Immunogenicity and Protective Capacity of CpG ODN Adjuvanted Alum Adsorbed Bivalent Meningococcal Outer Membrane Vesicle Vaccine.

Invasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X, and Y. To date, several vaccines targeting N.meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal assays (SBA) based protective coverage of OMV vaccine to X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W+B OMV vaccine, either adjuvanted with Alum, CpG ODN or their combinations and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer) and a MenB OMV-based vaccine (Bexsero®, GSK), The immune responses were assessed through ELISA and SBA. Antibody responses and SBA titers were significantly higher in the W+B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W+B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W+B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.

Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes.

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an "inflammatory signaling cascade" in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris.

Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 106 and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.

Gene network landscape of mouse splenocytes reveals integrin complex as the A151 ODN-responsive hub molecule in the immune transcriptome.

Homeostatic restoration of an inflammatory response requires quenching of the immune system after pathogen threats vanish. A continued assault orchestrated by host defense results in tissue destruction or autoimmunity. A151 is the epitome of synthetic oligodeoxynucleotides (ODNs) that curb the immune response by a subset of white corpuscles through repetitive telomere-derived TTAGGG sequences. Currently, the genuine effect of A151 on the immune cell transcriptome remains unknown. Here, we leveraged an integrative approach where weighted gene co-expression network analysis (WGCNA), differential gene expression analysis, and gene set enrichment analysis (GSEA) of our in-house microarray datasets aided our understanding of how A151 ODN suppresses the immune response in mouse splenocytes. Our bioinformatics results, together with experimental validations, indicated that A151 ODN acts on components of integrin complexes, Itgam and Itga6, to interfere with immune cell adhesion and thereby suppresses the immune response in mice. Moreover, independent lines of evidence in this work converged on the observation that cell adhesion by integrin complexes serves as a focal point for cellular response to A151 ODN treatment in immune cells. Taken together, the outcome of this study sheds light on the molecular basis of immune suppression by a clinically useful DNA-based therapeutic agent.

Comparative analysis of magnetically activated cell sorting and ultracentrifugation methods for exosome isolation.

Mesenchymal stem cell-derived exosomes regulate cell migration, proliferation, differentiation, and synthesis of the extracellular matrix, giving great potential for the treatment of different diseases. The ultracentrifugation method is the gold standard method for exosome isolation due to the simple protocol, and high yield, but presents low purity and requires specialized equipment. Amelioration of technical optimization is required for quick and reliable confinement of exosomes to translate them to the clinic as cell therapeutics In this study, we hypothesized that magnetically activated cell sorting may provide, an effective, reliable, and rapid tool for exosome isolation when compared to ultracentrifugation. We, therefore, aimed to compare the efficiency of magnetically activated cell sorting and ultracentrifugation for human mesenchymal stem cell-derived exosome isolation from culture media by protein quantification, surface biomarker, size, number, and morphological analysis. Magnetically activated cell sorting provided a higher purity and amount of exosomes that carry visible magnetic beads when compared to ultracentrifugation. The particle number of the magnetically activated cell sorting group was higher than the ultracentrifugation. In conclusion, magnetically activated cell sorting presents a quick, and reliable method to collect and present human mesenchymal stem cell exosomes to clinics at high purity for potential cellular therapeutic approaches. The novel isolation and purification method may be extended to different clinical protocols using different autogenic or allogeneic cell sources.

The effect of exosomes released from apheresis platelet concentrates under the impact of gamma irradiation and storage time upon platelet aggregation and hemostasis.

BACKGROUND: Blood components should be gamma-irradiated (γ-IR) in order to prevent transfusion-associated graft-versus-host disease. The aim of this study is to determine the effect of γ-IR and storage time on the exosomes released from apheresis platelet concentrates (aPC) and to investigate their impact on the maximum platelet aggregation (MPA) and hemostasis. MATERIALS AND METHODS: Eight units of aPC were included in this study. These were divided into four equal portions. Two portions were irradiated before storage while the other two were not. Thus, irradiated and non-irradiated aPC samples for storage Days 0 (D0) and 5 (D5) were obtained. Exosomes were isolated from these samples using a commercial kit and were evaluated to ascertain their parent cells by flow cytometry. For the following steps, exosomes were pooled according to their features. Pooled exosomes were then used for aggregometry and thromboelastography. RESULTS: Platelet-derived exosome (PD-EX) levels decreased in D5 compared to D0 in NI-aPC, whereas granulocyte-derived exosome (GD-EX) levels increased. Exosome pools had no effect on MPA compared to saline groups. Exosome pools decreased the time to initial fibrin formation (R), whereas they increased the rate of clot formation (α-angle) and coagulation index (CI) compared to saline groups. DISCUSSION: Storage time and γ-IR each have almost the opposite effects on PD-EX and GD-EX. Exosomes have no impact on MPA, but enhance the clot strength. The impact of exosomes on aPC quality and effectiveness can be ignored or considered as a positive effect.

3D-MSCs A151 ODN-loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution.

INTRODUCTION: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. OBJECTIVES: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. METHODS: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. RESULTS: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-ß, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-ß, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. CONCLUSION: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines.

Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia.

Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.

Low Density Granulocytes and Dysregulated Neutrophils Driving Autoinflammatory Manifestations in NEMO Deficiency.

NF-κB essential modulator (NEMO, IKK-γ) deficiency is a rare combined immunodeficiency caused by mutations in the IKBKG gene. Conventionally, patients are afflicted with life threatening recurrent microbial infections. Paradoxically, the spectrum of clinical manifestations includes severe inflammatory disorders. The mechanisms leading to autoinflammation in NEMO deficiency are currently unknown. Herein, we sought to investigate the underlying mechanisms of clinical autoinflammatory manifestations in a 12-years old male NEMO deficiency (EDA-ID, OMIM #300,291) patient by comparing the immune profile of the patient before and after hematopoietic stem cell transplantation (HSCT). Response to NF-kB activators were measured by cytokine ELISA. Neutrophil and low-density granulocyte (LDG) populations were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) transcriptome before and after HSCT and transcriptome of sorted normal-density neutrophils and LDGs were determined using the NanoString nCounter gene expression panels. ISG15 expression and protein ISGylation was based on Immunoblotting. Consistent with the immune deficiency, PBMCs of the patient were unresponsive to toll-like and T cell receptor-activators. Paradoxically, LDGs comprised 35% of patient PBMCs and elevated expression of genes such as MMP9, LTF, and LCN2 in the granulocytic lineage, high levels of IP-10 in the patient's plasma, spontaneous ISG15 expression and protein ISGylation indicative of a spontaneous type I interferon (IFN) signature were observed, all of which normalized after HSCT. Collectively, our results suggest that type I IFN signature observed in the patient, dysregulated LDGs and spontaneously activated neutrophils, potentially contribute to tissue damage in NEMO deficiency.

Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes.

Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with steroid-sensitive NS in relapse and remission, 10 healthy controls, and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry, and Western blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. Gene Ontology classification analysis and Ingenuity Pathway Analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho-p38 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro.NEW & NOTEWORTHY Up to now, the role of extracellular vesicles (EVs) in the pathogenesis of steroid-sensitive nephrotic syndrome (NS) has not been studied. Here, we found that relapse NS EVs contain significantly increased active RAC1, induce enhanced podocyte motility, and increase expression of RAC-GTP and phospho-p38 expression in vitro. These results suggest that plasma EVs are biologically active molecules in the pathogenesis of NS.

TLR ligand loaded exosome mediated immunotherapy of established mammary Tumor in mice.

Tumor-derived exosomes (TEXs) could be harnessed as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. Engineering TEXs to harbor immunostimulatory molecules still remains a challenge. Previously, we demonstrated that nucleic acid ligand encapsulated liposomes could trigger synergistic strong humoral, and cell mediated immune responses and provokes tumor regression to that of their standalone counterparts. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). Engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic cancer model, administration of4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regress tumor growth in 4T1 tumor-bearing mice. Taken together this work implicated that an exosome-based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. This approach offers a personalized tumor therapy strategy that could be implemented in the clinic.

The role of bcsE gene in the pathogenicity of Salmonella.

The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the 'rdar' (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains.

Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation.

Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases.

Effects of Obesity on Airway and Systemic Inflammation in Asthmatic Children.

BACKGROUND: Obese asthma is a complex syndrome with certain phenotypes that differ in children and adults. There is no clear evidence regarding the presence of additive or synergistic pathological interaction between obesity and asthma in children. OBJECTIVES: Our aim was to demonstrate the interaction of obesity and asthma in children in terms of airway and systemic inflammation by a controlled observational study. METHODS: Four groups were formed: asthma obese (AO), asthma nonobese (ANO), non-AO (NAO), nonasthma nonobese (NANO). Spirometry test, fractional exhaled nitric oxide (FeNO) test, skin prick test, serum inflammatory biomarkers (C-reactive protein, C3, C4, adiponectin, leptin, resistin, periostin, YKL-40, Type 1, and Type 2 cytokines) were conducted and evaluated in all participants. Sputum inflammatory cells (sputum eosinophils and neutrophils) were evaluated in patients who could produce induced sputum and obesity-asthma interactions were determined. RESULTS: A total of 153 participants aged 6-18 years were included in the study, including the AO group (n = 46), the ANO group (n = 45), the NAO group (n = 30), and the NANO group (n = 32). IL-4 (p < 0.001), IL-5 (p < 0.001), IL-13 (p < 0.001), resistin (p < 0.001), and YKL-40 (p < 0.001) levels were higher in patients with asthma independent of obesity. The lowest adiponectin level was found in the AO group and obesity-asthma interaction was detected (p < 0.001). Sputum eosinophilia (p < 0.01), sputum neutrophilia (p < 0.01), and FeNO levels (p = 0.07) were higher in asthmatic patients independent of obesity. In the group with paucigranulocytic inflammation, resistin and YKL-40 levels were significantly lower than in the group without paucigranulocytic inflammation (p < 0.01). CONCLUSION: No interaction was found between obesity and asthma in terms of airway inflammation. Interaction between obesity and asthma was shown in terms of adiponectin level and resistin/adiponectin and leptin/adiponectin ratios. It was found that serum YKL-40 and resistin levels could be associated with airway inflammation.

Human Gut Commensal Membrane Vesicles Modulate Inflammation by Generating M2-like Macrophages and Myeloid-Derived Suppressor Cells.

Immunomodulatory commensal bacteria modify host immunity through delivery of regulatory microbial-derived products to host cells. Extracellular membrane vesicles (MVs) secreted from symbiont commensals represent one such transport mechanism. How MVs exert their anti-inflammatory effects or whether their tolerance-inducing potential can be used for therapeutic purposes remains poorly defined. In this study, we show that MVs isolated from the human lactic acid commensal bacteria Pediococcus pentosaceus suppressed Ag-specific humoral and cellular responses. MV treatment of bone marrow-derived macrophages and bone marrow progenitors promoted M2-like macrophage polarization and myeloid-derived suppressor cell differentiation, respectively, most likely in a TLR2-dependent manner. Consistent with their immunomodulatory activity, MV-differentiated cells upregulated expression of IL-10, arginase-1, and PD-L1 and suppressed the proliferation of activated T cells. MVs' anti-inflammatory effects were further tested in acute inflammation models in mice. In carbon tetrachloride-induced fibrosis and zymosan-induced peritonitis models, MVs ameliorated inflammation. In the dextran sodium sulfate-induced acute colitis model, systemic treatment with MVs prevented colon shortening and loss of crypt architecture. In an excisional wound healing model, i.p. MV administration accelerated wound closure through recruitment of PD-L1-expressing myeloid cells to the wound site. Collectively, these results indicate that P. pentosaceus-derived MVs hold promise as therapeutic agents in management/treatment of inflammatory conditions.

Dual-adjuvant effect of pH-sensitive liposomes loaded with STING and TLR9 agonists regress tumor development by enhancing Th1 immune response.

Nucleic acid-based pattern recognition receptor agonists are effective adjuvants and immunotherapeutic agents. Rather than single applications, ligand combinations could synergistically potentiate immune responses by elevating cytokine and chemokine production via triggering multiple signaling pathways. However, short half-lives of such labile ligands due to nuclease attack and limited cellular uptake due to their structure significantly hamper their in vivo performances. More importantly, simultaneous delivery and activity presentation of protein antigen and nucleic acid ligands critically limit the clinical development of these constructs. In this work, we approached this problem by co-encapsulating a model antigen ovalbumin along with TLR9 and STING ligands within liposomes, a well-established drug delivery system that enables payload stability and enhanced cellular activity upon internalization. Moreover, by loading dual ligands we postulated to achieve heightened Th-1 immune response that would yield pronounced protective vaccine efficacy. We show that, pH-sensitive liposomes co-encapsulating CpG ODN and cGAMP induced synergistic innate immune response by elevating type I and type II interferon levels. Most importantly, this vaccine formulation led to ~70% regression of established melanoma tumor. pH-sensitive liposomal vaccine administration elevated IgG2c/IgG1 antibody ratio, indicative of augmented OVA-specific Th1-biased immunity. Importantly, while the frequency of tumor-specific IFN-γ producing CD8+ T-cells was significantly increased, the M2-type anti-inflammatory macrophage levels were decreased in the tumor bed. In conclusion, our strategy induces reversal of immunosuppressive tumor microenvironment, while enhancing effective anti-tumor immune-response. We propose that this could be coupled with standard therapies during combating tumor eradication.

Modulation of immune responses using adjuvants to facilitate therapeutic vaccination.

Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies.

Mesenchymal stem cell derived extracellular vesicles: promising immunomodulators against autoimmune, autoinflammatory disorders and SARS-CoV-2 infection.

Discovery of novel and broad-acting immunomodulators is of critical importance for the prevention and treatment of disorders occurring due to overexuberant immune responseincluding SARS-CoV-2 triggered cytokine storm leading to lung pathology and mortality during the ongoing viral pandemic. Mesenchymal stem/stromal cells (MSCs), highly regarded for their regenerative capacities, also possessesremarkable immunoregulatory functions affecting all types of innate and adaptive immune cells. Owing to that, MSCs have been heavily investigated in clinic for the treatment of autoimmune and inflammatory diseases along with transplant rejection. Extensive research in the last decaderevealed that MSCs carry out most of their functions through paracrine factors which are soluble mediators and extracellular vesicles (EVs). EVs, including exosomes and microvesicles, are an efficient way of intercellular communication due to their unique ability to carry biological messages such as transcription factors, growth factors, cytokines, mRNAs and miRNAs over long distances. EVs originate through direct budding of the cell membrane or the endosomal secretion pathway and they consist of the cytosolic and membrane components of their parent cell. Therefore, they are able to mimic the characteristics of the parent cell, affecting the target cells upon binding or internalization. EVs secreted by MSCs are emerging as a cell-free alternative to MSC-based therapies. MSC EVs are being tested in preclinical and clinical settings where they exhibit exceptional immunosuppressivecapacity. They regulate the migration, proliferation, activation and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory response. Being as effective immunomodulators as their parent cells, MSC EVs are also preferable over MSC-based therapies due to their lower risk of immunogenicity, tumorigenicity and overall superior safety. In this review, we present the outcomes of preclinical and clinical studies utilizing MSC EVs as therapeutic agents for the treatment of a wide variety of immunological disorders.