Lipid nanoparticle encapsulation of a Delta spike-CD40L DNA vaccine improves effectiveness against Omicron challenge in Syrian hamsters.

The effectiveness of mRNA vaccines largely depends on their lipid nanoparticle (LNP) component. Herein, we investigate the effectiveness of DLin-KC2-DMA (KC2) and SM-102-based LNPs for the intramuscular delivery of a plasmid encoding B.1.617.2 (Delta) spike fused with CD40 ligand. LNP encapsulation of this CD40L-adjuvanted DNA vaccine with either LNP formulation drastically enhanced antibody responses, enabling neutralization of heterologous Omicron variants. The DNA-LNP formulations provided excellent protection from homologous challenge, reducing viral replication, and preventing histopathological changes in the pulmonary tissues. Moreover, the DNA-LNP vaccines maintained a high level of protection against heterologous Omicron BA.5 challenge despite a reduced neutralizing response. In addition, we observed that DNA-LNP vaccination led to the pulmonary downregulation of interferon signaling, interleukin-12 signaling, and macrophage response pathways following SARS-CoV-2 challenge, shedding some light on the mechanisms underlying the prevention of pulmonary injury. These results highlight the potential combination of molecular adjuvants with LNP-based vaccine delivery to induce greater and broader immune responses capable of preventing inflammatory damage and protecting against emerging variants. These findings could be informative for the future design of both DNA and mRNA vaccines.

Novel recombinant vaccinia virus-vectored vaccine affords complete protection against homologous Borrelia burgdorferi infection in mice.

The rising prevalence of Lyme disease (LD) in North America and Europe has emerged as a pressing public health concern. Despite the availability of veterinary LD vaccines, no vaccine is currently available for human use. Outer surface protein C (OspC) found on the outer membrane of the causative agent, Borrelia burgdorferi, has been identified as a promising target for LD vaccine development due to its sustained expression during mammalian infection. However, the efficacy and immunological mechanisms of LD vaccines solely targeting OspC are not well characterized. In this study, we developed an attenuated Vaccinia virus (VV) vectored vaccine encoding type A OspC (VV-OspC-A). Two doses of the VV-OspC-A vaccine conferred complete protection against homologous B. burgdorferi challenge in mice. Furthermore, the candidate vaccine also prevented the development of carditis and lymph node hyperplasia associated with LD. When investigating the humoral immune response to vaccination, VV-OspC-A was found to induce a robust antibody response predominated by the IgG2a subtype, indicating a Th1-bias. Using a novel quantitative flow cytometry assay, we also determined that elicited antibodies were capable of inducing antibody-dependent cellular phagocytosis in vitro. Finally, we demonstrated that VV-OspC-A vaccination generated a strong antigen-specific CD4+ T-cell response characterized by the secretion of numerous cytokines upon stimulation of splenocytes with OspC peptides. This study suggests a promising avenue for LD vaccine development utilizing viral vectors targeting OspC and provides insights into the immunological mechanisms that confer protection against B. burgdorferi infection.

The Expression Kinetics and Immunogenicity of Lipid Nanoparticles Delivering Plasmid DNA and mRNA in Mice.

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.

DNA lipid nanoparticle vaccine targeting outer surface protein C affords protection against homologous Borrelia burgdorferi needle challenge in mice.

Introduction: The incidence of Lyme disease (LD) in Canada and the United States has risen over the last decade, nearing 480,000 cases each year. Borrelia burgdorferi sensu lato, the causative agent of LD, is transmitted to humans through the bite of an infected tick, resulting in flu-like symptoms and often a characteristic bull's-eye rash. In more severe cases, disseminated bacterial infection can cause arthritis, carditis and neurological impairments. Currently, no vaccine is available for the prevention of LD in humans. Methods: In this study, we developed a lipid nanoparticle (LNP)-encapsulated DNA vaccine encoding outer surface protein C type A (OspC-type A) of B. burgdorferi. Results: Vaccination of C3H/HeN mice with two doses of the candidate vaccine induced significant OspC-type A-specific antibody titres and borreliacidal activity. Analysis of the bacterial burden following needle challenge with B. burgdorferi (OspC-type A) revealed that the candidate vaccine afforded effective protection against homologous infection across a range of susceptible tissues. Notably, vaccinated mice were protected against carditis and lymphadenopathy associated with Lyme borreliosis. Discussion: Overall, the results of this study provide support for the use of a DNA-LNP platform for the development of LD vaccines.

Regulatory Verification by Health Canada of Content in Recombinant Human Insulin, Human Insulin Analog, and Porcine Insulin Drug Products in the Canadian Market Using Validated Pharmacopoeial Methods Over Nonvalidated Approaches.

BACKGROUND: For diabetes mellitus treatment plans, the consistency and quality of insulin drug products are crucial for patient well-being. Because biologic drugs, such as insulin, are complex heterogeneous products, the methods for drug product evaluation should be carefully validated for use. As such, these criteria are rigorously evaluated and monitored by national authorities. Consequently, reports that describe significantly lower insulin content than their label claims are a concern. This issue was raised by a past publication analyzing insulin drug products available in Canada, and, as a result, consumers and major patient organizations have requested clarification. METHODS: To address these concerns, this study independently analyzed insulin drug products purchased from local Canadian pharmacies-including human insulin, insulin analogs, and porcine insulin-by compendial and noncompendial reversed-phase high-performance liquid chromatography (RP-HPLC) methods. RESULTS: We demonstrated the importance of using methods fit for purpose when assessing insulin quality. In a preliminary screen, the expected insulin peak was seen in all products except two insulin analogs-insulin detemir and insulin degludec. Further investigation showed that this was not caused by low insulin content but insufficient solvent conditions, which demonstrated the necessity for methods to be adequately validated for product-specific use. When drug products were appropriately assessed for content using the validated type-specific compendial RP-HPLC methods for insulin quantitation, values agreed with the label claim content. CONCLUSIONS: Because insulin drug products are used daily by over a million Canadians, it is important that researchers and journals present data using methods fit for purpose and that readers evaluate such reports critically.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses.

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

Universal antibody targeting the highly conserved fusion peptide provides cross-protection in mice.

Influenza is a major public health concern causing millions of hospitalizations every year. The current vaccines need annual updating based on prediction of likely strains in the upcoming season. However, mismatches between vaccines and the actual circulating viruses can occur, reducing vaccine effectiveness significantly because of the remarkably high rate of mutation in the viral glycoprotein, hemagglutinin (HA). Clearly, it would be of great interest to determine the potential role of universally conserved epitopes in inducing protective immunity. Here, an antibody against the 14-aa fusion peptide sequence at the N-terminus of the HA2 subunit (Uni-1) was investigated for its ability to elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro and cross-protection against lethal infection in animals. Uni-1, known to neutralize influenza type A (IAV) in vitro, was found to induce strong ADCC against diverse influenza viruses, including human and avian IAVs and both lineages of type B (IBV). The ADCC effects against human IAVs by Uni-1 was comparable to ADCC induced by well-characterized antibodies, F10 and FI6V3. Importantly, mice treated with Uni-1 were protected against lethal challenge of IAV and IBV. These results revealed the versatile effector functions of this universal antibody against markedly diverse strains of both IAV and IBV.

The fusion peptide is the only universally conserved epitope in both IAV and IBVMono-specific universal antibody induces strong ADCC against human and avian IAVMono-specific universal antibody induces strong ADCC against IBV from both genetic lineages of IBVThe antibody has bi-functional effector functions against several influenza viruses.

Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages.

A quarter of all seasonal influenza cases are caused by type B influenza virus (IBV) that also dominates periodically. Here, we investigated a recombinant adenovirus vaccine carrying a synthetic HA2 representing the consensus sequence of all IBV hemagglutinins. The vaccine fully protected mice from lethal challenges by IBV of both genetic lineages, demonstrating its breadth of protection. The protection was not mediated by neutralizing antibodies but robust antibody-dependent cellular cytotoxicity and cell-mediated immune responses. Complete protection of the animals required the entire codon-optimized HA2 sequence that elicited a balanced immune response, whereas truncated vaccines without either the fusion peptide or the transmembrane domain reduced the efficacy of protection. Finally, the vaccines did not demonstrate any sign of disease exacerbation following lung pathology and morbidity monitoring. Collectively, these data suggest that it could be worth further exploring this prototype universal vaccine because of its considerable efficacy, safety, and breadth of protection.

Single Immunization of a Vaccine Vectored by a Novel Recombinant Vaccinia Virus Affords Effective Protection Against Respiratory Syncytial Virus Infection in Cotton Rats.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infections worldwide and disease management measures are hampered by the lack of a safe and effective vaccine against the infection. We constructed a novel recombinant RSV vaccine candidate based on a deletion mutant vaccinia virus platform, in that the host range genes E3L and K3L were deleted (designated as VACVΔE3LΔK3L) and a poxvirus K3L ortholog gene was used as a marker for the rapid and efficient selection of recombinant viruses. The safety of the modified vaccinia virus was investigated by intranasal administration of BALB/c mice with the modified vaccinia vector using a dose known to be lethal in the wild-type Western Reserve. Only a minor loss of body weight by less than 5% and mild pulmonary inflammation were observed, both of which were transient in nature following nasal administration of the high-dose modified vaccinia virus. In addition, the viruses were cleared from the lung in 2 days with no viral invasions of the brain and other vital organs. These results suggest that the virulence of the virus has been essentially abolished. We then investigated the efficiency of the vector for the delivery of vaccines against RSV through comparison with another RSV vaccine delivered by the widely used Modified Vaccinia virus Ankara (MVA) backbone. In the cotton rats, we found a single intramuscular administration of VACVΔE3LΔK3L-vectored vaccine elicited immune responses and protection at a level comparable to the MVA-vectored vaccine against RSV infection. The distinct features of this novel VACV vector, such as an E3L deletion for attenuation and a K3L ortholog for positive selection and high efficiency for vaccine delivery, could provide unique advantages to the application of VACV as a platform for vaccine development.

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell.

BACKGROUND: Extracellular vesicles (EVs) produced by human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are currently investigated for their clinical effectiveness towards immune-mediated diseases. The large amounts of stem cell-derived EVs required for clinical testing suggest that bioreactor production systems may be a more amenable alternative than conventional EV production methods for manufacturing products for therapeutic use in humans. METHODS: To characterize the potential utility of these systems, EVs from four hBM-MSC donors were produced independently using a hollow-fiber bioreactor system under a cGMP-compliant procedure. EVs were harvested and characterized for size, concentration, immunophenotype, and glycan profile at three separate intervals throughout a 25-day period. RESULTS: Bioreactor-inoculated hBM-MSCs maintained high viability and retained their trilineage mesoderm differentiation capability while still expressing MSC-associated markers upon retrieval. EVs collected from the four hBM-MSC donors showed consistency in size and concentration in addition to presenting a consistent surface glycan profile. EV surface immunophenotypic analyses revealed a consistent low immunogenicity profile in addition to the presence of immuno-regulatory CD40 antigen. EV cargo analysis for biomarkers of immune regulation showed a high abundance of immuno-regulatory and angiogenic factors VEGF-A and IL-8. CONCLUSIONS: Significantly, EVs from hBM-MSCs with immuno-regulatory constituents were generated in a large-scale system over a long production period and could be frequently harvested with the same quality and quantity, which will circumvent the challenge for clinical application.

DNA Based Vaccine Expressing SARS-CoV-2 Spike-CD40L Fusion Protein Confers Protection Against Challenge in a Syrian Hamster Model.

SARS-CoV-2 infections present a tremendous threat to public health. Safe and efficacious vaccines are the most effective means in preventing the infections. A variety of vaccines have demonstrated excellent efficacy and safety around the globe. Yet, development of alternative forms of vaccines remains beneficial, particularly those with simpler production processes, less stringent storage conditions, and the capability of being used in heterologous prime/boost regimens which have shown improved efficacy against many diseases. Here we reported a novel DNA vaccine comprised of the SARS-CoV-2 spike protein fused with CD40 ligand (CD40L) serving as both a targeting ligand and molecular adjuvant. A single intramuscular injection in Syrian hamsters induced significant neutralizing antibodies 3-weeks after vaccination, with a boost substantially improving immune responses. Moreover, the vaccine also reduced weight loss and suppressed viral replication in the lungs and nasal turbinates of challenged animals. Finally, the incorporation of CD40L into the DNA vaccine was shown to reduce lung pathology more effectively than the DNA vaccine devoid of CD40L. These results collectively indicate that this DNA vaccine candidate could be further explored because of its efficacy and known safety profile.

Resolving Isomeric Structures of Native Glycans by Nanoflow Porous Graphitized Carbon Chromatography-Mass Spectrometry.

Characterization of the structural diversity of glycans by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains an analytical challenge in large-scale glycomics applications because of the presence of heterogeneous composition, ubiquitous isomers, lability of post-translational glycan modifications, and complexity of data interpretation. High-resolution separation of glycan isomers differentiating from positional, linkage, branching, and anomeric structures is often a prerequisite to ensure the comprehensive glycan identification. Here, we developed a straightforward method using self-packed capillary porous graphitic carbon (PGC) columns for nanoflow LC-MS/MS analyses of native glycans released from glycoproteins. The technique enables highly resolved chromatographic separation of over 20 high-mannose glycan isomers in ribonuclease B and a diverse range of hybrid and complex-type sialoglycoforms of fetuin. The distinct structures of anomeric glycans and linkage sialoglycan isomers, α2,3 and α2,6, were identified by the characteristic MS/MS fragment ions. A glycan sequencing strategy utilizing diagnostic ions and complementary fragments specific to branching residues was established to simplify the MS/MS data interpretation of closely related isomeric structures. To promote the PGC-LC-MS/MS-based method for glycome-wide applications, we extended analyses to native sulfoglycans from the egg-propagated and cell culture-derived influenza vaccines and demonstrate the high-resolution separation and structural characterization of underivatized neutral and anionic glycoforms including oligomannosidic glycan anomers, sialoglycan linkage isomers, and regioisomers of afucosylated and fucosylated sulfoglycans containing sulfated-6-GlcNAc and sulfated-4-GalNAc residues.

Dysregulation of Ephrin receptor and PPAR signaling pathways in neural progenitor cells infected by Zika virus.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles.

BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. METHODS: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. RESULTS: Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. CONCLUSION: The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

PD-1 of Sigmodon hispidus: Gene identification, characterization and preliminary evaluation of expression in inactivated RSV vaccine-induced enhanced respiratory disease.

Sigmodon hispidus or cotton rat is an excellent animal model for studying human infections of respiratory viruses including respiratory syncytial virus (RSV), which is the leading cause of hospitalization in infants and causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research, no vaccine has been licensed whereas inactivated vaccines have been shown to induce severe adverse reaction in a clinical trial, with other forms of RSV vaccine also found to induce enhanced disease in preclinical animal studies. While arguably the cotton rat is the best small animal model for evaluation of RSV vaccines and antivirals, many important genes of the immune system remain to be isolated. Programmed cell death-1 (PD-1) plays an integral role in regulating many aspects of immunity by inducing suppressive signals. In this study, we report the isolation of mRNA encoding the cotton rat PD-1 (crPD-1) and characterization of the PD-1 protein. crPD-1 bound to its cognate ligand on dendritic cells and effectively suppressed cytokine secretion. Moreover, using the newly acquired gene sequence, we observed a decreased level of crPD-1 levels in cotton rats with enhanced respiratory disease induced by inactivated RSV vaccine, unraveling a new facet of vaccine-induced disease.

Chitosan alters inactivated respiratory syncytial virus vaccine elicited immune responses without affecting lung histopathology in mice.

Chitosan is a polysaccharide capable of augmenting immune responses with a proven safety record in animals and humans. These properties make it a potentially attractive agent for the prevention and treatment of infectious disease. Infection by respiratory syncytial virus (RSV) is the leading cause of serious lower respiratory disease in young children throughout the world. There is no licensed vaccine available against RSV whereas inactivated vaccine is known to cause enhanced respiratory disease instead of protection. Here, we investigated whether chitosan administered one or three days post-infection could protect animals against RSV infection and whether it could alter immune responses or immunopathology induced by inactivated RSV vaccine when administered twice before RSV infection. We found chitosan could modestly protect animals against RSV infection when given post-infection, while, in conjunction with inactivated RSV vaccine when given pre-infection, it could significantly reduce RSV infection in mice. Further mechanistic investigation revealed that chitosan enhanced antigen-specific immune responses through augmenting the induction of regulatory T cells, lung resident T cells and neutralizing antibodies while reversing Th2-skewed immune responses induced by inactivated RSV vaccine but, surprisingly, failing to reverse lung histopathology. Overall, this study sheds more light on the molecular mechanisms underlying inactivated RSV vaccine-induced disease.

Unveiling Integrated Functional Pathways Leading to Enhanced Respiratory Disease Associated With Inactivated Respiratory Syncytial Viral Vaccine.

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

Targeting CD40 enhances antibody- and CD8-mediated protection against respiratory syncytial virus infection.

Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. Here, the RSV fusion protein (F) was fused with CD40 ligand and delivered by an adenoviral vector into BALB/c mice where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule. In contrast to a formaldehyde-inactivated vaccine, the vectored vaccine effectively protected animals against RSV without inducing enhanced respiratory disease. This protection involved a robust induction of neutralizing antibodies and memory CD8 T cells, which were not observed in the inactivated vaccine group. Finally, the vectored vaccine was able to elicit long-lasting protection against RSV, one of the most challenging issues in RSV vaccine development. Further studies indicate that the long lasting protection elicited by the CD40 ligand targeted vaccine was mediated by increased levels of effector memory CD8 T cell 3 months post-vaccination.

Micro-RNA Profiling of Exosomes from Marrow-Derived Mesenchymal Stromal Cells in Patients with Acute Myeloid Leukemia: Implications in Leukemogenesis.

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n = 3) and from healthy controls (n = 3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n = 69) and healthy donors (n = 40). Two molecules with decreased gene expression in AML (EZH2 and GSK3ß) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis (KRBA2, RRBP1 and HIST2H 2BE). In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies.