A chimeric haemagglutinin-based universal influenza virus vaccine boosts human cellular immune responses directed towards the conserved haemagglutinin stalk domain and the viral nucleoprotein.

BACKGROUND: The development of a universal influenza virus vaccine, to protect against both seasonal and pandemic influenza A viruses, is a long-standing public health goal. The conserved stalk domain of haemagglutinin (HA) is a promising vaccine target. However, the stalk is immunosubdominant. As such, innovative approaches are required to elicit robust immunity against this domain. In a previously reported observer-blind, randomised placebo-controlled phase I trial (NCT03300050), immunisation regimens using chimeric HA (cHA)-based immunogens formulated as inactivated influenza vaccines (IIV) -/+ AS03 adjuvant, or live attenuated influenza vaccines (LAIV), elicited durable HA stalk-specific antibodies with broad reactivity. In this study, we sought to determine if these vaccines could also boost T cell responses against HA stalk, and nucleoprotein (NP). METHODS: We measured interferon-γ (IFN-γ) responses by Enzyme-Linked ImmunoSpot (ELISpot) assay at baseline, seven days post-prime, pre-boost and seven days post-boost following heterologous prime:boost regimens of LAIV and/or adjuvanted/unadjuvanted IIV-cHA vaccines. FINDINGS: Our findings demonstrate that immunisation with adjuvanted cHA-based IIVs boost HA stalk-specific and NP-specific T cell responses in humans. To date, it has been unclear if HA stalk-specific T cells can be boosted in humans by HA-stalk focused universal vaccines. Therefore, our study will provide valuable insights for the design of future studies to determine the precise role of HA stalk-specific T cells in broad protection. INTERPRETATION: Considering that cHA-based vaccines also elicit stalk-specific antibodies, these data support the further clinical advancement of cHA-based universal influenza vaccine candidates. FUNDING: This study was funded in part by the Bill and Melinda Gates Foundation (BMGF).

Memory discrimination is promoted by the expression of the transcription repressor WT1 in the dentate gyrus.

The hippocampus is critical for the precise formation of contextual memories. Overlapping inputs coming from the entorhinal cortex are processed by the trisynaptic pathway to form distinct memories. Disruption in any step of the circuit flow can lead to a lack of memory precision, and to memory interference. We have identified the transcriptional repressor Wilm's Tumor 1 (WT1) as an important regulator of synaptic plasticity involved in memory discrimination in the hippocampus. In male mice, using viral and transgenic approaches, we showed that WT1 deletion in granule cells of the dentate gyrus (DG) disrupts memory discrimination. With electrophysiological methods, we then identified changes in granule cells' excitability and DG synaptic transmission indicating that WT1 knockdown in DG granule cells disrupts the inhibitory feedforward input from mossy fibers to CA3 by decreasing mIPSCs and shifting the normal excitatory/inhibitory (E/I) balance in the DG → CA3 circuit in favor of excitation. Finally, using a chemogenetic approach, we established a causal link between granule cell hyperexcitability and memory discrimination impairments. Our results suggest that WT1 enables a circuit-level computation that drives pattern discrimination behavior.

Transcriptome signatures preceding the induction of anti-stalk antibodies elicited after universal influenza vaccination.

A phase 1 clinical trial to test the immunogenicity of a chimeric group 1 HA (cHA) universal influenza virus vaccine targeting the conserved stalk domain of the hemagglutinin of influenza viruses was carried out. Vaccination with adjuvanted-inactivated vaccines induced high anti-stalk antibody titers. We sought to identify gene expression signatures that correlate with such induction. Messenger-RNA sequencing in whole blood was performed on the peripheral blood of 53 vaccinees. We generated longitudinal data on the peripheral blood of 53 volunteers, at early (days 3 and 7) and late (28 days) time points after priming and boosting with cHAs. Differentially expressed gene analysis showed no differences between placebo and live-attenuated vaccine groups. However, an upregulation of genes involved in innate immune responses and type I interferon signaling was found at day 3 after vaccination with inactivated adjuvanted formulations. Cell type deconvolution analysis revealed a significant enrichment for monocyte markers and different subsets of dendritic cells as mediators for optimal B cell responses and significant increase of anti-stalk antibodies in sera. A significant upregulation of immunoglobulin-related genes was only observed after administration of adjuvanted vaccines (either as primer or booster) with specific induction of anti-stalk IGVH1-69. This approach informed of specific immune signatures that correlate with robust anti-stalk antibody responses, while also helping to understand the regulation of gene expression induced by cHA proteins under different vaccine regimens.

A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial.

Seasonal influenza viruses constantly change through antigenic drift and the emergence of pandemic influenza viruses through antigenic shift is unpredictable. Conventional influenza virus vaccines induce strain-specific neutralizing antibodies against the variable immunodominant globular head domain of the viral hemagglutinin protein. This necessitates frequent re-formulation of vaccines and handicaps pandemic preparedness. In this completed, observer-blind, randomized, placebo-controlled phase I trial (NCT03300050), safety and immunogenicity of chimeric hemagglutinin-based vaccines were tested in healthy, 18-39-year-old US adults. The study aimed to test the safety and ability of the vaccines to elicit broadly cross-reactive antibodies against the hemagglutinin stalk domain. Participants were enrolled into five groups to receive vaccinations with live-attenuated followed by AS03-adjuvanted inactivated vaccine (n = 20), live-attenuated followed by inactivated vaccine (n = 15), twice AS03-adjuvanted inactivated vaccine (n = 16) or placebo (n = 5, intranasal followed by intramuscular; n = 10, twice intramuscular) 3 months apart. Vaccination was found to be safe and induced a broad, strong, durable and functional immune response targeting the conserved, immunosubdominant stalk of the hemagglutinin. The results suggest that chimeric hemagglutinins have the potential to be developed as universal vaccines that protect broadly against influenza viruses.

Activation of the histaminergic H3 receptor induces phosphorylation of the Akt/GSK-3 beta pathway in cultured cortical neurons and protects against neurotoxic insults.

Stimulation of histamine H(3) receptors (H(3)R) activates G(i/o)-proteins that inhibit adenylyl cyclase and triggers MAPK and phospholipase A(2). In a previous study, we showed that H(3)R-mediated phosphorylation of Akt at Ser473 occurs in primary cultures of rat cortical neurons, but neither the downstream targets nor the function of such activation were explored. In this report we address these questions. Western blotting experiments showed that H(3)R-mediated activation of Akt in cultured rat cortical neurons was inhibited by LY 294004 and U0126, suggesting that it depends on phosphoinositide-3-kinase and mitogen-activated protein kinase kinase. H(3)R activation phosphorylated, hence inactivated, the Akt downstream effector glycogen synthase kinase-3beta, increased the expression of the antiapoptotic protein Bcl-2 and protected cultured rat and mouse cortical neurons from neurotoxic insults in a dose-dependent manner. All these effects were inhibited by the H(3)R antagonist inverse/agonist thioperamide. Mouse cortical cells expressed H(3)R as revealed by immunostaining experiments, and stimulation of H(3)R phoshorylated Akt and decreased caspase 3 activity. Hence, we uncovered a yet unexplored action of the H(3)R that may help understand the impact of H(3)R signaling in the CNS.

Genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer identified by systems therapeutics analyses.

The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop transcriptomic signatures for specific drugs. We analyzed lung adenocarcinoma patient data from The Cancer Genome Atlas and identified a subset of patients in which xanthine dehydrogenase (XDH) expression correlated with decreased survival. We tested allopurinol, an FDA-approved drug that inhibits XDH, on human non-small-cell lung cancer (NSCLC) cell lines obtained from the Broad Institute Cancer Cell Line Encyclopedia and identified sensitive and resistant cell lines. We utilized the transcriptomic profiles of these cell lines to identify six-gene signatures for allopurinol-sensitive and allopurinol-resistant cell lines. Transcriptomic networks identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 was verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinol-sensitive NSCLC cell lines and four allopurinol-resistant cell lines susceptible to combination therapy. We searched the transcriptomic data from a library of patient-derived NSCLC tumors from the Jackson Laboratory to identify tumors that would be predicted to be sensitive to allopurinol or allopurinol + CEP-33779 treatment. Patient-derived tumors showed the predicted drug sensitivity in vivo. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

Wilm's tumor 1 promotes memory flexibility.

Under physiological conditions, strength and persistence of memory must be regulated in order to produce behavioral flexibility. In fact, impairments in memory flexibility are associated with pathologies such as post-traumatic stress disorder or autism; however, the underlying mechanisms that enable memory flexibility are still poorly understood. Here, we identify transcriptional repressor Wilm's Tumor 1 (WT1) as a critical synaptic plasticity regulator that decreases memory strength, promoting memory flexibility. WT1 is activated in the hippocampus following induction of long-term potentiation (LTP) or learning. WT1 knockdown enhances CA1 neuronal excitability, LTP and long-term memory whereas its overexpression weakens memory retention. Moreover, forebrain WT1-deficient mice show deficits in both reversal, sequential learning tasks and contextual fear extinction, exhibiting impaired memory flexibility. We conclude that WT1 limits memory strength or promotes memory weakening, thus enabling memory flexibility, a process that is critical for learning from new experiences.

Differential roles of the basolateral amygdala and nucleus basalis magnocellularis during post-reactivation contextual fear conditioning reconsolidation in rats.

The roles of the basolateral amygdala and nucleus basalis magnocellularis in fear conditioning reconsolidation were investigated by means of tetrodotoxin bilateral inactivation performed 96 h after conditioning, immediately after reactivation training. Footshocks of 1.2 mA intensity were employed to induce the generalization phenomenon. Basolateral amygdala inactivation disrupts the contextual fear response and its generalization but not acoustic CS trace retention, when measured 72 and 96 h after tetrodotoxin administration. Nucleus basalis magnocellularis functional inactivation does not affect memory post-reactivation phase of any of the three conditioned fear responses. The present findings show a differential role of the two structures in fear memory reconsolidation and can be a starting point for future investigation of the neural circuits subserving generalization.

The role of the nucleus basalis magnocellularis in fear conditioning consolidation in the rat.

The nucleus basalis magnocellularis (NBM) is known to be involved in the memorization of several conditioned responses. To investigate the role of the NBM in fear conditioning memorization, this neural site was subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that had undergone fear training to acoustic conditioned stimulus (CS) and context. TTX was stereotaxically administered to different groups of rats at increasing intervals after the acquisition session. Memory was assessed as the conditioned freezing duration measured during retention testing, always performed 72 and 96 h after TTX administration. In this way, there was no interference with normal NBM function during either acquisition or retrieval phases, allowing any amnesic effect to be due only to consolidation disruption. The results show that for contextual fear response memory consolidation, NBM functional integrity is necessary up to 24 h post-acquisition. On the other hand, NBM functional integrity was shown to be necessary for memory consolidation of the acoustic CS fear response only immediately after acquisition and not 24-h post-acquisition. The present findings help to elucidate the role of the NBM in memory consolidation and better define the neural circuits involved in fear memories.

Role of the endothelin-1 system in the luteolytic process of pseudopregnant rabbits.

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 microg iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ET(A)/ET(B) receptor antagonist, or cyclooxygenase (COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F2alpha (PGF2alpha). In CL cultured in vitro, ET-1 increased (P

Substantia nigra role in fear conditioning consolidation.

The substantia nigra (SN) is known to be involved in the memorization of several conditioned responses. To investigate the role of the SN in fear conditioning consolidation this neural site was subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats which had undergone fear training to acoustic CS and context. TTX was stereotaxically administered to different groups of rats at increasing intervals after the acquisition session. Memory was assessed as conditioned freezing duration measured during retention testing, always performed 72 and 96 h after TTX administration. In this way there was no interference with normal SN function during either acquisition or retrieval phases, so that any amnesic effect could be due only to consolidation disruption. The results show that SN functional integrity is necessary for contextual fear response consolidation up to the 24-h after-acquisition delay. On the contrary SN functional integrity was shown not to be necessary for the consolidation of acoustic CS fear responses. The present findings help to elucidate the role of the SN in memory consolidation and better define the neural circuits involved in fear memories.

The Akt/GSK-3beta axis as a new signaling pathway of the histamine H(3) receptor.

Drugs targeting the histamine H(3) receptor (H(3)R) are suggested to be beneficial for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. The H(3)R activates G(i/o)-proteins to inhibit adenylyl cyclase activity and modulates phospholipase A(2) and MAPK activity. Herein we show that, in transfected SK-N-MC cells, the H(3)R modulates the activity of the Akt/Glycogen synthase kinase 3beta (GSK-3beta) axis both in a constitutive and agonist-dependent fashion. H(3)R stimulation with the H(3)R agonist immepip induces the phosphorylation of both Ser473 and Thr308 on Akt, a serine/threonine kinase that is important for neuronal development and function. The H(3)R-mediated activation of Akt can be inhibited by the H(3)R inverse agonist thioperamide, and by Wortmannin, LY294002 and PTX, suggesting the observed Akt activation occurs via a G(i/o)-mediated activation of phosphoinositide-3-kinase. H(3)R activation also results in the phosphorylation of Ser9 on GSK-3beta, which acts downstream of Akt and has a prominent role in brain function. In addition, we show the H(3)R-mediated phosphorylation of Akt at Ser473 to occur in primary rat cortical neurons and in rat brain slices. The discovery of this signaling property of the H(3)R adds new understanding to the roles of histamine and the H(3)R in brain function and pathology.

Aversive memory reactivation engages in the amygdala only some neurotransmitters involved in consolidation.

Consolidation refers to item stabilization in long-term memory. Retrieval renders a consolidated memory sensitive, and a "reconsolidation" process has been hypothesized to keep the original memory persistent. Some authors could not detect this phenomenon. Here we show that retrieved contextual fear memory is vulnerable to amnesic treatments and that the amygdala is critically involved. Cholinergic and histaminergic systems seem to modulate only consolidation, whereas cannabinoids are involved in both consolidation and reactivation. The lability of retrieved memory affords opportunities to treat disorders such as phobias, post-traumatic stress, or chronic pain, and these results help searching for appropriate therapeutic targets.