Search Results (503)

Background/Objective: Francisella tularensis is a highly infectious intracellular pathogen that causes severe pulmonary tularemia following aerosol exposure, yet no licensed vaccine exists. Because infection initiates at the respiratory mucosa, understanding mechanisms of protective pulmonary immunity is critical for mucosal vaccine development. This study investigated the role of lung-resident γδ T cells following intranasal immunization with inactivated F. tularensis (iFt) and subsequent lethal challenge with live vaccine strain (LVS). Methods: Mice were intranasally immunized with iFt and later challenged with lethal LVS. Pulmonary immune responses were evaluated using flow cytometry and cytokine analysis. Recruitment of γδ and αβ T cells, production of IL-17 and IFN-γ, neutrophil infiltration, and γδ T cell memory phenotypes were assessed in naïve and immunized mice following infection. Results: Primary LVS infection induced rapid recruitment of γδ T cells to the lung beginning on Day 2 post-infection, preceding significant αβ T cell accumulation. Increased pulmonary IL-17 and IFN-γ correlated with expansion of IL-17– and IFN-γ–associated γδ T cell populations. Following iFt immunization, mice demonstrated enhanced survival after lethal LVS challenge, accompanied by early increases in pulmonary IL-17 and IL-17 producing γδ T cells. Immunized mice also exhibited expansion of effector memory and central memory γδ T cell populations associated with IL-17 production. Conclusions: These findings identify IL-17 producing γδ T cells as contributors to early mucosal immunity following intranasal vaccination against F. tularensis and suggest that targeting lung-resident γδ T cells may support the development of next-generation mucosal vaccines against respiratory pathogens. Full article

Air pollutants can cause respiratory diseases, highlighting the need for effective preventive and therapeutic strategies. We investigated the protective effects of heat-killed Lacticaseibacillus paracasei ATG-E1 against particulate matter plus diesel exhaust particle (PM₁₀D)-induced airway inflammation. BALB/c mice were intranasally injected with PM₁₀D and treated with heat-killed L. paracasei ATG-E1 via oral gavage for 5 days. In the bronchoalveolar lavage fluid (BALF) and lungs, inflammatory mediators, immune cell subtypes, and histological changes were analyzed, while gut microbiota composition was analyzed in the cecum. Heat-killed L. paracasei ATG-E1 suppressed the infiltration of immune cells, including neutrophils, T cells, and B cells. Furthermore, it decreased various inflammatory mediators, such as C-X-C Motif chemokine ligand (CXCL)-1, macrophage inflammatory protein (MIP)-2, interleukin (IL)-1α, and tumor necrosis factor (TNF)-α, in the BALF and lung tissue, as well as serum symmetric dimethylarginine (SDMA) levels in the PM₁₀D-induced airway inflammation model. Heat-killed L. paracasei ATG-E1 also exhibited a protective effect against lung damage induced by PM₁₀D. Furthermore, heat-killed L. paracasei ATG-E1 treatment shifted the gut microbiota composition, increasing several bacterial genera. The data demonstrate that heat-killed L. paracasei ATG-E1 acts as a protective agent against air pollutant-induced lung injury, suggesting its potential as a candidate adjunctive strategy for prevention. Full article

Background: The COVID-19 pandemic exposed critical gaps in pandemic preparedness and highlighted the need for vaccine platforms capable of rapid adaptation. Outer membrane vesicle (OMV)-based platforms utilizing vesicles derived from genetically detoxified Neisseria meningitidis serogroup B (Nm-nOMV) represent a promising plug-and-play approach. Methods: This Phase I, first-in-human, randomized, double-blind, placebo- and OMV-controlled trial, evaluated safety, tolerability, and immunogenicity of intranasally administered OMVs combined with SARS-CoV-2 Spike protein in healthy SARS-CoV-2 seropositive adults aged 18–55 years. Forty participants were enrolled across two cohorts: a low-dose cohort receiving 140 μg OMV/70 μg Spike (OMV + Spike, n = 13; OMV alone, n= 3; Placebo, n = 5) and a high-dose cohort receiving 280 μg of OMV/140 μg of Spike (OMV + Spike, n = 13; OMV alone, n = 3; Placebo, n = 3), administered on Days 1 and 22. Safety was assessed through adverse events, vital signs, laboratory parameters, ECG, and pulse oximetry. Immunogenicity was evaluated via systemic SARS-CoV-2 neutralizing antibodies, antigen-specific antibodies (IgG and IgA), and mucosal antibodies (IgA in nasal wash). Results: Intranasal administration of OMVs combined with SARS-CoV-2 Spike protein was safe, well-tolerated, and immunogenic. No serious adverse events were reported, and adverse events were predominantly mild and transient. Dose-dependent increases in systemic and mucosal immune responses were observed, with statistically significant enhanced serum IgG and nasal wash IgA antibodies in the high-dose group. Conclusions: The current clinical data confirm key aspects of the preclinical profile, which demonstrate the potential of the Nm-nOMV platform as a strong adjuvant for mucosal vaccines. These findings support the broader application of the Nm-nOMV vaccine platform in pandemic preparedness. Full article

Radix pseudostellariae saponins (RPS) enhance immune responses in animals; however, the regulatory mechanisms of these effects remain unclear. This study observed that 14 days post-intranasal immunization with RPS and a Mycoplasma gallisepticum-attenuated vaccine (MGAV), MGAV-specific antibody titers were significantly increased in the blood, and chemokine (C-C motif) ligand 5 (CCL5) messenger RNA expression was significantly increased in the trachea and blood of chickens. Transcriptomic analysis demonstrated that RPS treatment significantly upregulated specific Kyoto Encyclopedia of Genes and Genomes pathways, notably the cytokine–cytokine receptor interaction pathway, which is linked to immune cell migration and involves chemokine receptor chemokine (C-C motif) receptor 4 (CCR4). This finding was corroborated at the protein level by immunohistochemical evidence showing increased CCL5 expression in tracheal tissue. In vitro studies showed that RPS enhanced the phagocytic capacity of RAW264.7 macrophages against ovalbumin, with immunofluorescence revealing time-dependent and dose-dependent CCL5 in these cells. Transwell and scratch-healing assays confirmed that RPS promoted this migration of both RAW264.7 cells and CCR4-positive lymphocytes. Collectively, the findings revealed that RPS modulated the activation, chemotaxis, and migration of macrophages and lymphocytes and is associated with the promotion of the CCL5/CCR4 signaling axis, providing novel evidence for the immune-enhancing effects of RPS by enhancing immunogenicity. Full article

This study aimed to determine whether intranasal hinokitiol modulates short-term salivary secretory IgA (SIgA) secretion dynamics and IgA antibody-forming cell (AFC) activity in the submandibular glands of aged mice, a model of age-associated mucosal immune decline. Aged BALB/c mice received intranasal hinokitiol (50 μg) once weekly for 4 weeks. Saliva was collected on days 0, 7, 14, and 21 at baseline, 0.5 h, 1.5 h, 3 h, and 6 h after each administration. SIgA levels were measured using an enzyme-linked immunosorbent assay. On day 21, IgA AFCs were enumerated using an enzyme-linked immunosorbent spot assay, and their viability and proliferative activity were assessed using the MTT assay. Salivary SIgA rose transiently after each dose, peaking at 1.5 h and returning to baseline by 6 h. By day 21, baseline SIgA secretion was significantly higher than at day 0, indicating a cumulative effect. IgA AFCs were unchanged in number, but viability and proliferation increased at 0.5 and 1.5 h, coinciding with SIgA peaks. Flow cytometry revealed significant expansion of B220⁺CD38⁺ memory B-cells; B220⁺CD138⁺ plasma cells were unaffected. Intranasal hinokitiol transiently enhances salivary SIgA secretion in aged mice, likely through short-term modulation of salivary gland immune activity. This non-invasive approach may aid mucosal defense in aging populations. These findings suggest that intranasal HNK may represent a novel non-invasive approach for enhancing mucosal immune function during aging and may provide a basis for future preventive strategies against oral and respiratory infections. Full article

Background: Toxoplasmosis is a prevalent zoonotic disease worldwide, affecting approximately one-third of the global human population. Primary infection with Toxoplasma gondii during pregnancy can induce miscarriage or congenital infection, leading to irreversible damage to the foetus. Moreover, reactivation of T. gondii infection in immunosuppressed individuals can result in fatal outcomes. No vaccine exists to prevent human disease caused by this parasite. Thus, a vaccine that could induce complete and lasting protection against human toxoplasmosis is an unmet need. Method: In this work, BALB/cByJ mice were intranasally immunised with a subunit vaccine consisting of T. gondii membrane proteins (TGMP) from the T. gondii Me49 strain plus CpG-oligodeoxynucleotide adjuvant (CpG). Antibody responses were analysed by ELISA, while T-cell responses were evaluated by flow cytometry. The immunogenic proteins present in TGMP were identified by mass spectrometry, and parasite burden was quantified by qPCR. Result: The results showed raised TGMP-specific serum IgG and intestinal IgA antibody levels, and parasite-specific IFN-γ-producing CD4⁺ and CD8⁺ memory T cells. Dense granule proteins (GRA) 2 and 7, surface antigen (SAG)-related sequences 25, 29B, and 34A, microneme protein (MIC) 10, toxofilin, nascent polypeptide-associated complex (NAC) domain-containing protein, and NAC subunit beta were identified as immunogenic proteins. Mice immunised with TGMP+CpG were challenged with T. gondii tachyzoites and showed a significant reduction in the parasitic burden in the peritoneal exudate, spleen, and lungs, compared to mice sham-immunised with CpG alone. Conclusions: Altogether, these results indicate that mucosal immunisation with TGMP plus CpG adjuvant is worth exploring as a vaccination approach to prevent toxoplasmosis. Full article

Background/Objectives: Although influenza A viruses with partially truncated NS1 proteins are substantially attenuated and immunogenic due to enhanced innate immune activation; residual NS1-mediated antagonism of antiviral innate responses may support viral replication in the lower respiratory tract and constrain optimal immune responses. Strategies to further improve their immunogenicity and protective efficacy by incorporating immunomodulatory cytokines, such as IL-2, have been successfully explored. Methods: Here, we extended this approach to chemokine expression by engineering an NS1-truncated PR8-based virus (PR8/NS124) to express the immunomodulatory chemokine CXCL10 from the NS segment and compared it with the parental vector. Results: The recombinant NS124_SS_CXCL10 virus replicated to high titers in embryonated chicken eggs and MDCK cells. In vivo, however, CXCL10 expression reduced viral replication in mouse lungs by ~10⁴-fold, resulting in a near-non-replicating phenotype. In contrast to the parental virus, the vector did not induce weight loss and exhibited a strongly attenuated phenotype. This effect was associated with altered innate immune signaling, including increased IRF7 expression and early induction of IFN-α responses in the lungs, together with modulation of TLR-dependent sensing pathways in the upper respiratory tract. Despite severely impaired replication, intranasal immunization induced antigen-specific T-cell responses comparable to those elicited by the parental vector. Following intraperitoneal immunization, when replication of both vectors was minimal, the CXCL10-expressing vector induced significantly higher frequencies of antigen-specific CD8⁺ and CD4⁺ effector-memory T cells. This was accompanied by enhanced antigen-specific T-cell recall responses in the lungs following intranasal challenge. Importantly, the CXCL10-expressing vector demonstrated protective efficacy comparable to that of the parental NS124 vector against heterologous H3N2 challenge while exhibiting an improved safety profile. Conclusions: These findings support the incorporation of CXCL10 as a strategy to improve the safety and T-cell immunogenicity of NS1-truncated influenza vectors. Full article

Background: This study aimed to construct a recombinant Pseudomonas aeruginosa outer membrane vesicle (OMV) vector vaccine delivering pcrV and compare the immunological impacts of OMVs as carriers versus as adjuvants. Methods: The recombinant plasmid pBBRMCS5-pcrV was constructed and transformed into P. aeruginosa. Recombinant OMVs (OMV_PcrV) were prepared via ultracentrifugation and characterized in terms of their morphology and particle size by means of transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). After a biosafety evaluation, mice were intramuscularly immunized with PcrV or OMV_PcrV, followed by a booster immunization on day 21. On day 42, the mice were challenged subcutaneously and intranasally with PAO1. Bacterial loads in tissues and blood, pulmonary T-cell subsets, and serum antibody levels were assessed. Results: The recombinant plasmid was successfully constructed, and Western blotting confirmed the delivery of PcrV into OMVs. TEM revealed typical spherical nanostructures, and NTA showed a median particle size of 127.4 ± 5.3 nm. Upon subcutaneous challenge, the OMV, OMV_PcrV, and OMV + PcrV groups all achieved 100% protection. Both the OMV_PcrV and OMV + PcrV groups exhibited increased CD4⁺ and CD8⁺ T-cell counts and higher induction levels of specific IgM, IgG1, and IgG2a antibodies. The OMV_PcrV group showed superior clearance of respiratory bacterial colonization and reduced inflammatory injury compared with the PBS control group. Conclusions: The constructed vector successfully delivered the PcrV antigen, and the OMVPcrV vaccine induced effective immune responses. Compared with wild-type outer membrane vesicles (OMVs) and the strategy of directly mixing free PcrV antigen with OMVs (OMV + PcrV), the recombinant OMV_PcrV vaccine exhibited superior immunoprotective efficacy in terms of bacterial clearance and tissue protection, providing experimental evidence for the development of a Pseudomonas aeruginosa vaccine. Full article

Respiratory syncytial virus (RSV) is considered the leading causative agent of acute lower respiratory infections in infants and young children worldwide, which makes it a major contributor to pediatric morbidity and mortality. Infants are especially susceptible to severe disease in early life, which underlines the urgent need for developing effective immunization strategies against this virus. However, the development of vaccines against RSV has long been associated with significant challenges. For example, initial attempts, especially those involving formalin-inactivated RSV, resulted in vaccine-enhanced respiratory disease upon subsequent infection, which set a significant safety obstacle for future vaccine candidates. Other challenges facing vaccine development against RSV include the short-lived immunity induced by natural infection, lack of clear correlates of immunity, and immune naivety in infants. Recent breakthroughs in structural virology and immunology have provided insights into protective immunity against RSV, especially regarding neutralizing antibodies that recognize the virus in its prefusion conformation of the viral F protein. Among promising vaccine candidates, intranasal live-attenuated vaccines have emerged as especially promising for infant immunization, especially considering their close mimicry of natural infection that can elicit systemic as well as mucosal immunity in the respiratory tract. A newly emerging approach for live-attenuated virus vaccine development is codon-pair deoptimization (CPD), which is based on synthetic recoding that reduces viral replicative capacity while maintaining intact protein sequences and structure. The preclinical results of CPD-based RSV candidates have provided evidence of such vaccines’ ability to elicit robust immunity while maintaining favorable safety profiles. This review addresses the major challenges associated with the development of effective RSV vaccines for infant immunization, with particular emphasis on lessons learned from previous vaccine failures and recent advances in RSV vaccine development, particularly CPD-based attenuation strategies. Full article

Background: Avian influenza is a critical zoonotic infection threatening both the poultry industry and global public health. While traditional intramuscular vaccines elicit systemic immunity, they often fail to provide robust local protection at mucosal surfaces. There is thus significant interest in the development of mucosal avian influenza vaccines administered via the intranasal route. However, in humans, this approach is significantly hampered by the availability of safe and effective adjuvants. Methods: This study investigated the immunogenicity of a modified recombinant influenza A/H5 hemagglutinin (rHA/H5-modif) formulated with Novochizol, a novel chitosan-derived delivery system, administered intranasally to laboratory animals. Results: Our results demonstrate that mucosal immunization with the rHA/H5-modif/Novochizol complex induces potent humoral (IgG and IgA) and cell-mediated immune responses. Crucially, the formulation provided 100% survival in mice following a lethal challenge with highly pathogenic avian influenza A/H5. Conclusions: These findings position the rHA/H5-modif/Novochizol complex as a promising candidate for next-generation mucosal vaccines, in particular against highly pathogenic avian influenza A/H5 subtype. Full article

Vaccination represents the cornerstone of Newcastle disease control. Nanotechnology offers a promising approach to improve the effectiveness of DNA vaccines, supporting their use as an alternative to conventional platforms. Herein, the Avian Orthoavulavirus 1 (AOAV-1) fusion (F) gene was cloned into a DNA expression plasmid (pDNA). After validating the constructed pDNA-F and confirming robust intracellular protein expression in vitro, three polymeric nanoparticles (NPs)-based formulations were generated using Chitosan (Cs), poly(lactic-co-glycolic) (PLGA), and poly(amidoamine) (PAMAM)-Dendrimers. Physicochemical characterization, stability assessment, and in vitro release analysis confirmed nanoparticle formation and effective DNA incorporation. In vivo experiments were conducted to comparatively evaluate the immunogenicity, particularly the immune priming capacity, and protective efficacy of nanoparticle-based formulations and naked pDNA-F, all tested in parallel at standardized pDNA doses via intranasal (IN) and intramuscular routes. PAMAM-Dendrimers-pDNA-F IM group demonstrated superior efficacy, with 100% survival, the highest post-challenge anamnestic antibody titers, and a pronounced reduction in viral RNA shedding. PLGA-NPs-pDNA-F IN group demonstrated enhanced efficacy, with 90% survival. Naked pDNA-F surpassed the Cs-NPs-pDNA-F in both immune priming and clinical protection, with Cs-NPs-pDNA-F exhibiting the lowest overall performance. These findings highlight that DNA vaccine performance depends on both carrier type and administration route, with PAMAM dendrimers and PLGA enhancing efficacy, whereas chitosan demonstrated reduced efficacy under the tested conditions. Full article

Highly pathogenic avian influenza A (H7N9) remains a threat to poultry health and poses a zoonotic risk, highlighting the need for vaccine antigens capable of inducing both systemic and mucosal immunity. In this study, we evaluated X33CLS-H7, a clarified cell-lysate supernatant derived from glycoengineered Pichia pastoris expressing H7 hemagglutinin, in BALB/c mice following intramuscular(i.m.) injection, nebulized inhalation, or intranasal instillation. H7 expression and hemagglutination activity were confirmed by Western blotting and hemagglutination assay, respectively. Serum HA7-specific IgG and IgA responses, hemagglutination inhibition(HI) activity, H7N9 pseudovirus neutralization, bronchoalveolar lavage fluid (BALF) antibodies, and safety readouts were assessed. After two i.m. immunizations, X33CLS-H7 induced the strongest systemic antibody responses, with an HI geometric mean titer of 1:1622 95% CI, 1:1108–1:2348 and a mean log10 NT₅₀ of 4.62. Respiratory immunization also elicited antibody responses. After four doses, high-dose nebulized delivery produced the strongest responses among the respiratory delivery regimens, with serum IgG and IgA titers of 1.02 × 10⁵ and 2.24 × 10³, respectively, an endpoint HI GMT r of 1:457 95% CI, 1:211–1:971, and a mean log10 NT₅₀ of 3.77 compared with 2.02 in saline controls. High-dose nebulized delivery also generated detectable HA7-specific IgG and IgA responses in bronchoalveolar lavage fluid. No overt local or systemic toxicity signals were observed under the tested conditions. These findings indicate that X33CLS-H7 retains HA7-associated antigenicity and can induce systemic and respiratory mucosal antibody responses, supporting its further evaluation as a simplified and scalable H7N9 vaccine antigen candidate. Full article

Influenza A virus (IAV) remains a major global health threat despite available vaccines and antiviral agents, while current therapies are limited by drug resistance and safety concerns. Curcuminoids exhibit antiviral and anti-inflammatory activities but are constrained by poor water solubility and low bioavailability. To address these limitations, we investigated the antiviral and immunomodulatory properties of a water-solubilized curcuminoid nanoparticle formulation (C–S/M) in both in vitro and in vivo models of IAV infection. To evaluate the potential antiviral and anti-inflammatory effects of C–S/M, we performed a cytopathic effect (CPE) reduction assay in triplicate at 0.001 MOI and quantitative real-time PCR (qRT-PCR) targeting viral NS1 transcripts in MDCK cells. C–S/M suppressed viral NS1 vRNA levels in MDCK cells at lower curcuminoid-equivalent concentrations than native curcuminoids and attenuated IAV-induced TNF-α, IL-6, and IL-8 production. Furthermore, in vivo antiviral efficacy was evaluated in female C57BL/6 mice intranasally infected with IAV and treated orally with C–S/M. Survival, lung viral loads, pulmonary cytokine levels, and splenic immune cell phenotypes were analyzed. In IAV-infected mice, oral administration of C–S/M modestly improved survival and significantly reduced lung viral burden and pulmonary proinflammatory cytokine levels. In addition, in vivo C–S/M treatment was associated with recovery of virus-suppressed T-cell immune responses, including increased Th1 and activated CD8⁺ T cells, reduced regulatory T-cell expansion, and restoration of multifunctional CD4⁺ and CD8⁺ T cells. These findings suggest that C–S/M exerts antiviral and immunomodulatory effects in experimental IAV infection and may serve as a potential adjunctive candidate for further investigation against influenza-associated inflammation. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major challenge to swine production worldwide. Current vaccines have limited efficacy against genetically diverse PRRSV strains. Therefore, strategies with alternative modes of action—such as antiviral approaches that target conserved virus–host interactions, including viral attachment and entry, rather than relying solely on adaptive immune responses—are needed. We first evaluated the in vitro effect of griffithsin (GRFT), a high-mannose-binding lectin, in the monkey kidney cell line MARC-145. Cells were pre-treated with GRFT (50–200 µg/mL) prior to PRRSV infection, after which cell morphology and viral RNA replication (measured by RT-qPCR) were assessed. Pre-treatment with 100–200 µg/mL GRFT, followed by PRRSV inoculation at a multiplicity of infection of 1 or 10, reduced viral replication in MARC145 cells in a dose-dependent manner, achieving almost 100% inhibition of ORF5 and ORF7 RNA compared with untreated controls (p < 0.0001). We next investigated the in vivo effects of intranasal GRFT administration (7.5 or 15 mg/day) in pigs (n = 56). Pigs treated with 15 mg/day GRFT exhibited significantly reduced (p < 0.05) viremia 2, 4 and 7 days post-challenge, compared with untreated, challenged, and controls (log₁₀ 8.1 ± 0.2 vs. 9.0 ± 0.25, 8.2 ± 0.1 vs. 9.1 ± 0.2, and 8.9 ± 0.2 vs. 9.3 ± 0.2, respectively), along with earlier resolution of fever and a trend toward increased average daily gain over 42 days (p < 0.1). These findings are the first report of GRFT efficacy in pigs and support its potential as an antiviral strategy against PRRSV, alongside existing interventions. Full article

Most infectious pathogens enter the host through mucosal surfaces, yet conventional injectable vaccines primarily induce systemic immunity without eliciting robust secretory immunoglobulin A (SIgA) responses at mucosal sites. The COVID-19 pandemic highlighted this limitation, as intramuscular mRNA vaccines failed to establish durable mucosal immunity in the upper respiratory tract. This review covers recent progress in mucosal vaccine development. We first discuss the organization of the mucosal immune system, focusing on SIgA induction, tissue-resident memory T (${\mathrm{T}}_{\mathrm{RM}}$) cells, and resident memory B (${\mathrm{B}}_{\mathrm{RM}}$) cells. We then examine mucosal adjuvants, from cholera toxin and heat-labile enterotoxin derivatives to stimulator of interferon gene (STING) agonists and a strategy to enhance alum adjuvanticity through neutrophil elastase inhibition. Delivery routes including intranasal, oral, and sublingual administration are reviewed alongside viral vectors, nanoparticles, mRNA-lipid nanoparticles, virus-like particles, and engineered bacterial platforms. The roles of innate immune cells, T helper cell subsets, and the microbiota in shaping vaccine responses are discussed. Finally, we survey licensed mucosal vaccines and the COVID-19 mucosal vaccine pipeline, analyze persistent barriers to clinical translation including the absence of validated mucosal correlates of protection, and outline future directions for thermostable formulations and systems biology-driven vaccine design. Full article