers and around blood vessels, adipocytes, and sweat glands. They were also observed in contact with mast cells. TCs around sweat glands were CD34⁺, Vimentin⁺, and α-SMA⁺, whereas TCs at other sites were CD34⁺, Vimentin⁺, and α-SMA⁻. After JNFI, inflammatory cell infiltration into the skin was observed; TCs were in contact with these cells, and TCs surrounding adipocytes redistributed into the adjacent loose connective tissue. Quantitative TEM analysis showed that TC profile density and visible telopod length did not differ significantly between normal skin and JNFI 24 h skin (p ≥ 0.05). In contrast, vesicle profiles per TC increased significantly in both perivascular and adipose-associated compartments (p < 0.05). These findings provide morphological evidence suggesting that TCs may participate in early cutaneous responses after JNFI vaccine delivery.

Conventional needle-based vaccination may cause tissue injury and increase the risk of cross-contamination between animals. This study evaluated the immunological effects of delivering an inactivated porcine circovirus type 2 (PCV2) vaccine using a needle-free jet injection system compared with needle injection in pigs. Immune responses were assessed using enzyme-linked immunosorbent assay (ELISA) for PCV2-specific antibody levels on days 7, 14, and 28 post-vaccination. Cellular architecture at injection sites and differential gene expression were analyzed using immunohistochemistry and transcriptome sequencing. Results showed that pigs receiving needle-free vaccination at 0.50 mL and 0.75 mL injection volumes exhibited higher PCV2-specific antibody responses than pigs receiving 1.0 mL needle injection. Needle-free vaccination also preserved dermal collagen fiber architecture, altered antigen-presenting cell distribution, and was associated with distinct activation of immune-related pathways, particularly those involved in leukocyte migration and cytokine-cytokine receptor interactions. These findings indicate that needle-free vaccination was associated with distinct PCV2-specific antibody responses and local immune microenvironment changes, supporting its potential as an alternative vaccine delivery strategy for swine PCV2 vaccination.

Intradermal (ID) immunization is a potent vaccination strategy, but its potential is limited by the lack of adjuvants that can effectively enhance immune responses while minimizing local skin reactogenicity. To develop and evaluate a novel composite nano-emulsion adjuvant, designated VP, for enhancing the efficacy of intradermal vaccination against swine influenza virus (SIV). A novel adjuvant VP, formulated with squalene, sea buckthorn oil, and the TLR3 agonist Poly(I: C), was designed. Its physicochemical properties (particle size, zeta potential, viscosity) were characterized. The immunogenicity and protective efficacy of the VP-adjuvanted inactivated H3N2 vaccine were systematically evaluated in mice, comparing intradermal (ID) and intramuscular (IM) routes. Assessments included humoral immunity (serum and bronchoalveolar lavage fluid (BALF) IgG/HAI titers, duration), cellular immunity (splenic T cell subsets, cytokine profiles), and protection against homologous viral challenge (viral load, lung pathology, weight loss). Local skin reactogenicity and inflammatory responses were also investigated. The VP adjuvant exhibited a potent immune-stimulatory effect, comparable to commercial adjuvants. The VP/H3N2 formulation formed a stable nano-emulsion with an average particle size of ~ 137 nm, a near-neutral surface charge, and low viscosity (~ 1.3cP), facilitating antigen transport to skin-draining lymph nodes. Immunization induced a robust, Th1-biased immune response characterized by high IgG2a/IgG1 ratios and elevated IFN-γ secretion. Notably, the VP-adjuvanted vaccine elicited the highest proportions of IFN-γ⁺ CD4⁺, IFN-γ⁺ CD8⁺and memory-type CD44⁺ CD4⁺ T cells among all experimental groups. ID administration was superior to IM, yielding significantly higher and more durable serum/BALF antibody titers and longer-lasting immunity. VP-adjuvanted vaccination provided protection against challenge, with minimal lung pathology and markedly reduced viral loads. Crucially, ID immunization with VP elicited only a moderate, transient local inflammatory response with rapid absorption and no significant adverse skin reactions. Mechanistic studies revealed that VP functioned by upregulating key chemokines (e.g., CX3CL1, CCL19) at the injection site, significantly enhancing antigen uptake by dendritic cells in vitro, and promoting targeted antigen delivery to draining lymph nodes in vivo. This study successfully developed a novel composite nano-emulsion adjuvant, VP, which demonstrated high efficacy in intradermal SIV vaccination by potently enhancing both humoral and cellular immunity, providing durable protection, and maintaining an acceptable safety profile. The adjuvant mechanism involves enhanced local immune activation, promotion of Dendritic cells (DCs) phagocytosis, and improved lymphoid trafficking. These findings provide a theoretical and practical foundation for developing next-generation influenza vaccine adjuvants.