A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses.

Vaccinia viruses (VACV) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified Vaccinia virus Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and therefore are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic Vaccinia virus Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

The Spreading and Effects of Human Recombinant α-Synuclein Preformed Fibrils in the Cerebrospinal Fluid of Mice.

Parkinson's disease (PD) patients harbor seeding-competent α-synuclein (α-syn) in their cerebrospinal fluid (CSF), which is mainly produced by the choroid plexus (ChP). Nonetheless, little is known about the role of the CSF and the ChP in PD pathogenesis. To address this question, we used an intracerebroventricular (icv) injection mouse model to assess CSF α-syn spreading and its short- and long-term consequences on the brain. Hereby, we made use of seeding-competent, recombinant α-syn preformed fibrils (PFF) that are known to induce aggregation and subsequent spreading of endogenous α-syn in stereotactic tissue injection models. Here, we show that icv-injected PFF, but not monomers (Mono), are rapidly removed from the CSF by interaction with the ChP. Additionally, shortly after icv injection both Mono and PFF were detected in the olfactory bulb and striatum. This spreading was associated with increased inflammation and complement activation in these tissues as well as leakage of the blood-CSF barrier. Despite these effects, a single icv injection of PFF didn't induce a decline in motor function. In contrast, daily icv injections over the course of 5 days resulted in deteriorated grip strength and formation of phosphorylated α-syn inclusions in the brain 2 months later, whereas dopaminergic neuron levels were not affected. These results point toward an important clearance function of the CSF and the ChP, which could mediate removal of PFF from the brain, whereby chronic exposure to PFF in the CSF may negatively impact blood-CSF barrier functionality and PD pathology.

An immunological puzzle: The adaptive immune system fuels Alzheimer's disease pathology.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by a concerning rise in prevalence. It is projected that the number of affected individuals will reach a staggering 150 million by 2050. While recent advancements in monoclonal antibodies targeting Aß have shown some clinical effects, there is an urgent need for improved therapies to effectively address the impeding surge of AD patients worldwide. To achieve this, a deeper understanding of the intricate mechanisms underlying the disease is crucial. In recent years, mounting evidence has underscored the vital role of the innate immune system in AD pathology. However, limited findings persist regarding the involvement of the adaptive immune system. Here, we report on the impact of the adaptive immune system on various aspects of AD by using AppNL-G-F mice crossed into a Rag2-/- background lacking mature adaptive immune cells. In addition, to simulate the continuous exposure to various challenges such as infections that is commonly observed in humans, the innate immune system was activated through the repetitive induction of peripheral inflammation. We observed a remarkably improved performance on complex cognitive tasks when a mature adaptive immune system is absent. Notably, this observation is pathologically associated with lower Aß plaque accumulation, reduced glial activation, and better-preserved neuronal networks in the mice lacking a mature adaptive immune system. Collectively, these findings highlight the detrimental role of the adaptive immune system in AD and underscore the need for effective strategies to modulate it for therapeutic purposes.

Limitations of PLX3397 as a microglial investigational tool: peripheral and off-target effects dictate the response to inflammation.

Microglia, the resident macrophages of the central nervous system (CNS), play a critical role in CNS homeostasis and neuroinflammation. Pexidartinib (PLX3397), a colony-stimulating factor 1 (CSF1) receptor inhibitor, is widely used to deplete microglia, offering flexible options for both long-term depletion and highly versatile depletion-repopulation cycles. However, the potential impact of PLX3397 on peripheral (immune) cells remains controversial. Until now, the microglia-specificity of this type of compounds has not been thoroughly evaluated, particularly in the context of peripherally derived neuroinflammation. Our study addresses this gap by examining the effects of PLX3397 on immune cells in the brain, liver, circulation and bone marrow, both in homeostasis and systemic inflammation models. Intriguingly, we demonstrate that PLX3397 treatment not only influences the levels of tissue-resident macrophages, but also affects circulating and bone marrow immune cells beyond the mononuclear phagocyte system (MPS). These alterations in peripheral immune cells disrupt the response to systemic inflammation, consequently impacting the phenotype irrespective of microglial depletion. Furthermore, we observed that a lower dose of PLX3397, which does not deplete microglia, demonstrates similar (non-)MPS effects, both in the periphery and the brain, but fails to fully replicate the peripheral alterations seen in the higher doses, questioning lower doses as a 'peripheral control' strategy. Overall, our data highlight the need for caution when interpreting studies employing this compound, as it may not be suitable for specific investigation of microglial function in the presence of systemic inflammation.

Bacterial extracellular vesicles: an emerging avenue to tackle diseases.

A growing body of research, especially in recent years, has shown that bacterial extracellular vesicles (bEVs) are one of the key underlying mechanisms behind the pathogenesis of various diseases like pulmonary fibrosis, sepsis, systemic bone loss, and Alzheimer's disease. Given these new insights, bEVs are proposed as an emerging vehicle that can be used as a diagnostic tool or to tackle diseases when used as a therapeutic target. To further boost the understanding of bEVs in health and disease we thoroughly discuss the contribution of bEVs in disease pathogenesis and the underlying mechanisms. In addition, we speculate on their potential as novel diagnostic biomarkers and how bEV-related mechanisms can be exploited as therapeutic targets.

Experimental hepatic encephalopathy causes early but sustained glial transcriptional changes.

Hepatic encephalopathy (HE) is a common complication of liver cirrhosis, associated with high morbidity and mortality, for which no brain-targeted therapies exist at present. The interplay between hyperammonemia and inflammation is thought to drive HE development. As such, astrocytes, the most important ammonia-metabolizing cells in the brain, and microglia, the main immunomodulatory cells in the brain, have been heavily implicated in HE development. As insight into cellular perturbations driving brain pathology remains largely elusive, we aimed to investigate cell-type specific transcriptomic changes in the HE brain. In the recently established mouse bile duct ligation (BDL) model of HE, we performed RNA-Seq of sorted astrocytes and microglia at 14 and 28 days after induction. This revealed a marked transcriptional response in both cell types which was most pronounced in microglia. In both cell types, pathways related to inflammation and hypoxia, mechanisms commonly implicated in HE, were enriched. Additionally, astrocytes exhibited increased corticoid receptor and oxidative stress signaling, whereas microglial transcriptome changes were linked to immune cell attraction. Accordingly, both monocytes and neutrophils accumulated in the BDL mouse brain. Time-dependent changes were limited in both cell types, suggesting early establishment of a pathological phenotype. While HE is often considered a unique form of encephalopathy, astrocytic and microglial transcriptomes showed significant overlap with previously established gene expression signatures in other neuroinflammatory diseases like septic encephalopathy and stroke, suggesting common pathophysiological mechanisms. Our dataset identifies key molecular mechanisms involved in preclinical HE and provides a valuable resource for development of novel glial-directed therapeutic strategies.

Helicobacter pylori-derived outer membrane vesicles contribute to Alzheimer's disease pathogenesis via C3-C3aR signalling.

The gut microbiota represents a diverse and dynamic population of microorganisms that can influence the health of the host. Increasing evidence supports the role of the gut microbiota as a key player in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Unfortunately, the mechanisms behind the interplay between gut pathogens and AD are still elusive. It is known that bacteria-derived outer membrane vesicles (OMVs) act as natural carriers of virulence factors that are central players in the pathogenesis of the bacteria. Helicobacter pylori (H. pylori) is a common gastric pathogen and H. pylori infection has been associated with an increased risk to develop AD. Here, we are the first to shed light on the role of OMVs derived from H. pylori on the brain in healthy conditions and on disease pathology in the case of AD. Our results reveal that H. pylori OMVs can cross the biological barriers, eventually reaching the brain. Once in the brain, these OMVs are taken up by astrocytes, which induce activation of glial cells and neuronal dysfunction, ultimately leading to exacerbated amyloid-ß pathology and cognitive decline. Mechanistically, we identified a critical role for the complement component 3 (C3)-C3a receptor (C3aR) signalling in mediating the interaction between astrocytes, microglia and neurons upon the presence of gut H. pylori OMVs. Taken together, our study reveals that H. pylori has a detrimental effect on brain functionality and accelerates AD development via OMVs and C3-C3aR signalling.

A mouse model of hepatic encephalopathy: bile duct ligation induces brain ammonia overload, glial cell activation and neuroinflammation.

Hepatic encephalopathy (HE) is a common complication of chronic liver disease, characterized by an altered mental state and hyperammonemia. Insight into the brain pathophysiology of HE is limited due to a paucity of well-characterized HE models beyond the rat bile duct ligation (BDL) model. Here, we assess the presence of HE characteristics in the mouse BDL model. We show that BDL in C57Bl/6j mice induces motor dysfunction, progressive liver fibrosis, liver function failure and hyperammonemia, all hallmarks of HE. Swiss mice however fail to replicate the same phenotype, underscoring the importance of careful strain selection. Next, in-depth characterisation of metabolic disturbances in the cerebrospinal fluid of BDL mice shows glutamine accumulation and transient decreases in taurine and choline, indicative of brain ammonia overload. Moreover, mouse BDL induces glial cell dysfunction, namely microglial morphological changes with neuroinflammation and astrocyte reactivity with blood-brain barrier (BBB) disruption. Finally, we identify putative novel mechanisms involved in central HE pathophysiology, like bile acid accumulation and tryptophan-kynurenine pathway alterations. Our study provides the first comprehensive evaluation of a mouse model of HE in chronic liver disease. Additionally, this study further underscores the importance of neuroinflammation in the central effects of chronic liver disease.

Choroid plexus-derived extracellular vesicles exhibit brain targeting characteristics.

The brain is protected against invading organisms and other unwanted substances by tightly regulated barriers. However, these central nervous system (CNS) barriers impede the delivery of drugs into the brain via the blood circulation and are therefore considered major hurdles in the treatment of neurological disorders. Consequently, there is a high need for efficient delivery systems that are able to cross these strict barriers. While most research focuses on the blood-brain barrier (BBB), the design of drug delivery platforms that are able to cross the blood-cerebrospinal fluid (CSF) barrier, formed by a single layer of choroid plexus epithelial cells, remains a largely unexplored domain. The discovery that extracellular vesicles (EVs) make up a natural mechanism for information transfer between cells and across cell layers, has stimulated interest in their potential use as drug delivery platform. Here, we report that choroid plexus epithelial cell-derived EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles are able to functionally deliver cargo into the brain. Our findings underline the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface.

Recent insights into viral infections as a trigger and accelerator in alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which only symptomatic medication is available, except for the recently FDA-approved aducanumab. This lack of effective treatment urges us to investigate alternative paths that might contribute to disease development. In light of the recent SARS-CoV-2 pandemic and the disturbing neurological complications seen in some patients, it is desirable to (re)investigate the viability of the viral infection theory claiming that a microbe could affect AD initiation and/or progression. Here, we review the most important evidence for this theory with a special focus on two viruses, namely HSV-1 and SARS-CoV-2. Moreover, we discuss the possible involvement of extracellular vesicles (EVs). This overview will contribute to a more rational approach of potential treatment strategies for AD patients.

The tremendous biomedical potential of bacterial extracellular vesicles.

Bacterial extracellular vesicles (bEVs) are nano-sized, lipid membrane-delimited particles filled with bacteria-derived components. They have important roles in the physiology and pathogenesis of bacteria, and in bacteria-bacteria and bacteria-host interactions. Interestingly, recent advances in biotechnology have made it possible to engineer the bEV surface and decorate it with diverse biomolecules and nanoparticles (NPs). bEVs have been the focus of significant interest in a range of biomedical fields and are being evaluated as vaccines, cancer immunotherapy agents, and drug delivery vehicles. However, significant hurdles in terms of their safety, efficacy, and mass production need to be addressed to enable their full clinical potential. Here, we review recent advances and remaining obstacles regarding the use of bEVs in different biomedical applications and discuss paths toward clinical translation.

Fighting fire with fire: The immune system might be key in our fight against Alzheimer's disease.

The ultimate cause of Alzheimer's disease (AD) is still unknown and no disease-modifying treatment exists. Emerging evidence supports the concept that the immune system has a key role in AD pathogenesis. This awareness leads to the idea that specific parts of the immune system must be engaged to ward off the disease. Immunotherapy has dramatically improved the management of several previously untreatable cancers and could hold similar promise as a novel therapy for treating AD. However, before potent immunotherapies can be rationally designed as treatment against AD, we need to fully understand the dynamic interplay between AD and the different parts of our immune system. Accordingly, here we review the most important aspects of both the innate and adaptive immune system in relation to AD pathology.

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus.

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

Anti-Inflammatory Mesenchymal Stromal Cell-Derived Extracellular Vesicles Improve Pathology in Niemann-Pick Type C Disease.

Niemann-Pick type C (NPC) disease is a rare neurovisceral lipid storage disease with progressive neurodegeneration, leading to premature death. The disease is caused by loss-of-function mutations either in the NPC1 or NPC2 gene which results in lipid accumulation in the late endosomes and lysosomes. The involved disease mechanisms are still incompletely understood, making the design of a rational treatment very difficult. Since the disease is characterized by peripheral inflammation and neuroinflammation and it is shown that extracellular vesicles (EVs) obtained from mesenchymal stromal cells (MSCs) provide immunomodulatory capacities, we tested the potential of MSC-EV preparations to alter NPC1 disease pathology. Here, we show that the administration of an MSC-EV preparation with in vitro and in vivo confirmed immune modulatory capabilities is able to reduce the inflammatory state of peripheral organs and different brain regions of NPC1-diseased mice almost to normal levels. Moreover, a reduction of foamy cells in different peripheral organs was observed upon MSC-EV treatment of NPC1-/- mice. Lastly, the treatment was able to decrease microgliosis and astrogliosis, typical features of NPC1 patients that lead to neurodegeneration. Altogether, our results reveal the therapeutic potential of MSC-EVs as treatment for the genetic neurovisceral lipid storage disease NPC, thereby counteracting both central and peripheral features.

Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C.

Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the Npc1 or Npc2 gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment. As NPC patients, who carry NPC1 mutations, have shown to share several pathological features with Alzheimer's disease (AD) and we and others have previously shown that AD is associated with a dysfunctionality of the blood-cerebrospinal fluid (CSF) barrier located at choroid plexus, we investigated the functionality of this latter barrier in NPC1 pathology. Using NPC1-/- mice, we show that despite an increase in inflammatory gene expression in choroid plexus epithelial (CPE) cells, the blood-CSF barrier integrity is not dramatically affected. Interestingly, we did observe a massive increase in autophagosomes in CPE cells and enlarged extracellular vesicles (EVs) in CSF upon NPC1 pathology. Additionally, we revealed that these EVs exert toxic effects on brain tissue, in vitro as well as in vivo. Moreover, we observed that EVs derived from the supernatant of NPC1-/- choroid plexus explants are able to induce typical brain pathology characteristics of NPC1-/-, more specifically microgliosis and astrogliosis. Taken together, our data reveal for the first time that the choroid plexus and CSF EVs might play a role in the brain-related pathogenesis of NPC1.

Low-grade peripheral inflammation affects brain pathology in the AppNL-G-Fmouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by the accumulation of amyloid ß (Aß) and neurofibrillary tangles. The last decade, it became increasingly clear that neuroinflammation plays a key role in both the initiation and progression of AD. Moreover, also the presence of peripheral inflammation has been extensively documented. However, it is still ambiguous whether this observed inflammation is cause or consequence of AD pathogenesis. Recently, this has been studied using amyloid precursor protein (APP) overexpression mouse models of AD. However, the findings might be confounded by APP-overexpression artifacts. Here, we investigated the effect of low-grade peripheral inflammation in the APP knock-in (AppNL-G-F) mouse model. This revealed that low-grade peripheral inflammation affects (1) microglia characteristics, (2) blood-cerebrospinal fluid barrier integrity, (3) peripheral immune cell infiltration and (4) Aß deposition in the brain. Next, we identified mechanisms that might cause this effect on AD pathology, more precisely Aß efflux, persistent microglial activation and insufficient Aß clearance, neuronal dysfunction and promotion of Aß aggregation. Our results further strengthen the believe that even low-grade peripheral inflammation has detrimental effects on AD progression and may further reinforce the idea to modulate peripheral inflammation as a therapeutic strategy for AD.

The neurogliovascular unit in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neurological complication of hepatic dysfunction and portosystemic shunting. It is highly prevalent in patients with cirrhosis and is associated with poor outcomes. New insights into the role of peripheral origins in HE have led to the development of innovative treatment strategies like faecal microbiota transplantation. However, this broadening of view has not been applied fully to perturbations in the central nervous system. The old paradigm that HE is the clinical manifestation of ammonia-induced astrocyte dysfunction and its secondary neuronal consequences requires updating. In this review, we will use the holistic concept of the neurogliovascular unit to describe central nervous system disturbances in HE, an approach that has proven instrumental in other neurological disorders. We will describe HE as a global dysfunction of the neurogliovascular unit, where blood flow and nutrient supply to the brain, as well as the function of the blood-brain barrier, are impaired. This leads to an accumulation of neurotoxic substances, chief among them ammonia and inflammatory mediators, causing dysfunction of astrocytes and microglia. Finally, glymphatic dysfunction impairs the clearance of these neurotoxins, further aggravating their effect on the brain. Taking a broader view of central nervous system alterations in liver disease could serve as the basis for further research into the specific brain pathophysiology of HE, as well as the development of therapeutic strategies specifically aimed at counteracting the often irreversible central nervous system damage seen in these patients.

In Vivo Therapy with M2e-Specific IgG Selects for an Influenza A Virus Mutant with Delayed Matrix Protein 2 Expression.

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.

mRNA in cancer immunotherapy: beyond a source of antigen.

mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.