Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

MHC Class I Enables MSCs to Evade NK-Cell-Mediated Cytotoxicity and Exert Immunosuppressive Activity.

Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.

Inhibitory Effects of Extracellular Vesicles from iPS-Cell-Derived Mesenchymal Stem Cells on the Onset of Sialadenitis in Sjögren's Syndrome Are Mediated by Immunomodulatory Splenocytes and Improved by Inhibiting miR-125b.

Extracellular vesicles (EVs) from allogeneic-tissue-derived mesenchymal stem cells (MSCs) are promising to improve Sjögren's syndrome (SS) treatment, but their application is hindered by high variations in and limited expandability of tissue MSCs. We derived standardized and scalable MSCs from iPS cells (iMSCs) and reported that EVs from young but not aging iMSCs (iEVs) inhibited sialadenitis onset in SS mouse models. Here, we aim to determine cellular mechanisms and optimization approaches of SS-inhibitory effects of iEVs. In NOD.B10.H2b mice at the pre-disease stage of SS, we examined the biodistribution and recipient cells of iEVs with imaging, flow cytometry, and qRT-PCR. Intravenously infused iEVs accumulated in the spleen but not salivary glands or cervical lymph nodes and were mainly taken up by macrophages. In the spleen, young but not aging iEVs increased M2 macrophages, decreased Th17 cells, and changed expression of related immunomodulatory molecules. Loading miR-125b inhibitors into aging iEVs significantly improved their effects on repressing sialadenitis onset and regulating immunomodulatory splenocytes. These data indicated that young but not aging iEVs suppress SS onset by regulating immunomodulatory splenocytes, and inhibiting miR-125b in aging iEVs restores such effects, which is promising to maximize production of effective iEVs from highly expanded iMSCs for future clinical application.

Comprehensive Molecular Profiles of Functionally Effective MSC-Derived Extracellular Vesicles in Immunomodulation.

Accumulating evidence indicates that mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) exhibit immunomodulatory effects by delivering therapeutic RNAs and proteins; however, the molecular mechanism underlying the EV-mediated immunomodulation is not fully understood. In this study, we found that EVs from early-passage MSCs had better immunomodulatory potency than did EVs from late-passage MSCs in T cell receptor (TCR)- or Toll-like receptor 4 (TLR4)-stimulated splenocytes and in mice with ocular Sjögren's syndrome. Moreover, MSC-EVs were more effective when produced from 3D culture of the cells than from the conventional 2D culture. Comparative molecular profiling using proteomics and microRNA sequencing revealed the enriched factors in MSC-EVs that were functionally effective in immunomodulation. Among them, manipulation of transforming growth factor ß1 (TGF-ß1), pentraxin 3 (PTX3), let-7b-5p, or miR-21-5p levels in MSCs significantly affected the immunosuppressive effects of their EVs. Furthermore, there was a strong correlation between the expression levels of TGF-ß1, PTX3, let-7b-5p, or miR-21-5p in MSC-EVs and their suppressive function. Therefore, our comparative strategy identified TGF-ß1, PTX3, let-7b-5p, or miR-21-5p as key molecules mediating the therapeutic effects of MSC-EVs in autoimmune disease. These findings would help understand the molecular mechanism underlying EV-mediated immunomodulation and provide functional biomarkers of EVs for the development of robust EV-based therapies.

Outbreak of African Swine Fever, Vietnam, 2019.

African swine fever is one of the most dangerous diseases of swine. We confirmed the 2019 outbreak in Vietnam by real-time reverse transcription PCR. The causative strain belonged to p72 genotype II and was 100% identical with viruses isolated in China (2018) and Georgia (2007). International prevention and control collaboration is needed.

Effects of multigenerational exposure to elevated temperature on reproduction, oxidative stress, and Cu toxicity in Daphnia magna.

This study evaluated the effect of temperature (20 and 25°C) on reproduction, oxidative stress, and copper (Cu) toxicity in Daphnia magna across three generations (F0, F1, and F2). Exposing D. magna to elevated temperature significantly decreased the number of offspring per female per day, the time to first brood, and body length compared to exposure to the optimal temperature (p<0.05). In addition, elevated temperature induced a significantly higher production of reactive oxygen species and lipid peroxidation (p<0.05). These findings suggest that D. magna likely responded to thermal stress by investing more energy into defense mechanisms, rather than growth and reproduction. In addition, oxidative stress at the elevated temperature gradually increased with each generation, possibly owing to the reduced fitness of the offspring. Exposing D. magna to 25°C (EC50=34±3µgL(-1)) substantially increased the median effective concentration of Cu in all generations compared to exposure to 20°C (EC50=25±3µgL(-1)), indicating a decrease in acute toxicity at elevated temperature. However, elevated temperature significantly increased the oxidative stress induced by a sublethal concentration of Cu (10µgL(-1)). The interaction between elevated temperature and Cu exposure appears to be synergistic; however, this needs to be confirmed using multiple generations in a long-term experiment.

Serum Extracellular Vesicle Protein Profiling for Prediction of Corneal Transplant Rejection.

BACKGROUND: Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. METHODS: In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. RESULTS: Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. CONCLUSIONS: Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.

Biopotency and surrogate assays to validate the immunomodulatory potency of extracellular vesicles derived from mesenchymal stem/stromal cells for the treatment of experimental autoimmune uveitis.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been recognized as promising cytotherapeutics due to their demonstrated immunomodulatory effects in various preclinical models. The immunomodulatory capabilities of EVs stem from the proteins and genetic materials they carry from parent cells, but the cargo contents of EVs are significantly influenced by MSC tissues and donors, cellular age and culture conditions, resulting in functional variations. However, there are no surrogate assays available to validate the immunomodulatory potency of MSC-EVs before in vivo administration. In previous work, we discovered that microcarrier culture conditions enhance the immunomodulatory function of MSC-EVs, as well as the levels of immunosuppressive molecules such as TGF-ß1 and let-7b in MSC-EVs. Building on these findings, we investigated whether TGF-ß1 levels in MSC-EVs could serve as a surrogate biomarker for predicting their potency in vivo. Our studies revealed a strong correlation between TGF-ß1 and let-7b levels in MSC-EVs, as well as their capacity to suppress IFN-γ secretion in stimulated splenocytes, establishing biopotency and surrogate assays for MSC-EVs. Subsequently, we validated MSC-EVs generated from monolayer cultures (ML-EVs) or microcarrier cultures (MC-EVs) using murine models of experimental autoimmune uveoretinitis (EAU) and additional in vitro assays reflecting the Mode of Action of MSC-EVs in vivo. Our findings demonstrated that MC-EVs carrying high levels of TGF-ß1 exhibited greater efficacy than ML-EVs in halting disease progression in mice with EAU as well as inducing apoptosis and inhibiting the chemotaxis of retina-reactive T cells. Additionally, MSC-EVs suppressed the MAPK/ERK pathway in activated T cells, with treatment using TGF-ß1 or let-7b showing similar effects on the MAPK/ERK pathway. Collectively, our data suggest that MSC-EVs directly inhibit the infiltration of retina-reactive T cells toward the eyes, thereby halting the disease progression in EAU mice, and their immunomodulatory potency in vivo can be predicted by their TGF-ß1 levels.

Rescuing Photoreceptors in RPE Dysfunction-Driven Retinal Degeneration: The Role of Small Extracellular Vesicles Secreted from Retinal Pigment Epithelium.

Dysfunction of the retinal pigment epithelium (RPE) is a common shared pathology in major degenerative retinal diseases despite variations in the primary etiologies of each disease. Due to their demanding and indispensable functional roles throughout the lifetime, RPE cells are vulnerable to genetic predisposition, external stress, and aging processes. Building upon recent advancements in stem cell technology for differentiating healthy RPE cells and recognizing the significant roles of small extracellular vesicles (sEV) in cellular paracrine and autocrine actions, we investigated the hypothesis that the RPE-secreted sEV alone can restore essential RPE functions and rescue photoreceptors in RPE dysfunction-driven retinal degeneration. Our findings support the rationale for developing intravitreal treatment of sEV. We demonstrate that intravitreally delivered sEV effectively penetrate the full thickness of the retina. Xenogenic intraocular administration of human-derived EVs did not induce acute immune reactions in rodents. sEV derived from human embryonic stem cell (hESC)-derived fully differentiated RPE cells, but not sEV-depleted conditioned cell culture media (CCM minus sEV), rescued photoreceptors and their function in a Royal College of Surgeons (RCS) rat model. This model is characterized by photoreceptor death and retinal degeneration resulting from a mutation in the MerTK gene in RPE cells. From the bulk RNA sequencing study, we identified 447 differently expressed genes in the retina after hESC-RPE-sEV treatment compared with the untreated control. Furthermore, 394 out of 447 genes (88%) showed a reversal in expression toward the healthy state in Long-Evans (LE) rats after treatment compared to the diseased state. Particularly, detrimental alterations in gene expression in RCS rats, including essential RPE functions such as phototransduction, vitamin A metabolism, and lipid metabolism were partially reversed. Defective photoreceptor outer segment engulfment due to intrinsic MerTK mutation was partially ameliorated. These findings suggest that RPE-secreted sEV may play a functional role similar to that of RPE cells. Our study justifies further exploration to fully unlock future therapeutic interventions with sEV in a broad array of degenerative retinal diseases.

Preoperative risk evaluation and perioperative management of patients with obstructive sleep apnea: a narrative review.

Obstructive sleep apnea (OSA) is a common sleep-breathing disorder associated with significant comorbidities and perioperative complications. This narrative review is aimed at comprehensively overviewing preoperative risk evaluation and perioperative management strategies for patients with OSA. OSA is characterized by recurrent episodes of upper airway obstruction during sleep leading to hypoxemia and arousal. Anatomical features, such as upper airway narrowing and obesity, contribute to the development of OSA. OSA can be diagnosed based on polysomnography findings, and positive airway pressure therapy is the mainstay of treatment. However, alternative therapies, such as oral appliances or upper airway surgery, can be considered for patients with intolerance. Patients with OSA face perioperative challenges due to difficult airway management, comorbidities, and effects of sedatives and analgesics. Anatomical changes, reduced upper airway muscle tone, and obesity increase the risks of airway obstruction, and difficulties in intubation and mask ventilation. OSA-related comorbidities, such as cardiovascular and respiratory disorders, further increase perioperative risks. Sedatives and opioids can exacerbate respiratory depression and compromise airway patency. Therefore, careful consideration of alternative pain management options is necessary. Although the association between OSA and postoperative mortality remains controversial, concerns exist regarding adverse outcomes in patients with OSA. Understanding the pathophysiology of OSA, implementing appropriate preoperative evaluations, and tailoring perioperative management strategies are vital to ensure patient safety and optimize surgical outcomes.

TWIST1 and TSG6 are coordinately regulated and function as potency biomarkers in human MSCs.

Mesenchymal stem/stromal cells (MSCs) have been evaluated in >1500 clinical trials, but outcomes remain suboptimal because of knowledge gaps in quality attributes that confer potency. We show that TWIST1 directly represses TSG6 expression that TWIST1 and TSG6 are inversely correlated across bone marrow-derived MSC (BM-MSC) donor cohorts and predict interdonor differences in their proangiogenic, anti-inflammatory, and immune suppressive activity in vitro and in sterile inflammation and autoimmune type 1 diabetes preclinical models. Transcript profiling of TWIST1HiTSG6Low versus TWISTLowTSG6Hi BM-MSCs revealed previously unidentified roles for TWIST1/TSG6 in regulating cellular oxidative stress and TGF-ß2 in modulating TSG6 expression and anti-inflammatory activity. TWIST1 and TSG6 levels also correlate to donor stature and predict differences in iPSC-derived MSC quality attributes. These results validate TWIST1 and TSG6 as biomarkers that predict interdonor differences in potency across laboratories and assay platforms, thereby providing a means to manufacture MSC products tailored to specific diseases.

Association between timing of intubation and mortality in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Delayed intubation is associated with poor prognosis in patients with respiratory failure. However, the effect of delayed intubation in patients with idiopathic pulmonary fibrosis (IPF) remains unknown. This study aimed to analyze whether timing of intubation after high-concentration oxygen therapy was associated with worse clinical outcomes in IPF patients. METHODS: This retrospective propensity score-matched study enrolled adult patients with IPF who underwent mechanical ventilation between January 2011 and July 2021. Patients were divided into early and delayed intubation groups. Delayed intubation was defined as use of high-concentration oxygen therapy for at least 48 hours before tracheal intubation. The primary outcome was intensive care unit (ICU) mortality, and a conditional logistic regression model was used to evaluate the association between timing of intubation and clinical outcomes. RESULTS: The median duration of high-concentration oxygen therapy before intubation was 0.5 days in the early intubation group (n=60) and 5.1 days in the delayed intubation group (n=36). The ICU mortality rate was 56.7% and 75% in the early and delayed intubation groups, respectively, before propensity matching (P=0.075). After matching for demographic and clinical covariates, 33 matched pairs were selected. In the propensity-matched cohort, delayed intubation significantly increased the risk of ICU mortality (adjusted odds ratio, 3.99; 95% confidence interval, 1.02-15.63; P=0.046). However, in-hospital mortality did not differ significantly between the groups. CONCLUSIONS: In patients with IPF, delayed intubation after initiation of high-concentration oxygen therapy was significantly associated with increased risk of ICU mortality compared to early intubation.

Association of natural light exposure and delirium according to the presence or absence of windows in the intensive care unit.

BACKGROUND: Patients in the intensive care unit (ICU) have increased risks of delirium, which is associated with worse outcomes. As pharmacologic treatments for delirium are ineffective, prevention is important. Nonpharmacologic preventive strategies include exposure to natural light and restoring circadian rhythm. We investigated the effect of exposure to natural light through windows on delirium in the ICU. METHODS: This retrospective cohort study assessed all patients admitted to the medical ICU of a university-affiliated hospital between January and June 2020 for eligibility. The ICU included 12 isolation rooms, six with and six without windows. Patients with ICU stays of >48 hours were included and were divided into groups based on their admission to a single room with (window group) or without windows (windowless group). The primary outcome was the cumulative incidence of delirium. The secondary outcomes were the numbers of delirium- and mechanical ventilation-free days, ICU and hospital length of stay, and in-ICU and 28-day mortalities. RESULTS: Of the 150 included patients (window group: 83 [55.3%]; windowless group: 67 [44.7%]), the cumulative incidence of delirium was significantly lower in the window group than in the windowless group (21.7% vs. 43.3%; relative risk, 1.996; 95% confidence interval [CI], 1.220-3.265). Other secondary outcomes did not differ between groups. Admission to a room with a window was independently associated with a decreased risk of delirium (adjusted odds ratio, 0.318; 95% CI, 0.125-0.805). CONCLUSIONS: Exposure to natural light through windows was associated with a lower incidence of delirium in the ICU.

Mortality prediction in chronic obstructive pulmonary disease and obstructive sleep apnea.

BACKGROUND: We aimed to assess mortality in chronic obstructive pulmonary disease (COPD), obstructive sleep apnea (OSA), and overlap syndrome, and evaluate which polysomnographic indices-apnea-hypopnea index (AHI) or hypoxemic load measurements-better predict mortality within 10 years. METHODS: Adults with symptoms suggestive of sleep apnea and airway disease who underwent both polysomnography and spirometry plus bronchodilator response tests between 2000 and 2018 were included and divided into four groups according to presence of COPD and moderate-to-severe OSA (AHI ≥15/h). We estimated mortality using a Cox model adjusted for demographic/anthropometric covariates and comorbidities; this was called clinical model. To evaluate prognostic performance, we compared the concordance index (C-index) between clinical model and extended models, which incorporated one of polysomnographic indices-AHI, sleep time spent with SpO2 < 90% (TS90), and mean and lowest SpO2. RESULTS: Among 355 participants, patients with COPD alone (57/355, 16.1%) and COPD-OSA overlap syndrome (37/355, 10.4%) had increased all-cause mortality than those who had neither disease (152/355, 42.8%) (adjusted HR, 2.98 and 3.19, respectively). The C-indices of extended models with TS90 (%) and mean SpO2 were significantly higher than that of clinical model (0.765 vs. 0.737 and 0.756 vs. 0.737, respectively; all P < 0.05); however, the C-index of extended model with AHI was not (0.739 vs. 0.737; P = 0.15). CONCLUSIONS: In this cohort with symptoms of sleep apnea and airway disease, patients with overlap syndrome had increased mortality, but not higher than in those with COPD alone. The measurement of hypoxemic load, not AHI, better predicted mortality.

Internal motion of an electronically excited molecule in viscoelastic media.

The twisting motion of trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (4-DASPI) in the excited state was investigated in solutions and various polymers in order to understand dependence of molecular rotor dynamics on viscoelasticity. It was observed that the internal motion of electronically excited 4-DASPI correlates strongly with dynamic viscosity and elastic modulus. Our results also showed that condensed phase dynamics of 4-DASPI are governed by the explicit mode coupling between the rotamerizing coordinate and mechanical properties of viscoelastic media.

Outer membrane vesicle increases the efficacy of an influenza vaccine in a diet-induced obese mouse model.

Obesity has been associated with increased symptoms and mortality in influenza patients and impaired immune responses to the influenza vaccine. To date, however, there is no effective adjuvant to improve vaccine efficacy for the obese population. To address this issue, we generated a modified outer membrane vesicle with attenuated endotoxicity (fmOMV) and tested its adjuvant effect on the influenza vaccine in comparison with a squalene-based oil-in-water adjuvant (AddaVax) using a diet-induced obese (DIO) mouse model. Although coadministration of fmOMV did not affect neutralizing antibody (Ab) response, it preferentially induced IgG2c antibody response and significantly increased the vaccine-induced T cell response. More importantly, fmOMV conferred significant protection against homologous and heterologous influenza virus challenge, whereas AddaVax showed marginal protection irrespective of the strongest Ab and T cell responses in DIO mice. These results indicate that fmOMV improves the antigen-specific T cell response and the efficacy of an influenza vaccine, suggesting a potential influenza vaccine adjuvant for the obese population.

Dengue Virus-Polymersome Hybrid Nanovesicles for Advanced Drug Screening Using Real-Time Single Nanoparticle-Virus Tracking.

Dengue virus (DENV) is a major infectious viral pathogen that affects millions of individuals worldwide every year, causing a potentially fatal syndrome, while no commercial antiviral drugs are yet available. To develop an antiviral against dengue fever, it is necessary to understand the relationship between DENV and host cells, which could provide a basis for viral dynamics and identification of inhibitory drug targets. In this study, we designed DiD-loaded and BODIPY-ceramide-encapsulated DENV-polymersome hybrid nanovesicles (DENVSomes) prepared by an extrusion method, which trigger red fluorescence in the endosome and green in the Golgi. DENVSome monitors the dynamics of host cell-virus interaction and tracking in living cells with novel state-of-the-art imaging technologies that show images at high resolution. Also, DENVSome can be exploited to screen whether candidate antiviral drugs interact with DENVs. Consequently, we successfully demonstrated that DENVSome is an efficient tool for tracking and unraveling the mechanisms of replication and drug screening for antiviral drugs of DENV.

In Vitro Macrophage Assay Predicts the In Vivo Anti-inflammatory Potential of Exosomes from Human Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes.

Bacterial Outer Membrane Vesicles Provide Broad-Spectrum Protection against Influenza Virus Infection via Recruitment and Activation of Macrophages.

Influenza A virus (IAV) poses a constant worldwide threat to human health. Although conventional vaccines are available, their protective efficacy is type or strain specific, and their production is time-consuming. For the control of an influenza pandemic in particular, agents that are immediately effective against a wide range of virus variants should be developed. Although pretreatment of various Toll-like receptor (TLR) ligands have already been reported to be effective in the defense against subsequent IAV infection, the efficacy was limited to specific subtypes, and safety concerns were also raised. In this study, we investigated the protective effect of an attenuated bacterial outer membrane vesicle -harboring modified lipid A moiety of lipopolysaccharide (fmOMV) against IAV infection and the underlying mechanisms. Administration of fmOMV conferred significant protection against a lethal dose of pandemic H1N1, PR8, H5N2, and highly pathogenic H5N1 viruses; this broad antiviral activity was dependent on macrophages but independent of neutrophils. fmOMV induced recruitment and activation of macrophages and elicited type I IFNs. Intriguingly, fmOMV showed a more significant protective effect than other TLR ligands tested in previous reports, without exhibiting any adverse effect. These results show the potential of fmOMV as a prophylactic agent for the defense against influenza virus infection.

Outer membrane vesicles harboring modified lipid A moiety augment the efficacy of an influenza vaccine exhibiting reduced endotoxicity in a mouse model.

Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103+ dendritic cells in the mediastinal lymph nodes was significantly increased after fmOMV co-administration. Notably, the mice co-immunized with fmOMV showed a significantly higher protection rate against challenge with a lethal dose of homologous or heterologous influenza viruses without adverse effects. These results show the potential of fmOMV as an effective mucosal adjuvant for intranasal vaccines.