Evaluation of immune protective efficacy of recombinant adenovirus vector vaccine containing RBS of influenza virus subtype H1N1.

Hyaluronic acid-based dissolving microneedles regulate antigen release and route-dependent immunogenicity of adenovirus-based vaccines.

Improving cholera vaccination impact through advances in gut mucosal immunology: outcomes of a 2025 expert consultation.

African swine fever (ASF) is a highly fatal viral disease that poses a severe threat to the global swine industry. Developing a safe and effective vaccine remains an urgent and complex challenge. Lactobacillus plantarum (L. plantarum), recognized as a food-grade antigen delivery vehicle, has demonstrated great promise in vaccine development and application for various infectious diseases. To overcome the weak intestinal immunogenicity and short mucosal residence of conventional vaccines, we employed the antibiotic-free selection system of L. plantarum NC8. By fusing the nontoxic, intestinal-epithelium-targeting cholera toxin B subunit (CTB) with key ASFV antigens (p30, p54, p49, p22, pK205R, and pE248R), we constructed five newly engineered lactic acid bacteria (LAB) strains which were subsequently administered to mice orally, either individually or as a multivalent cocktail. Experimental findings revealed that oral administration of the newly engineered LAB vaccine significantly promoted the activation of DCs within the intestinal mucosa. Moreover, this oral vaccine effectively triggered T-cell immune responses in both the mucosal and systemic immune compartments and markedly enhanced B-cell activation in the intestinal mucosa. Further immunological analyses confirmed that the vaccine significantly elevated serum antigen-specific IgG and intestinal antigen-specific IgA levels. Collectively, these results indicate that the cocktail of newly engineered LAB strains elicits potent humoral, cellular, and mucosal immune responses against ASFV in mice. This novel oral immunization strategy not only provides a strong theoretical foundation for developing an oral ASFV vaccine but also contributes valuable insights for advancing oral vaccines capable of inducing systemic immune protection through mucosal immunity.IMPORTANCEAfrican swine fever virus (ASFV) is a highly virulent pathogen that continues to inflict severe economic losses on the global swine industry. In this study, we developed an environmentally friendly oral vaccine candidate utilizing an antibiotic-free selection system based on L. plantarum. A cocktail of newly engineered LAB strains, each expressing distinct ASFV antigenic proteins (p30, p54, p49, p22, pK205R, and pE248R), was administered to mice via oral gavage to evaluate its protective potential against ASFV infection. The oral delivery of this newly engineered LAB cocktail elicited robust humoral, cellular, and mucosal immune responses, demonstrating significant enhancement of the host's immunological defense mechanisms against ASFV. Overall, this strategy presents a promising approach for the prevention of ASFV infection while effectively eliminating the risks associated with antibiotic residues, thereby offering a safer and more sustainable vaccination platform for the swine industry.

Advanced age is one of the greatest risk factors for a severe outcome of COVID-19. Although mRNA vaccines were highly successful in protecting the elderly, the strongest increase in morbidity and mortality upon infection with emerging SARS-CoV-2 variants was among the elderly. To better understand SARS-CoV-2 pathogenicity and to thoroughly evaluate novel vaccination strategies, better models reliably reproducing human SARS-CoV-2 pathogenicity are needed. Here, we generated mice expressing a human-mouse chimera of ACE2 (chACE2) by CRISPR/Cas9-mediated gene editing in C57BL/6 mouse zygotes. ChACE2 mice express the chimeric viral receptor at physiological levels, enabling efficient SARS-CoV-2 infection without the heightened mortality seen in K18-hACE2 mice due to neuroinvasion. We used the chACE2 model to analyze SARS-CoV-2 infection as well as antiviral immune responses in vitro and in vivo. Similar to SARS-CoV-2 in elderly humans, aged chACE2 mice suffered from a highly aggravated disease. In addition, we found that a live attenuated vaccine candidate, LAVNsp16, induces robust mucosal and systemic immune responses in these mice despite being highly attenuated. The immunization with LAVNsp16 protected aged chACE2 mice from otherwise severe pathogenicity of SARS-CoV-2 by blocking viral replication of homologous and heterologous SARS-CoV-2 variants. The newly developed chACE2 model allows for longer observation periods of SARS-CoV-2 infection in mice, which is essential for assessing the immunogenicity of novel vaccine designs or monitoring viral pathogenicity over time. Immunization with LAVNsp16 induced robust and protective immune responses in young and aged mice, making viruses lacking 2'-O-methyltrasferse activity promising candidates for future live attenuated vaccine development.

Inhalational anthrax is a deadly disease caused by inhalation of Bacillus anthracis spores. Current anthrax vaccines for human use have limitations including undefined components, limited mucosal immunity, and suboptimal adjuvant delivery. This study introduces 2 polymers, MP1 and MP2, each containing disulfide bonds. MP1 incorporates tertiary amines to induce proton sponge effect, while MP2 features terminal phenylboronic acid moieties for protein conjugation. Nanoparticle vaccine YM1.7 is created through self-assembly of MP1, MP2, and STING agonist MSA-2, followed by N─B coordination of recombinant protein antigen (rPA) on its surface. When administered via aerosolized intratracheal inoculation into the lung, YM1.7 is internalized by antigen-presenting celland trafficked to the lysosome, where acidic environment dissociates N─B bond, releasing rPA for antigen presentation. Proton sponge effect allows nanoparticle to escape into the cytosol, and then disulfide bond cleavage triggered by cytosolic glutathionecauses dissociation of nanoparticle and release of MSA-2 within the cytosol, significantly enhancing bioavailability of MSA-2 as an adjuvant. This spatiotemporal delivery mechanism elicits a coordinated innate, humoral, mucosal, and cell-mediated immune response in mice, providing strong protection against inhalational anthrax. Given its modular design nature, YM1.7 represents a promising platform for developing next-generation mucosal vaccines against infections and cancers.

Conjugated linoleic acid (CLA) is a dietary lipid that modulates host–microbiota–immune interactions, yet its mechanistic impact on mucosal defense remains unclear. Here, we show that oral CLA supplementation enhances resistance to Salmonella Typhimurium infection and is associated with coordinated changes in gut microbial composition and mucosal immune responses. CLA-enriched commensals, including Dubosiella and Lactobacillus, were associated with increased production of CLA-derived oxylipins and activation of immune surveillance genes. Functionally, CLA pretreatment reduced Salmonella colonization, preserved epithelial integrity, and decreased neutrophilic inflammation without direct antibacterial effects. Single-cell RNA sequencing of ileal intraepithelial lymphocytes revealed that CLA predominantly reprogrammed intestinal CD8⁺ T cells toward an oxidative phenotype and enhanced effector activity. ATAC-seq revealed increased chromatin accessibility at loci associated with metabolic regulation, consistent with transcriptional reprogramming toward oxidative fitness. Mechanistically, CLA directly activated PPARγ signaling to promote mitochondrial biogenesis, oxidative phosphorylation, and the production of IFN-γ and granzyme B in CD8⁺ T cells; pharmacologic inhibition of PPARγ attenuated these effects both in vitro and in vivo. Notably, depletion of CD8⁺ T cells eliminated CLA-mediated protection and abolished early restriction of bacterial dissemination at Peyer’s patches and mesenteric lymph nodes. Although CLA enhanced CD8⁺ T-cell effector programs, antibiotic depletion and fecal microbiota transplantation experiments demonstrated that an intact gut microbiota is necessary for effective protection in vivo. Together, these findings identify CLA as a dietary modulator that strengthens mucosal resistance to Salmonella by promoting PPARγ-mediated metabolic reprogramming and enhanced effector fitness in intestinal CD8⁺ T cells.

The inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) are disorders that cause chronic inflammation of the gastrointestinal tract. Both genetic and environmental factors contribute to the pathogenesis of IBD. There are currently >200 known genetic susceptibility loci for the development of IBD. The physiological impact of the majority of these loci remain a gap in our knowledge. One such locus is the single nucleotide polymorphism rs1077773, located ~56 kbp downstream from the aryl hydrocarbon receptor (AHR) gene. AHR is a ligand-activated transcription factor that is crucial to maintaining intestinal homeostasis. We hypothesized that rs1077773 enhances AHR activity to regulate mucosal immune response and maintain intestinal homeostasis. All study procedures and reagents were approved by the Washington University Institutional Review Board (#202011003). Patient biopsies were collected at Barnes Jewish Hospital and genotyped using the IBD Genetics Consortium custom GSA SNP chip (Broad Institute) followed by imputation using TopMed Imputation Server at University of Michigan. Patient derived organoids (PDOs; N=3 G/G, N=4 G/A, N=5 A/A) were derived and maintained in 3D culture and supplemented with 50% L-WRN conditioned medium with passage every 3-4 days as previously described. PDOs were treated with AHR agonist 6-Formylindolo[3,2-b]carbazole (FICZ) or vehicle for 48 h. Expression of AHR and its transcriptional targets Cytochrome P450 1A1 (CYP1A1) and CYP1B1 was assessed by RT-qPCR. Blood was collected from pediatric patients undergoing intestinal resection at St. Louis Children's Hospital and was genotyped with custom TaqMan SNP assay (N=3 G/G, N=5 G/A). Peripheral blood monocyte-derived macrophages (MDMΦs) were treated with lipopolysaccharide in the presence or absence of AHR ligands FICZ or indole-3-carboxaldehyde for 24 h. Cytokine levels in culture supernatant were measured via using the ProcartaPlex human cytokine, chemokine, and growth factor 45-plex (ThermoFisher) on a Luminex FLEXMAP3D instrument. AHR expression was similar across genotypes and treatments. PDOs homozygous for rs1077773 demonstrate enhanced CYP1A1 expression in response to AHR activation. In MDMΦs, cytokine secretion was stimulated by LPS treatment and was abrogated by FICZ treatment. MDMΦs with rs1077773 alternate allele demonstrated significant reduction in secretion of 12 cytokines and chemokines. This work demonstrates that rs1077773 enhances AHR activity and modulates epithelial and immune cell function in vitro. Further mechanistic understanding of this locus and its correlates could improve our understanding of the molecular mechanisms of IBD susceptibility and may lead to novel personalized therapeutic approaches in IBD.

Periodontitis is a chronic inflammatory disease driven by oral microbial dysbiosis and dysregulated host immunity, in which Porphyromonas gingivalis (Pg) and its lysine-specific gingipain (Kgp) are key pathogenic factors. Although immune targeting of Kgp holds promise for interrupting disease progression, inefficient transmucosal antigen delivery and limited dendritic cell (DC) activation hinder effective mucosal and humoral immune responses. Here, we report a sublingual mucosal nanovaccine based on tetrahedral framework nucleic acids (tFNAs) for precise DC-directed immunomodulation. The vaccine integrates a DC-targeting aptamer, a Kgp-specific antigenic peptide (KAS1), and Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODNs) adjuvant within a programmable tFNAs scaffold and is embedded in a biodegradable mixed polyethylene glycol-based (MixPEG) hydrogel to enable spatially controlled assembly and sustained sublingual delivery. Leveraging the intrinsic transmucosal transport capability of tFNAs and the bioadhesive properties of the hydrogel, this system achieves noninvasive sublingual administration and efficient uptake by local DCs, inducing notable Kgp-specific immune responses. In a murine periodontitis model, sublingual immunization enhances salivary IgA production, suppresses Pg colonization, attenuates periodontal inflammation, and mitigates alveolar bone loss, representing a potentially promising strategy for periodontitis.

The FDA approved the first monovalent XBB1.5 booster vaccine in September 2023. However, whether this vaccine stimulates mucosal immune responses in the oropharynx, especially neutralizing antibodies, remains understudied. We analyzed serum and saliva samples from 28 participants, collected one week before and two to three weeks after the XBB1.5 mRNA vaccination, for neutralizing and binding antibodies to Wuhan and XBB1.5 Spike (S) protein. We observed a 2.9-fold increase in Wuhan and a 5.0-fold rise in XBB1.5 serum neutralization titers after mRNA vaccination, which correlated with increased binding antibody levels against the S protein variants in serum. We also examined saliva to assess oral mucosal immune responses to vaccination. Vaccination caused a 1.7-fold increase in ACE2 neutralization in saliva against XBB1.5 (p<0.0001) and a 1.4-fold increase against Wuhan (p=0.03). This rise in neutralizing antibody levels in saliva was not associated with the number and timing of previous COVID-19 infections, vaccination status, vaccine type, age, sex, or increases in S-specific IgG in either saliva or serum. Individuals with the largest increase in XBB1.5 neutralization in saliva tended to show a greater rise in S-specific sIgA levels. Depletion of salivary IgA1 or IgG after vaccination revealed that IgA1 was mainly responsible for the increase in ACE2 blocking activity (average reduction in blocking activity with IgA1 depletion=68%, range 58-81%) compared to IgG (average reduction=27%, range 0-71%). The XBB1.5 monovalent vaccine boosts neutralizing antibody levels in blood and mucous membranes, with the largest increase observed against the XBB1.5 variant. This likely reflects the enhancement of cross-reactive antibodies influenced by prior exposure and hybrid immunity, rather than the production of entirely XBB1.5-specific responses. Improved oral mucosal immune responses may reduce the risk of COVID-19 infection and severe illness.

Development of a chitosan-based acid-responsive mucoadhesive mRNA vaccine for eliciting mucosal and systemic immunity.

Current coronavirus disease 2019 (COVID-19) vaccines effectively prevent severe disease but induce primarily systemic immunity without mucosal protection in the respiratory tract, which is mandatory for protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the prevention of viral transmission. Vaccination strategies capable of inducing local immunity at the site of infection are therefore needed. Here, we evaluated a poly (lactic-co-glycolic acid) (PLGA) microparticle-based vaccine co-encapsulating the receptor binding domain (RBD) of SARS-CoV-2 Spike protein with the TLR3/RIG-1 agonist Riboxxim, employing a subcutaneous prime and intranasal boost immunization schedule. BALB/c mice received subcutaneous prime immunization followed by intranasal boost with PLGA microparticles containing RBD/Riboxxim. Antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), neutralization by competitive ELISA and T-cell responses by enzyme-linked immune spot assay, intracellular cytokine staining and flow cytometry. Memory responses were evaluated 30 days post boost immunization. Vaccination induced robust RBD-specific IgG and IgA antibody titres in both serum and bronchoalveolar lavage fluid, with neutralizing capacity against the Wuhan-Hu-1 strain. Strong CD4+ and CD8+ T-cell responses were detected systemically and in the respiratory tract. Importantly, the vaccine generated durable immunological memory, including tissue-resident memory T-cells in the respiratory tract and long-lived IgG and IgA memory B-cells in secondary lymphoid organs. PLGA microparticle-based vaccination induces potent systemic and mucosal immune responses against SARS-CoV-2 RBD. This adaptable platform represents a promising approach for mucosal vaccination strategies, with potential for rapid adaptation to emerging variants.

SARS-CoV-2 viral load in the upper respiratory tract (URT) typically peaks and declines within days of infection, even in individuals without prior infection or vaccination. Although this implicates the URT innate immune response in effectively restricting viral replication, the nature of the protective responses and how they are affected by demographic factors is poorly defined. We recruited 54 seronegative household contacts of recently diagnosed COVID-19 cases and prospectively collected URT samples during and after exposure. Among the 39 individuals who became infected, we quantified airway mucosal cytokine and chemokine responses and virus-specific nasal IgA using Meso Scale Discovery assays, and assessed associations with demographic factors, viral load, and symptoms. Participants with higher BMI had higher URT viral loads and more marked symptoms. This was significantly associated with delayed induction of protective inflammatory mediators in the airway mucosa but not in blood. Induction of virus-specific nasal IgA at 1-week post-infection also correlated with lower viral load. Elevated BMI retards initial airway mucosal innate immune responses to infection, which may partially explain the pronounced adverse impact of higher BMI on clinical and virological outcomes in COVID-19. This work is supported by the NIHR Health Protection Research Unit in Respiratory Infections, Imperial College London in partnership with the UK Health Security Agency (Grant number: NIHR200927; AL) and the Medical Research Council (Grant number: MR/X004058/1). Infrastructure support for this research was provided by the NIHR Imperial Biomedical Research Centre (BRC).

A semi-replicating VSV (srVSV)-based platform for developing broad-spectrum mucosal vaccines against influenza A viruses.

Antimicrobial peptides (AMPs) are essential components of the innate immune system, exhibiting diverse mechanisms of action. This study investigates the roles of cathelicidin (LL-37), alpha-defensins, and the S100 proteins S100A8 and S100A9 in systemic inflammation associated with sepsis, severe COVID-19, and acute pancreatitis using whole-blood bulk RNA-sequencing data. Gene co-expression network analysis revealed that during septic shock and severe COVID-19, cathelicidin and alpha-defensins act synergistically in innate immune responses, while S100A8 and S100A9 function through distinct pathways related to mitochondrial metabolism and ubiquitin ligase binding. In contrast, the acute pancreatitis network displayed a different pattern, with CAMP co-expressed alongside S100A8 and S100A9, whereas alpha-defensins were downregulated and associated with inhibited mucosal immune responses. These findings suggest that antimicrobial peptides contribute variably to systemic inflammation depending on the underlying insult, underscoring their complex, context-dependent roles in critical illness.

Time-dependent imprinting of gut homing receptors on lymphocytes by migratory antigen presenting cells.

Oral lichen planus (OLP) is a common T-cell-mediated inflammatory disease affecting the oral mucosa. Intraepithelial lymphocytes (IELs), a unique subset of T cells, play a crucial role in regulating mucosal immune responses. However, the mechanisms by which keratinocytes (KCs) regulate the homing migration of OLP IELs and their involvement in mucosal barrier disruption remain unclear. This study conducted colocalization and quantitative analysis of the expression of E-cadherin, CD103, CD8α, ZO-1, and Occludin. A three-dimensional simulation homing model of oral mucosal tissue was constructed. Short hairpin RNAs (shRNAs) were designed to inhibit E-cadherin and CD103 of KCs and OLP IELs. The JAK/STAT pathway was inhibited using AG490 and ruxolitinib (RPM). The expression levels of ZO-1 and occludin were detected. CD8α and CD103 were highly expressed in OLP, while the expression of E-cadherin, ZO-1, and Occludin was decreased. Silencing KCs' E-cadherin and IELs' CD103 significantly inhibited the homing migration of OLP IELs. After inhibiting the JAK/STAT pathway, KCs proliferation was reduced, while Bax and caspase-3 expression were upregulated and Bcl-2 expression was downregulated. The homing migration of OLP IELs was inhibited, with decreased expression of p-JAK2/JAK2 and p-STAT3/STAT3. Furthermore, ZO-1 and Occludin were upregulated. The regulation of KCs on homing migration of OLP IELs depended on KCs' E-cadherin and IELs' CD103. By downregulating JAK2/STAT3 phosphorylation, KCs proliferation was inhibited and apoptosis was induced, which has therapeutic benefits for OLP epithelial dysplasia. Meanwhile, upregulation of mucosal barrier molecule expression helps maintain the integrity of the mucosal barrier.

Background/Objectives: Hand, foot, and mouth disease (HFMD) is a major public health concern primarily caused by human enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and certain coxsackievirus B serotypes. Currently available EV-A71 vaccines lack cross-protective efficacy against other serotypes, highlighting the urgent need for multivalent and broadly effective enterovirus vaccines. Methods: Immunoinformatics approaches were used to predict highly immunogenic B-cell and T-cell epitopes, which were assembled to construct a novel multivalent epitope vaccine, rCV-A3V, followed by in silico validation. Recombinant protein expression was confirmed by Western blotting and immunofluorescence assays. The immunogenicity was evaluated in Balb/c mice following intranasal immunization. Results: A preliminary safety evaluation demonstrated that the rCV-A3V vaccine was well tolerated in the mouse model, with no abnormal changes in body weight observed after immunization. In addition, the target protein was successfully expressed. Intranasal immunization induced a strong Th1-biased immune response, robust serum neutralizing and IgG antibody responses, and pronounced mucosal immunity, including elevated sIgA and IgG levels in nasal lavage fluid, sIgA in feces, and substantial sIgA responses in milk. Dominant epitope peptides were also identified. Conclusions: The intranasal live attenuated rCV-A3V vaccine successfully induced humoral, mucosal, and cellular immune responses against EV-A71, CVA16, CVA6, and CVB3, demonstrating broad immunogenicity. These findings provide experimental evidence supporting its potential as a candidate vaccine for HFMD.

ABSTRACTFibrinogen‐like protein 2 (Fgl2) is a critical immunoregulatory factor, yet its precise roles in B‐cell biology and mucosal immunity remain largely undefined. In this study, utilizing Fgl2‐knockout (KO) mice, we identified novel B cell subsets in the spleen (SPL), predominantly characterized by IGHA clonal dominance. Employing an intestinal Trichinella spiralis (T. s) infection model and samples from patients exhibiting mucosal immune responses (the early stage of COVID‐19 infection), we investigated the function of Fgl2 in mucosal immunity. We demonstrate that Fgl2 directly interacts with Receptor for activated C‐kinase 1 (Rack1), thereby attenuating B cell receptor (BCR) signaling and metabolic activity by inhibiting AKT phosphorylation. Furthermore, the Fgl2 deficiency‐induced expansion of marginal zone (MZ) B cells, germinal center (GC) B cells, and IgA+ plasma cells was effectively counteracted by in vivo Rack1 inhibition. Consistently, a Rack1 inhibitor also abrogated the enhanced activation of Fgl2‐deficient B cells in vitro. Fgl2 deficiency also augmented early B cell activation, including B cell spreading, clustering, and signalosome recruitment, through upregulation of the DOCK8‐WASP‐actin axis. Our research uncovers an intrinsic role for Fgl2 in regulating BCR signaling, B cell differentiation, and mucosal immunity, elucidating a key underlying molecular mechanism.

Smoking is considered a global pandemic with more than 1.3 billion people being active smokers. Increasing evidence suggests that nicotine exposure can lead to changes in the gut microbiome, increases in permeability, and impaired mucosal immune responses in the gastrointestinal tract. However, the literature on behavioral aspects of nicotine-microbiome interaction, such as dependence and withdrawal, is limited. In this study, we used homologous fecal material transplants (FMT) to modify the gut microbiome and its impact on the intensity of nicotine withdrawal in mice. We used osmotic minipumps as an application of chronic nicotine for 15days and orally gavaged FMT 2x a day to the mice. We assessed the nicotine withdrawal by measuring the number of somatic signs and anxiety-like behaviors at 24h and 1week after the mini pump removal. Fecal samples were also collected points to identify the gut microbiome changes. Fecal transplants reduced the number of somatic signs and anxiety-like behaviors in nicotine-treated mice up to a week after the removal of minipumps. The shotgun metagenomic results of the fecal samples from 24h after minipumps removal time point show altered gut microbiome with a significant shift in the species composition between the nicotine treated and its homologous FMT treatment. Our results indicate that under our experimental conditions fecal transplant can reduce the severity of nicotine withdrawal. This suggests that interactions along the gut-brain axis are important for the development of nicotine dependence and might help lower the risk of cancer and other serious health problems in humans.