Search Results (150)

Background: Vaccines that stimulate systemic and mucosal immunity to a level required to prevent SARS-CoV-2 infection and transmission are an unmet need. Highly protective hepatitis B and human papillomavirus nanoparticle vaccines highlight the potential of multivalent nanoparticle vaccine platforms to provide enhanced immunity. Here, we report the construction and characterization of self-assembling 60-subunit icosahedral nanoparticle SARS-CoV-2 vaccines using the bacterial enzyme lumazine synthase (LuS). Methods and Results: Nanoparticles displaying prefusion-stabilized SARS-CoV-2 spike ectodomains fused to the surface-exposed amino terminus of LuS were designed using structure-guided approaches. Negative stain-electron microscopy studies of purified nanoparticles were consistent with self assembly into 60-mer nanoparticles displaying 20 spike trimers. After two intramuscular doses, these purified spike-LuS nanoparticles elicited significantly higher SARS-CoV-2 neutralizing activity than spike trimers in vaccinated mice. Furthermore, intramuscular DNA priming and intranasal boosting with a SARS-CoV-2 LuS nanoparticle vaccine stimulated mucosal IgA responses. Conclusion: These data identify LuS nanoparticles as highly immunogenic SARS-CoV-2 vaccine candidates and support the further development of this platform against SARS-CoV-2 and its emerging variants. Full article

Background/Objectives: The ongoing evolution of SARS-CoV-2 has highlighted the limitations of parenteral vaccines in preventing viral transmission, largely due to their failure to elicit robust mucosal immunity. Methods: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic Bacillus subtilis DG101 spores. Results: In BALB/c mice, IN spore-RBD immunization induced strong systemic and mucosal humoral responses, including elevated specific IgG, IgM, and IgA levels in serum, bronchoalveolar lavage fluid (BALF), nasal-associated lymphoid tissue (NALT), and saliva. It further promoted mucosal B cell and T cell memory, along with a Th1/Tc1-skewed T cell response, characterized by increased IFN-γ-expressing CD4⁺ and CD8⁺ T cells in the lungs. Conclusions: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic B. subtilis spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission. Full article

Background:Salmonella Typhimurium is a major zoonotic pathogen, in which type 1 fimbriae play a crucial role in intestinal colonization and immune modulation. This study aimed to improve the protective immunity of a previously developed growth-deficient strain—a double auxotroph for D-glutamate and D-alanine—by engineering the inducible expression of type 1 fimbriae. Methods: P_tetA-driven expression of the fim operon was achieved by λ-Red mutagenesis. fimA expression was quantified by qRT-PCR, and fimbriation visualized by transmission electron microscopy. Adhesive properties were evaluated through FimH sequence analysis, yeast agglutination, mannose-binding/inhibition assays, and HT-29 cell adherence. BALB/c mice were immunized orogastrically with IRTA ΔΔΔ or IRTA ΔΔΔ P_tetA::fim. Safety and immunogenicity were assessed by clinical monitoring, bacterial load, fecal shedding, ELISA tests, and adhesion/blocking assays using fecal extracts. Protection was evaluated after challenging with wild-type and heterologous strains. Results: IRTA ΔΔΔ P_tetA::fim showed robust fimA expression, dense fimbrial coverage, a marked mannose-sensitive adhesive phenotype and enhanced HT-29 attachment. Fimbrial overexpression did not alter intestinal colonization or translocation to mesenteric lymph nodes (mLNs). Immunization elicited a mixed IgG1/IgG2a, significantly increased IgA and IgG against type 1 fimbriae-expressing Salmonella, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P_tetA::fim reduced infection burden in the cecum (−1.46/1.47-log), large intestine (−1.35/2.17-log), mLNs (−1.32/0.98-log) and systemic organs more effectively than IRTA ΔΔΔ. Conclusions: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation Salmonella vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery. Full article

Synbiotics can be used to reduce intestinal inflammation and mitigate dysbiosis in dogs with chronic inflammatory enteropathy (CIE). Prior research has not assessed the colonic mucosal ultrastructure of dogs with active CIE treated with synbiotics, nor has it determined a possible association between morphologic injury and signaling pathways. Twenty client-owned dogs diagnosed with CIE were randomized to receive either a hydrolyzed diet (placebo; PL) or a hydrolyzed diet supplemented with synbiotic-IgY (SYN) for 6 weeks. Endoscopic biopsies of the colon were obtained for histopathologic, ultrastructural, and molecular analyses and were compared before and after treatment. Using transmission electron microscopy (TEM), an analysis of the ultrastructural alterations in microvilli length (MVL), mitochondria (MITO), and rough endoplasmic reticulum (ER) was compared between treatment groups. To explore potential signaling pathways that might modulate MITO and ER stress, a transcriptomic analysis was also performed. The degree of mucosal ultrastructural pathology differed among individual dogs before and after treatment. Morphologic alterations in enterocytes, MVL, MITO, and ER were detected without significant differences between PL and SYN dogs prior to treatment. Notable changes in ultrastructural alterations were identified post-treatment, with SYN-treated dogs exhibiting significant improvement in MVL, MITO, and ER injury scores compared to PL-treated dogs. Transcriptomic profiling showed many pathways and key genes to be associated with MITO and ER injury. Multiple signaling pathways and their associated genes with protective effects, including fibroblast growth factor 2 (FGF2), fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), synaptic Ras GTPase activating protein 1 (SynGAP1), RAS guanyl releasing protein 2 (RASGRP2), RAS guanyl releasing protein 3 (RASGRP3), thrombospondin 1 (THBS1), colony stimulating factor 1 (CSF1), colony stimulating factor 3 (CSF3), interleukin 21 receptor (IL21R), collagen type VI alpha 6 chain (COL6A6), ectodysplasin A receptor (EDAR), forkhead box P3 (FoxP3), follistatin (FST), gremlin 1 (GREM1), myocyte enhancer factor 2B (MEF2B), neuregulin 1 (NRG1), collagen type I alpha 1 chain (COL1A1), hepatocyte growth factor (HGF), 5-hydroxytryptamine receptor 7 (HTR7), and platelet derived growth factor receptor beta (PDGFR-β), were upregulated with SYN treatment. Differential gene expression was associated with improved MITO and ER ultrastructural integrity and a reduction in oxidative stress. Conversely, other genes, such as protein kinase cAMP-activated catalytic subunit beta (PRKACB), phospholipase A2 group XIIB (PLA2G12B), calmodulin 1 (CALM1), calmodulin 2 (CALM2), and interleukin-18 (IL18), which have harmful effects, were downregulated following SYN treatment. In dogs treated with PL, genes including PRKACB and CALM2 were upregulated, while other genes, such as FGF2, FGF10, SynGAP1, RASGRP2, RASGRP3, and IL21R, were downregulated. Dogs with CIE have colonic ultrastructural pathology at diagnosis, which improves following synbiotic treatment. Ultrastructural improvement is associated with an upregulation of protective genes and a downregulation of harmful genes that mediate their effects through multiple signaling pathways. Full article

During 2022, AIDS claimed a life every minute and about 9.2 million HIV-infected people were not on treatment. In addition, a person living with HIV is estimated to be 20–30 times more susceptible to developing active tuberculosis. Every year, 130,000 infants are newly infected, with vertical transmission being the main cause of pediatric HIV infection. Thus, the development of an effective, safe, and accessible vaccine for neonates and/or adults is an urgent need to prevent or control HIV infection or progression to AIDS. An effective HIV vaccine should induce long-lasting mucosal immunity, broadly neutralizing antibodies, innate immunity, and robust stimulation of CD4+ and CD8+ T-cell responses. Recombinant BCG is a promising live-attenuated bacterial vaccine vector because of its capacity to stimulate T-cell immunity. As a slow-growing microorganism, it provides prolonged low-level antigenic exposure upon infecting macrophages and APCs, potentially stimulating both effector and memory T-cell responses. BCG is considered safe and is currently administered to 80% of infants in countries where it is part of the national immunization program. Additionally, BCG offers several benefits as a live vaccine vehicle since it is cost-effective, easy to mass-produce, and heat stable. It is also well-suited for newborns, as maternal antibodies do not interfere with its efficacy. Furthermore, BCG has a strong safety profile, having been administered to over three billion people as a TB vaccine. In this review, we provide an extensive summary of the literature relating to immunogenicity studies in animal models performed since 2011. Moreover, we provide a comprehensive analysis of the key factors influencing the design of recombinant BCG as a live vaccine vehicle: (i) expression vectors; (ii) selection of HIV immunogen; (iii) promoters to regulate gene expression; (iv) BCG strain and BCG codon optimization; (v) genetic plasmid stability; (vi) influence of preexisting immunity, route, and dose immunization; and (vii) safety profile. Full article

Intestinal development is a critical determinant of growth and overall health in pigs. Accumulating evidence underscores the significant influence of intestinal microbiota on essential physiological functions and systemic health. Dietary nutrients play a pivotal role in regulating both intestinal development and the composition of intestinal microbiota. Optimal early-life nutrient provision ensures proper intestinal growth and functional maturation, with maternal nutrition emerging as a key factor shaping intestinal development during fetal and neonatal stages. This review synthesizes recent studies on maternal nutrient intake—encompassing protein, energy, carbohydrates, minerals, vitamins, probiotics, and prebiotics—and their effects on intestinal growth and health of offspring. Emerging multi-omics evidence has revealed that gestational and lactational nutrition dynamically coordinates offspring intestinal development through vertical microbial transmission and epigenetic mechanisms, such as DNA methylation and histone acetylation. These processes further regulate intestinal barrier maturation, mucosal immunity, and enteroendocrine signaling. Collectively, this review emphasizes that enhancing maternal nutrition can promote postnatal growth by enhancing intestinal development and early microbial colonization in piglets. Further research is crucial to determining the optimal nutritional strategies during the perinatal period. Full article

Lassa mammarenavirus (LASV), a member of the family Arenaviridae, is a highly pathogenic virus capable of causing severe systemic infections in humans. The primary host reservoir is the Natal multimammate mouse (Mastomys natalensis), with human infections typically occurring through mucosal exposure to virus-containing aerosols from rodent excretions. To better understand the molecular mechanisms underlying LASV replication in the respiratory tract, we utilized differentiated primary human airway epithelial cells (HAECs) grown under air–liquid interface conditions, closely mimicking the bronchial epithelium in vivo. Our findings demonstrate that HAECs are permissive to LASV infection and support productive virus replication. While LASV entry into polarized HAECs occurred through both apical and basolateral surfaces, progeny virus particles were predominantly released from the apical surface, consistent with an intrinsic apical localization of the envelope glycoprotein GP. This suggests that apical virus shedding from infected bronchial epithelia may facilitate LASV transmission via airway secretions. Notably, limited basolateral release at later stages of infection was associated with LASV-induced rearrangement of the actin cytoskeleton, resulting in compromised epithelial barrier integrity. Finally, we demonstrate that LASV-infected HAECs exhibited a pronounced type III interferon response. A detailed understanding of LASV replication and host epithelial responses in the respiratory tract could facilitate the development of targeted future therapeutics. Full article

Vaccination has been instrumental in curbing the transmission of SARS-CoV-2 and mitigating the severity of clinical manifestations associated with COVID-19. Numerous COVID-19 vaccines have been developed to this effect, including BioNTech-Pfizer and Moderna’s mRNA vaccines, as well as adenovirus vector-based vaccines such as Oxford–AstraZeneca. However, the emergence of new variants and subvariants of SARS-CoV-2, characterized by enhanced transmissibility and immune evasion, poses significant challenges to the efficacy of current vaccination strategies. In this review, we aim to comprehensively outline the landscape of emerging SARS-CoV-2 variants of concern (VOCs) and sub-lineages that have recently surfaced in the post-pandemic years. We assess the effectiveness of existing vaccines, including their booster doses, against these emerging variants and subvariants, such as BA.2-derived sub-lineages, XBB sub-lineages, and BA.2.86 (Pirola). Furthermore, we discuss the latest advancements in vaccine technology, including multivalent and pan-coronavirus approaches, along with the development of several next-generation coronavirus vaccines, such as exosome-based, virus-like particle (VLP), mucosal, and nanomaterial-based vaccines. Finally, we highlight the key challenges and critical areas for future research to address the evolving threat of SARS-CoV-2 subvariants and to develop strategies for combating the emergence of new viral threats, thereby improving preparedness for future pandemics. Full article

Vaccine formulations are a successful strategy against pathogen transmission because vaccine candidates induce effective and long-lasting memory immune responses (B and CD4+ T cells) at systemic and mucosal sites. Extracellular vesicles of lipoproteins, bioactive compounds from plants and invertebrates (sponges) encapsulated in liposomes, and glycoproteins can target these sites. The vaccine candidates developed can mimic microbial pathogens in a way that successfully links the innate and adaptive immune responses. In addition, vaccines plus adjuvants promote and maintain an inflammatory response. In this review, we aimed to identify the host–pathogen interface as a rich source of candidate targets for vaccine-induced protective and long-lasting memory immune responses. Full article

Initial interactions between HIV-1 and the immune system at mucosal exposure sites play a critical role in determining whether the virus is eliminated or progresses to establish systemic infection. The virus that successfully crosses the mucosal barrier to establish infection in the new host is referred to as the transmitted/founder (TF) virus. Following mucosal HIV-1 transmission, type 1 interferons (IFN-I) are rapidly induced at sites of initial virus replication. The resistance of TF variants to these antiviral effects of the IFN-I has been studied among HIV-1 subtypes B and C. However, their role in restricting HIV-1 replication among subtypes D and AD recombinant remains unexplored. This study assessed the sensitivity of HIV-1 subtype D and AD recombinant TF viruses to IFN-I by infecting peripheral blood mononuclear cells in vitro with infectious molecular clones of these viruses. Cells were exposed to varying concentrations of interferon-α and interferon-β, and viral replicative capacity was measured using HIV-1 p24 antigen ELISA from culture supernatants. Sensitivity to IFN-I was quantified based on viral replication levels. The results showed that interferon-α was more effective in inhibiting viral replication than interferon-β, regardless of the varying amounts of IFN-I used. However, recombinant AD viruses were found to be more resistant to the antiviral effects of IFN-I compared to subtype D viruses. These findings highlight the differential sensitivity of HIV-1 subtypes AD recombinant and D TF viruses to IFN-I and underscore the potential of IFN-I as a therapeutic strategy to target TF viruses and reduce HIV-1 transmission, particularly in populations where subtype D is prevalent. Full article

Background: The rapid genetic evolution of SARS-CoV-2 has led to the emergence of immune-evading, highly transmissible variants of concern (VOCs). This prompts the need for next-generation vaccines that elicit robust mucosal immunity in the airways to directly curb viral infection. Objective: Here, we investigate the impact of heterologous variant prime–boost regimens on humoral responses, focusing on intramuscular (IM) and intranasal (IN) routes of administration. Using a murine model, we assessed the immunogenicity of unadjuvanted protein boosts with Wu-1, Omicron BA.4/5, or Wu-1 + BA.4/5 spike antigens following monovalent or bivalent IM priming with mRNA-LNP vaccines. Results: IM priming induced strong systemic total and neutralizing antibody responses that were further enhanced by IN boosts with BA.4/5. IN boosting achieved the broadest serum neutralization across all VOCs tested. Notably, bivalent mRNA-LNP IM priming induced robust, cross-variant serum neutralizing antibody production, independent of subsequent IN boost combinations. Conclusions: Our findings highlight the benefit of including distinct antigenic variants in the prime vaccination followed by a variant-tailored IN boost to elicit both systemic and mucosal variant-specific responses that are potentially capable of reducing SARS-CoV-2 transmission. Full article

Background: Live attenuated bacteria are promising candidates for mucosal vaccine delivery due to their ability to elicit robust immune responses. FimH is the adhesion protein of type 1 fimbriae, which is used as mucosal adjuvants. This study aims to develop a novel attenuated live bacterial vector via fimbriae recovery on Shigella flexneri. Methods: We generated pBAD-Fim/FWL01 by deleting IS elements in the fimbrial cluster of S. flexneri 2a strain T32. Transmission electron microscopy (TEM) and a mannose–sensitive agglutination assay were used to confirm that type 1 fimbriae were displayed on the recombinant strain. We then evaluated the immune induction of pBAD-Fim/FWL01 in J774A.1 murine macrophages and mice. Additionally, we used pBAD-Fim/FWL01 to deliver the neutrophil–activating protein A subunit (NapA) to assess immunogenicity. Results: Functional type 1 fimbriae on pBAD-Fim/FWL01 were confirmed using TEM and mannose–sensitive agglutination assays. Transcriptome analysis, qRT-PCR, and ELISA assays revealed that pBAD-Fim/FWL01 significantly stimulated mouse macrophages to release cytokines IL-1α, IL-1β, IL-6, and IL-10, inducing an immune response. Orally administrated pBAD-Fim-trc-napA-His/FWL01 elicited significant mucosal and humoral immune responses. Conclusions: The strain pBAD-Fim/FWL01, which expresses type 1 fimbriae, holds promise for development as an attenuated bacterial vaccine vehicle. Full article

Human milk-derived extracellular vesicles (HMEVs) are key components in breast milk, promoting infant health and development. Maternal conditions could affect HMEV cargo; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study investigated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. The median duration from SARS-CoV-2 test positivity to milk collection was 3 months. After defatting and casein micelle disaggregation, HMEVs were isolated from milk samples of nine mothers with prenatal SARS-CoV-2 and six controls by sequential centrifugation, ultrafiltration, and qEV-size exclusion chromatography. The presence of HMEV was confirmed via transmission electron microscopy. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1 × 10¹¹ particles per mL of milk. Western immunoblots detected ALIX, CD9, and HSP70, supporting the presence of HMEVs in the isolates. Cargo from thousands of HMEVs were analyzed using a multi-omics approach, including proteomics and microRNA sequencing, and predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming, mucosal tissue development, and immunomodulation. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era. Full article

Mycoplasma bovis (M. bovis) is capable of causing pneumonia, arthritis, mastitis, and various other ailments in cattle of all age groups, posing a significant threat to the healthy progression of the worldwide cattle industry. The invasion of non-phagocytic host cells serves as a pivotal mechanism enabling M. bovis to evade the immune system and penetrate mucosal barriers, thereby promoting its spread. To investigate the differences in M. bovis invasion into four types of non-phagocytic cells (Madin–Darby bovine kidney (MDBK) cells, embryonic bovine lung (EBL) cells, bovine embryo tracheal (EBTr) cells and bovine turbinate (BT) cells) and further elucidate its invasion mechanism, this study first optimized the experimental methods for M. bovis invasion into cells. Utilizing laser scanning confocal microscopy, transmission electron microscopy, and high-content live-cell imaging systems, the invasion process of M. bovis into four types of non-phagocytic cells was observed. The invasion rates of three different strains of M. bovis (PG45, 07801, 08M) were quantified through the plate counting method. In order to clarify the specific pathway of M. bovis invasion into cells, chlorpromazine (CPZ), amiloride (AMI), and methyl-β-cyclodextrin (M-β-CD) were used to inhibit CLR-mediated clathrin-dependent endocytosis (CDE) pathway, macropinocytosis, and lipid raft pathway, respectively. Subsequently, the invasion rates of PG45 into these four types of cells were measured. Using siRNA technology, the expression of clathrin (CLR) in EBL cells was knocked down to further verify the role of CLR in the invasion process of M. bovis. The results showed that the optimal conditions for M. bovis to invade non-phagocytic cells were a multiplicity of infection (MOI) of 1000 and an optimal invasion time of 4 h. All three strains of M. bovis have the ability to invade the four types of non-phagocytic cells, yet their invasion abilities vary significantly. Observations from transmission electron microscopy further confirmed that at 120 min post-infection, PG45 had successfully invaded EBL cells and was present within endocytic vesicles. It is noteworthy that almost all PG45 successfully escaped from the endocytic vesicles after 240 min of infection had passed. Through chemical inhibition experiments and CLR protein knockdown experiments, it was found that when the CDE and lipid raft pathways were blocked or CLR protein expression was reduced, the invasion rates of PG45, 07801, and 08M in MDBK, EBL, EBTr, and BT cells were significantly decreased (p < 0.05). The above results indicate that M. bovis can invade all types of non-phagocytic cells through endocytic pathways involving CDE (clathrin-dependent endocytosis) or lipid raft-mediated endocytosis, and possesses the ability to escape from phagosomes. Full article

Respiratory viruses present significant global health challenges due to their rapid evolution, efficient transmission, and zoonotic potential. These viruses primarily spread through aerosols and droplets, infecting respiratory epithelial cells and causing diseases of varying severity. While traditional intramuscular vaccines are effective in reducing severe illness and mortality, they often fail to induce sufficient mucosal immunity, thereby limiting their capacity to prevent viral transmission. Mucosal vaccines, which specifically target the respiratory tract’s mucosal surfaces, enhance the production of secretory IgA (sIgA) antibodies, neutralize pathogens, and promote the activation of tissue-resident memory B cells (BrMs) and local T cell responses, leading to more effective pathogen clearance and reduced disease severity. Bacillus subtilis spore surface display (BSSD) technology is emerging as a promising platform for the development of mucosal vaccines. By harnessing the stability and robustness of Bacillus subtilis spores to present antigens on their surface, BSSD technology offers several advantages, including enhanced stability, cost-effectiveness, and the ability to induce strong local immune responses. Furthermore, the application of BSSD technology in drug delivery systems opens new avenues for improving patient compliance and therapeutic efficacy in treating respiratory infections by directly targeting mucosal sites. This review examines the potential of BSSD technology in advancing mucosal vaccine development and explores its applications as a versatile drug delivery platform for combating respiratory viral infections. Full article