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Oncolytic virus therapy is a promising novel immunotherapy. In this report, we engineered a novel oncolytic influenza virus (IV) carrying an antihuman programmed cell death 1 (PD-1) monoclonal antibody utilizing reverse genetics. A reassortant chimeric IV, named rFlu-huPD1, was synthesized as follows: the heavy chain of the PD-1 antibody was encoded on the PB1 fragment, and the light chain of the PD-1 antibody was encoded on the polymerase acid protein fragment. rFlu-huPD1 antibodies were produced in infected ovalantoic eggs and could replicate to high titers. Moreover, selective cytotoxicity of rFlu-huPD1 was upregulated in multiple hepatocellular carcinoma (HCC) cell lines compared with a control, as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the activation of T cells in the spleen of tumor-bearing BALB/c mice treated with rFlu-huPD1 was observed, especially cytotoxic CD8

Avian influenza H7N9 virus has first emerged in 2013 and since then has spread in China in five seasonal waves. In humans, influenza H7N9 virus infection is associated with a high fatality rate; thus, an effective vaccine for this virus is needed. In the present study, we evaluated the usefulness of dissolving microneedles (MNs) loaded with influenza H7N9 vaccine in terms of the dissolution time, insertion capacity, insertion depth, and structural integrity of H7N9 virus in vitro. Our in vitro results showed MNs dissolved within 6 mins. The depth of skin penetration was 270 µm. After coating with a matrix material solution, the H7N9 proteins were agglomerated. We detected the H7N9 delivery time and humoral immune response in vivo. In a mouse model, the antigen retention time was longer for MNs than for intramuscular (IM) injection. The humoral response showed that similar to IM administration, MN administration increased the levels of functional and systematic antibodies and protection against the live influenza A/Anhui/01/2013 virus (Ah01/H7N9). The protection level was determined by the analysis of pathological sections of infected lungs. MN and IM administration yielded results superior to those in the control group. Taken together, these findings demonstrate that the use of dissolving MNs to deliver influenza H7N9 vaccines is a promising immunization approach.

Long noncoding RNAs (lncRNAs) are known to be the important regulators in cancer progression. However, the role of lncRNA FAM66C (FAM66C) is yet to be investigated in intrahepatic cholangiocarcinoma (ICC). This study aimed to investigate the effects and related mechanisms of FAM66C in ICC. Human ICC tissues and cell lines were collected. The expression levels of FAM66C, hsa-miR-23b-3p (miR-23b-3p), and KCND2 were detected by qRT-RCR. The transfection experiments were employed to measure the effect of FAM66C on cell viabilities, migration, and invasion in ICC cells by CCK-8, transwell assays. Glycolysis was investigated by glucose consumption, lactate production and ATP levels. The dual-luciferase reporter and RNA pull down assays were conducted as a means of confirming the interactions between FAM66C, miR-23b-3p, and KCND2. Furthermore, the levels of the EMT-associated proteins (KCND2, GLUT1, PKM2, and LDHA) in ICC cells were detected by western blot. FAM66C was increased in ICC tissues and cells, increased cell viability, glycolysis, migration and invasion, and decreased apoptosis were shown in FAM66C overexpressing cells. Mechanistic analyses revealed that FAM66C regulated the downstream target gene KCND2 by sponging miR-23b-3p. FAM66C effect on ICC was further validated in murine xenograft assays. FAM66C knockdown cells gave rise to tumors that were smaller in size, consistent with the role of FAM66C as a promoter of in vivo tumor growth. These data revealed that FAM66C was able to drive ICC tumor progression and glycolytic activity via the miR-23b-3p/KCND2 axis, indicating FAM66C may be a viable target for treating ICC.

The Plasmodium parasites that cause malaria are adept at developing resistance to antimalarial drugs, necessitating the search for new antiplasmodials. Although several amide analogs of pantothenate (pantothenamides) show potent antiplasmodial activity, hydrolysis by pantetheinases (or vanins) present in blood rapidly inactivates them. We report herein the facile synthesis and biological activity of a small library of pantothenamide analogs in which the labile amide group is replaced with a variety of heteroaromatic rings. Several of the new analogs display antiplasmodial activity in the nanomolar range against P. falciparum and/or P. knowlesi in the presence of pantetheinase. A previously reported triazole and an isoxazole derivative presented here were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although we show here that the two compounds fail to suppress proliferation of P. berghei in vivo, pharmacokinetic and contact time data presented provide a benchmark for the compound profile required to achieve antiplasmodial activity in mice and should facilitate lead optimization.

SARS-CoV-2 is the cause of the current global pandemic of COVID-19; this virus infects multiple organs, such as the lungs and gastrointestinal tract. The microbiome in these organs, including the bacteriome and virome, responds to infection and might also influence disease progression and treatment outcome. In a cohort of 13 COVID-19 patients in Beijing, China, we observed that the gut virome and bacteriome in the COVID-19 patients were notably different from those of five healthy controls. We identified a bacterial dysbiosis signature by observing reduced diversity and viral shifts in patients, and among the patients, the bacterial/viral compositions were different between patients of different severities, although these differences are not entirely distinguishable from the effect of antibiotics. Severe cases of COVID-19 exhibited a greater abundance of opportunistic pathogens but were depleted for butyrate-producing groups of bacteria compared with mild to moderate cases. We replicated our findings in a mouse COVID-19 model, confirmed virome differences and bacteriome dysbiosis due to SARS-CoV-2 infection, and observed that immune/infection-related genes were differentially expressed in gut epithelial cells during infection, possibly explaining the virome and bacteriome dynamics. Our results suggest that the components of the microbiome, including the bacteriome and virome, are affected by SARS-CoV-2 infections, while their compositional signatures could reflect or even contribute to disease severity and recovery processes.

The prevalence of atrial fibrillation (AF), which is one of the common arrhythmias in clinics, is increasing sharply and has affected millions of patients, which is expected to triple by 2050. The purpose of the study was to explore the regulatory relationship between Src-homology domain 2 containing protein tyrosine phosphatase-1 (SHP-1) and proto-oncogene tyrosine-protein kinase Src (c-Src) and the regulation of Connexins 43 (Cx43), and its effect on AF was also studied. Mouse atrial myocyte line (HL-1 cell line) was used as the research object. After overexpression of SHP-1, the expressions of p-c-Src, Cx43, and SHP-1 were detected by Western blot and cellular immunofluorescence, respectively. The location and interaction of SHP-1 and c-Src in the cells were detected by immunofluorescence co-localization and co-immunoprecipitation (Co-IP). The regulation of c-Src and Cx43 was detected by DNA pull down, chromatin co-immunoprecipitation (CHIP), and dual-luciferase reporter system. The results revealed that overexpression of SHP-1 could inhibit the phosphorylation and activation of c-Src and increase the expression of Cx43. Moreover, there was a direct binding between SHP-1 and c-Src, and c-Src could bind to the promoter region of Cx43 and inhibit the transcription of Cx43. In conclusion, SHP-1 could bind to c-Src and inhibit the activity of c-Src, thus enhancing the transcriptional activation of Cx43 and improving the function of gap junction.

Uncovering the tissue molecular architecture at single-cell resolution could help better understand organisms’ biological and pathological processes. However, bulk RNA-seq can only measure gene expression in cell mixtures, without revealing the transcriptional heterogeneity and spatial patterns of single cells. Herein, we introduce Bulk2Space (https://github.com/ZJUFanLab/bulk2space), a deep learning framework-based spatial deconvolution algorithm that can simultaneously disclose the spatial and cellular heterogeneity of bulk RNA-seq data using existing single-cell and spatial transcriptomics references. The use of bulk transcriptomics to validate Bulk2Space unveils, in particular, the spatial variance of immune cells in different tumor regions, the molecular and spatial heterogeneity of tissues during inflammation-induced tumorigenesis, and spatial patterns of novel genes in different cell types. Moreover, Bulk2Space is utilized to perform spatial deconvolution analysis on bulk transcriptome data from two different mouse brain regions derived from our in-house developed sequencing approach termed Spatial-seq. We have not only reconstructed the hierarchical structure of the mouse isocortex but also further annotated cell types that were not identified by original methods in the mouse hypothalamus.

Staphylococcal enterotoxin B (SEB) produced by the Staphylococcus aureus bacteriumis most commonly associated with food poisoning and is known to also cause toxic shock syndrome. Currently, no approved vaccine or specific drug is available to treat SEB intoxication. In this study, we fabricated dissolving microneedles (MNs) loaded with recombinant SEB (rSEB) protein, and evaluated its characteristics, including dissolution profile, protein particle size, insertion depth, antigen retention time in vivo, and skin irritation. Our results showed that rSEB protein-loaded dissolving MNs made of chondroitin sulfate (2%) and trehalose (0.8%) could easily penetrate into the mouse skin within 5 min. The rSEB particle size was unchanged before and after MN fabrication. The skin penetration depth of the MNs was 260 µm. Moreover, the MNs also significantly extended the antigen retention time in vivo. rSEB protein-loaded dissolving MNs also triggered slight erythema at the beginning of administration, but this erythema disappeared within a few hours. More importantly, we investigated the immunogenicity and protective efficacy of rSEB protein-loaded dissolving MNs. Challenge studies in mice revealed that mice in full-dose MN group had a high level of SEB specific antibody response thatprovided100% protection against a lethal SEB toxin challenge. However, there was only 60% protection observed in mice that were in the half-dose MN (dose sparing) group. We also determined the pathological alterations in the tissues of the immunized mice. Taken together, these dissolving MNs may present a promising transcutaneous immunization strategy for treating SEB intoxication.

Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma (HCC) cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy. This article is protected by copyright. All rights reserved.

Accumulating evidence has shown that long noncoding RNAs (lncRNAs) are involved in several biological processes, including immune responses. However, the role of lncRNAs in antiviral innate immune responses remains largely elusive. Here, we identify an uncharacterized human lncRNA AVAN from influenza A virus (IAV) infected patients, that is significantly upregulated following RNA virus infection. During IAV infection, AVAN play an indispensable role in antiviral immune responses. In vivo, we enforced the expression of AVAN in transgenic mice or adeno-associated virus encoding AVAN delivery system and found that AVAN significantly alleviated IAV virulence and virus replication. Mechanistically, nuclear AVAN positively regulates the transcription of forkhead box O3A (FOXO3a) by associating with its promoter and inducing chromatin remodeling to promote neutrophil chemotaxis. Meanwhile, cytoplasmic AVAN binds directly to the E3 ligase TRIM25 and enhances TRIM25-mediated K63-linked ubiquitination of RIG-I, thereby promoting TRIM25- and RIG-I-mediated antiviral innate immune responses, including the induction of type I interferon and ISGs. Moreover, AVAN binds to the B Box/CCD domain of TRIM25 and 1-200nt of AVAN were the functional moieties. Collectively, our findings highlight the potential clinical implications of human lncRNA AVAN as a key positive regulator of the antiviral innate immune response and a promising target for developing broad antiviral therapeutics.

Vaccination is the most effective method of preventing the spread of the influenza virus. However, the traditional intramuscular (IM) immunization causes fear, pain, and cross infection. In contrast, needle-free (NF) immunization is quick and easy for medical personnel and painless and safe for patients. In this study, we assessed the safety and protective efficacy of NF intradermal (ID) immunization with the influenza H7N9 split vaccine (Anhui H7N9/PR8). A preliminary safety evaluation showed that ID immunization with 15 μg of the H7N9 influenza vaccine was not toxic in rats. Moreover, the antigen was metabolized more rapidly after ID than after IM immunization, as determined by in vivo imaging, and ID immunization accelerated the generation of a specific immune response. Additionally, ID immunization with a 20% dose of the H7N9 split vaccine Anhui H7N9/PR8 offered complete protection against lethal challenge by the live H7N9 virus. Taken together, our findings suggest that NF ID immunization with the H7N9 influenza vaccine induces effective protection, has a good safety profile, requires little antigen, and elicits an immune response more rapidly than does IM immunization. This approach may be used to improve the control of influenza H7N9 outbreaks.

Myocardial infarction (MI) is strongly associated with the temporal regulation of cardiac immunity. However, a variety of current clinical trials have failed because of the lack of post-MI immunomodulating/anti-inflammatory targets. We performed single-cell RNA sequencing analysis of cardiac Cd45+ immune cell at 0, 3, 7, and 14 days after injury in a mouse left anterior descending coronary artery ligation model. Major immune cell populations, distinct subsets, and dynamic changes were identified. Macrophages (Mø)/monocytes were most abundant, peaking at 3 days after infarction. Mø-5 and Mø-6 were the predominant infiltrated subsets at this time point, with strong expression of inflammatory factors. Further analysis demonstrated that suppressing these sets attenuated pathological MI progression by preventing subsequent leukocyte extravasation and adverse remodeling. We also detected abundant apoptotic neutrophils and a profibrotic macrophage subset on days 7 and 14 respectively. These results provide a basis for developing cell type- and time-specific interventions in MI.Graphic abstractTemporal regulation of cardiac immunity is strongly associated with the onset, progression, and outcomes of MI. Our study of time-series scRNA-seq on the whole cardiac immune cells isolated from the LAD ligation mice and the tanshinone IIA treated mice shed light on the underlying pathology of MI.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies has impelled researchers to reconsider the importance of the choice of animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resolution.MethodsThis study aimed to follow the dynamic fibrotic features of bleomycin-treated mouse lungs over extended durations through a combination of the latest technologies (micro-computed tomography imaging and histological detection of degraded collagens) and traditional methods. In addition, we also applied immunohistochemistry to explore the distribution of all hydroxyproline-containing molecules.ResultsAs determined by classical biochemical methods, total lung hydroxyproline contents reached a peak at 4 weeks after bleomycin injury and maintained a steady high level thereafter until the end of the experiments (16 weeks). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degradation of collagens and a recovery of lung aeration after the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and fluorescent peptide staining against degraded collagen, together showing large amounts of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10 or 16 weeks rather than at 4 weeks after bleomycin treatment.ConclusionsOur present data not only offer respiratory researchers a new perspective towards the resolution nature of mouse lung fibrosis, but also remind them to be cautious when using the hydroxyproline content assay to evaluate the severity of fibrosis.

Advances in single-cell RNA sequencing (scRNA-seq) have furthered the simultaneous classification of thousands of cells in a single assay based on transcriptome profiling. In most analysis protocols, single-cell type annotation relies on marker genes or RNA-seq profiles, resulting in poor extrapolation. Still, the accurate cell-type annotation for single-cell transcriptomic data remains a great challenge. Here, we introduce scDeepSort (https://github.com/ZJUFanLab/scDeepSort), a pre-trained cell-type annotation tool for single-cell transcriptomics that uses a deep learning model with a weighted graph neural network (GNN). Using human and mouse scRNA-seq data resources, we demonstrate the high performance and robustness of scDeepSort in labeling 764 741 cells involving 56 human and 32 mouse tissues. Significantly, scDeepSort outperformed other known methods in annotating 76 external test datasets, reaching an 83.79% accuracy across 265 489 cells in humans and mice. Moreover, we demonstrate the universality of scDeepSort using more challenging datasets and using references from different scRNA-seq technology. Above all, scDeepSort is the first attempt to annotate cell types of scRNA-seq data with a pre-trained GNN model, which can realize the accurate cell-type annotation without additional references, i.e. markers or RNA-seq profiles.

Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2-PI3K-AKT-NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection.

Hepatitis C virus (HCV) has a devastating impact on human health, and infections can progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no effective HCV vaccine. In this study, we rescued a recombinant PR8 influenza viral vector, called rgFLU-HCVCE1E2, carrying the core and envelope glycoprotein (C/E1/E2) epitopes of HCV inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of rgFLU-HCVCE1E2 and the expression of the C/E1/E2 epitopes of HCV were examined. rgFLU-HCVCE1E2 replicated in various cell lines, including MDCK, A549, and Huh7.5 cells. More importantly, in BALB/c mice immunized intranasally twice at a 21-day interval with 104, 105, or 106 TCID50 rgFLU-HCVCE1E2, the viral vector induced a robust antibody response to influenza and HCV and potent IFN-γ and IL-4 secretion in response to HCV antigens in a dose-dependent manner. The rgFLU-HCVCE1E2 virus also stimulated IFN-γ production by virus-specific peripheral blood mononuclear cells in patients with chronic HCV infection. The study demonstrated that rgFLU-HCVCE1E2 carrying HCV antigens is immunogenic in vivo and has potential for the development of a HCV vaccine.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infection in infants and children, but there is still no licensed vaccine available. In this report, we developed virus-like particle (VLP) vaccines based on the Bac-to-Bac baculovirus expression system, consisting of an influenza virus matrix (M1) protein and the RSV fusion protein (F) or glycoprotein (G). These RSV VLPs were identified by western blot analysis and electron microscopy. Female BALB/c mice immunized intranasally (i.n.) with RSV-F VLPs, RSV-G VLPs, or both showed viral-specific antibody responses against RSV. Total IgG, IgG1, IgG2a, and mucosal IgA were detected in mice with RSV-F plus RSV-G VLPs, revealing potent cellular and mucosal immune responses. Moreover, we found that these mixed RSV VLPs conferred enhanced protection against live RSV challenges, showing significant decreases in lung viral replication and obvious attenuation of histopathological changes associated with viral infections. These results demonstrate that RSV-F plus RSV-G VLPs by intranasal vaccination is a promising vaccine candidate that warrants further evaluation using cotton rat and primate models.

Background and aims Cancer is typically considered as a genetic and epigenetic disease. Although numerous studies have indicated that an aberrant structure, function, or expression level of epigenetic enzymes contribute to many tumor types, precisely how the epigenetic mechanisms are involved in the hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remains unknown. Approach and results In this study, we found that the WD repeat domain 5 protein (WDR5)-a core subunit of histone H3 lysine 4 methyltransferase complexes, which catalyze the generation of histone H3 lysine 4 trimethylation (H3K4me3) modification-is highly expressed in HBV-related HCC and promotes HCC development. WDR5 plays a critical role in HBV-driven cell proliferation and tumor growth in mice, and the WDR5-0103 small-molecule inhibitor of WDR5 activity compromises HBV- and hepatitis B x protein (HBx)-driven tumor proliferation. The aberrantly high WDR5 protein level was found to involve HBx through its stabilization of the WDR5 protein by inhibiting the interaction between the damage-specific DNA-binding protein 1/cullin-4 and WDR5, causing decreased ubiquitination of the WDR5 protein. HBx was found to colocalize with WDR5 on chromatin genome wide and promotes genome-wide H3K4me3 modification by means of WDR5. Furthermore, the recruitment of HBx to promoters of target genes relied on its interaction with WDR5 through its α-helix domain. WDR5 was also found to promote HBV transcription through H3K4 modification of covalently closed circular DNA minichromosome, and WDR5-0103 was able to inhibit HBV transcription. Finally, the in vitro and in vivo data further proved that HBx exerted its tumor-promoting function in a WDR5-dependent manner. Conclusions Our data reveals that WDR5 is a key epigenetic determinant of HBV-induced tumorigenesis and that the HBx-WDR5-H3K4me3 axis may be a potential therapeutic target in HBV-induced liver pathogenesis.

Influenza A H7N9 virus has demonstrated considerable pandemic potential in China ever since early spring 2013. Until now, there have been no specific medicines to treat influenza A H7N9 virus infected patients. Development of a safe and effective H7N9 therapeutic preparation is urgently needed. To this end, we prepared and evaluated the pepsin-digested F(ab′) 2 fragments of serum IgGs from the horses inoculated with a inactivated influenza A H7N9 whole virus antigens. The protective effects of the F(ab′) 2 fragments against H7N9 virus infection were determined in cultured MDCK cells by cytopathic effect (CPE) and evaluated in a BALB/c mouse model by observing death, weight loss and viral load. The in vitro results showed that the F(ab′) 2 fragments had an HI titer of 1:2048 and a neutralization titer of 1: 31,623. The in vivo assays suggested that 600U of the preparations could efficiently protect BALB/c mice from a lethal dose of A/Anhui/01/2013 (H7N9) infection even when administered two days post infection. Thus, this highly purified preparation should be a potential candidate for treating severe patients suffering from influenza A H7N9.

Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and the elderly, and no vaccine against this virus has yet been licensed. Here, we report a recombinant PR8 influenza virus with the RSV fusion (F) protein epitopes of the subgroup A gene inserted into the influenza virus non-structural (NS) gene (rFlu/RSV/F) that was generated as an RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein (NEP) of rFlu/RSV/F was also investigated using an immunofluorescent assay. The rescued virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line and CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with rFlu/RSV/F had specific haemagglutination inhibition antibody responses against the PR8 influenza virus and RSV neutralization test proteins. Furthermore, intranasal immunization with rFlu/RSV/F elicited T helper type 1-dominant cytokine profiles against the RSV strain A2 virus. Taken together, our findings suggested that rFlu/RSV/F was immunogenic in vivo and warrants further development as a promising candidate vaccine.

Respiratory syncytial virus (RSV) is an important viral pathogen that causes life-threatening respiratory infections in both infants and the elderly; no vaccines are at present available. In this report, we examined the use of influenza virus as a vehicle for production of an experimental RSV vaccine. We used reverse genetics to generate a recombinant influenza A virus with epitopes from the RSV fusion (F) and attachment (G) proteins (rFlu/RSV/F+G) in the influenza virus nonstructural (NS1) protein gene. Expression of RSV F+G epitope proteins was confirmed by Western blotting, and no changes in viral morphology were evident following examination by electron microscopy. BALB/c mice immunized intranasally with rFlu/RSV/F+G showed viral-specific antibody responses against both influenza and RSV. Total IgG, IgG1, IgG2a and IgA were measured in mice immunized with rFlu/RSV/F+G, revealing robust cellular and mucosal immune responses. Furthermore, we found that rFlu/RSV/F+G conferred protection against subsequent influenza and RSV challenges, showing significant decreases in viral replication and obvious attenuation of histopathological changes associated with viral infections. These findings suggest that rFlu/RSV/F+G is a promising vaccine candidate, which should be further assessed using cotton rat and primate models.

Summary: Circular RNAs (circRNAs) are emerging as essential regulators of various biological and disease processes. To comprehensively understand the diversity of circRNAs and prioritize their importance, we present a large-scale study of circRNA repertoires from multiple tissues from human, macaque, and mouse. We discovered totals of 104,388, 96,675, and 82,321 circRNAs from the three species, respectively, with an average of 72.6% being successfully assembled into full-length transcripts for each species. Using these full-length circRNAs, we identified thousands of evolutionarily conserved circRNAs that were valuable for functional screening and prioritization. By constructing both species-specific and conserved gene co-expression networks, we inferred circRNA functions on a global scale and prioritized promising functional candidates. To illustrate how well-established prior knowledge facilitates to screen functional candidates, we used the circRNA co-expression networks to prioritize circRNAs that may be involved in liver tumorigenesis and experimentally validated their functions. : Ji et al. present a large-scale study of circRNA repertoires from multiple tissues of human, macaque, and mouse and propose a new approach to annotate and prioritize functional circRNAs. Keywords: circular RNA, circRNA, co-expression network, functional prioritization, noncoding RNA, RNA-binding protein, transcriptome

Monoclonal antibody against CXCL-10/IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury

A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials.

The avian H5N1 influenza virus has the potential to cause a new pandemic. The increasing number of recent outbreaks of highly pathogenic avian influenza H5N1 in birds and humans emphasizes the urgent need to develop a potent H5N1 vaccine. Here, we studied the immunogenicity and protective effect of a vaccine prepared from H5N1 inactivated whole virus. This vaccine was intranasally co-administered in mice with phosphate buffered saline, recombinant cholera toxin B subunit (rCTB), cholera toxin (CT), rCTB containing a trace amount of holotoxin (rCTB/CT), polyinosinic:polycytidylic acid double-stranded RNA (polyI:C), or MF59 as an adjuvant. Intranasal administration of H5N1 inactivated whole virus vaccine with rCTB, CT, rCTB/CT, polyI:C, and MF59 elicited an immunological response with both secretory IgA (sIgA) in nasal, lung, and vaginal lavage, and IgG antibody in serum, showing protective immunity against lethal H5N1 infection. Cross-clade protection was also observed in animals immunized with a vaccine derived from Anhui/01/2005(H5N1) with rCTB, CT, rCTB/CT, polyI:C, or MF59 as adjuvants that were subsequently challenged with the A/OT/SZ/097/03 influenza strain.

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory illness in infants and young children, but the underlying mechanisms responsible for viral pathogenesis have not been fully elucidated. To date, no drugs or vaccines have been employed to improve clinical outcomes for RSV-infected patients. In this paper, we report that angiotensin-converting enzyme-2 (ACE2) protected against severe lung injury induced by RSV infection in an experimental mouse model and in pediatric patients. Moreover, ACE2 deficiency aggravated RSV-associated disease pathogenesis, mainly by its action on the angiotensin II type 1 receptor (AT1R). Furthermore, administration of a recombinant ACE2 protein alleviated the severity of RSV-induced lung injury. These findings demonstrate that ACE2 plays a critical role in preventing RSV-induced lung injury and suggest that ACE2 is a promising potential therapeutic target in the management of RSV-induced lung disease.

Respiratory syncytial virus (RSV) is a major pathogen in infants and the elderly, causing pneumonia and bronchiolitis. Despite decades of research, to date there is still no approved RSV vaccine available. In this study, we developed RSV virus-like particle (VLP) vaccines containing an RSV fusion (F) and/or attachment (G) protein with Newcastle disease virus (NDV) as the platform. The VLPs were expressed in a baculovirus system and purified by sucrose gradient centrifugation. BALB/c mice immunized intranasally (i.n.) with rNDV/RSV/F plus rNDV/RSV/G developed robust humoral, mucosal RSV-specific antibodies and cellular immune responses. Furthermore, rNDV/RSV/F plus rNDV/RSV/G provided better protection than did rNDV/RSV/F or rNDV/RSV/G alone, as shown by an obvious decrease in viral replication together with alleviation of histopathological changes in the lungs of the challenged mice. Our data demonstrate that the intranasal vaccination of combined RSV virus-like particle vaccine candidates has great potential for protection against RSV infection.

The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates.

The matrix protein 2ectodomain (M2e) of the influenza A virus is a rational target antigen candidate for the development of a universal influenza virus-like particle (VLP) vaccine. In this study, a recombinant M2 protein with three tandem copies of M2e (3M2e), nucleoprotein (NP) epitopes and hepatitis B virus core (HBc), were expressed in Escherichia coli and purified by column chromatography. Mice immunized with 3M2e-NP-HBc in combination with an oil-in-water SP01 adjuvant produced robust M2e specific antibodies and cellular immune responses. Most importantly, the 3M2e-NP-HBc VLP vaccine provided enhanced protection against a lethal challenge with pandemic 2009 H1N1 and HPAI H5N1 virus through increased survival rates, a significant decrease in viral replication, and obvious alleviation of histopathological lung changes in challenged mice. Our results imply that a cellular immune response to NP is a plausible mechanism mediating this enhanced protection. These findings suggest that 3M2e-NP-HBc VLP has great potential as the basis development of a broadly protective influenza vaccine.

BackgroundThe increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.Methodology/principal findingsMice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group.Conclusion/significanceOur findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.

BACKGROUND: The 2009 influenza pandemic affected people in almost all countries in the world, especially in younger age groups. During this time, the debate over whether to use corticosteroid treatment in severe influenza H1N1 infections patients resurfaced and was disputed by clinicians. There is an urgent need for a susceptible animal model of 2009 H1N1 infection that can be used to evaluate the pathogenesis and the therapeutic effect of corticosteroid treatment during infection. METHODOLOGY/PRINCIPAL FINDINGS: We intranasally inoculated two groups of C57BL/6 and BALB/c mice (using 4- or 6-to 8-week-old mice) to compare the pathogenesis of several different H1N1 strains in mice of different ages. Based on the results, a very susceptible 4-week-old C57BL/6 mouse model of Beijing 501 strain of 2009 H1N1 virus infection was established, showing significantly elevated lung edema and cytokine levels compared to controls. Using our established animal model, the cytokine production profile and lung histology were assessed at different times post-infection, revealing increased lung lesions in a time-dependent manner. In additional,the mice were also treated with dexamethasone, which significantly improved survival rate and lung lesions in infected mice compared to those in control mice. Our data showed that corticosteroid treatment ameliorated acute lung injury induced by the 2009 A/H1N1 virus in mice and suggested that corticosteroids are valid drugs for treating 2009 A/H1N1 infection. CONCLUSIONS/SIGNIFICANCE: Using the established, very susceptible 2009 Pandemic Influenza A (H1N1) mouse model, our studies indicate that corticosteroids are a potential therapeutic remedy that may address the increasing concerns over future 2009 A/H1N1 pandemics.

Oil-in-water emulsions are potent human adjuvants commonly used in effective pandemic influenza vaccines; however, such emulsions that can induce both Th1-biased systemic immune responses and strong mucosal immune responses via an easy method of administration are lacking. To address this need for new adjuvants, we developed a novel oil/water emulsion, SPO1, which allows convenient mucosal immunization via an intranasal spray as well as by parenteral routes. Our report shows that SPO1 was able to boost up immunological resistance by inducing effective mucosal and serum antibodies, and the immune response was polarized to a Th1 pattern, as demonstrated by high IgG2α antibody levels and interferon-gamma production by splenocytes from intranasally (i.n.) immunized mice. Up-regulation of co-stimulatory and antigen-presenting molecules on dendritic cells was also observed in vivo after i.n. immunization, suggesting a possible mechanism for the adjuvant effects of SPO1. Another explanation may simply be a depot of antigen at the immunization site, as evidenced by in vivo imaging of i.n. immunized mice. In conclusion, our results demonstrate that a novel oil/water emulsion, SPO1, is a potent Th1 adjuvant for use in influenza and other vaccines, as it induces strong mucosal and systemic immune responses.

The 2009 flu pandemic involved the emergence of a new strain of a swine-origin H1N1 influenza virus (S-OIV H1N1) that infected almost every country in the world. Most infections resulted in respiratory illness and some severe cases resulted in acute lung injury. In this report, we are the first to describe a mouse model of S-OIV virus infection with acute lung injury and immune responses that reflect human clinical disease. The clinical efficacy of the antiviral oseltamivir (Tamiflu) administered in the early stages of S-OIV H1N1 infection was confirmed in the mouse model. Moreover, elevated levels of IL-17, Th-17 mediators and IL-17-responsive cytokines were found in serum samples of S-OIV-infected patients in Beijing. IL-17 deficiency or treatment with monoclonal antibodies against IL-17-ameliorated acute lung injury induced by the S-OIV H1N1 virus in mice. These results suggest that IL-17 plays an important role in S-OIV-induced acute lung injury and that monoclonal antibodies against IL-17 could be useful as a potential therapeutic remedy for future S-OIV H1N1 pandemics.

Background Highly pathogenic avian influenza H5N1 virus (HPAI H5N1) has the potential to cause a new pandemic, which may lead to disasters in the world. However, we cannot predict the HPAI H5N1 strain that might cause the pandemic. Therefore, broad-spectrum prophylactic or therapeutic preparations for containment of a possible future pandemic are urgently needed. Polyvalent equine immunoglobulin F(ab′)2 may be a promising candidate. Methods We prepared four pepsin digested immunoglobulin F(ab′)2 from the horses immunized with purified VNH5N1-Puerto Rico/8/34 (PR8)/CDC-RG (VNRG, Clade 1), A/Indonesia/05/2005(H5N1)-PR8-IBCDC-RG2 (INRG, Clade 2.1), and A/Anhui/01/2005(H5N1)-PR8-IBCDC-RG5 (AHRG, Clade 2.3.4) and PBS (negative control), respectively. The protective effect of the monovalent or polyvalent F(ab′)2 against A/Ostrich/SZ/097/04 (clade 0) infection was determined by cytopathic effect (CPE) in cultured Madin–Darby canine kidney (MDCK) cells. The prophylactic and therapeutic efficacy of the polyvalent F(ab′)2 was further evaluated by observing survival, weight loss and viral load when the polyvalent F(ab′)2 was introduced into mice one day prior to-, three days post-lethal challenge with A/Ostrich/SZ/097/04. Results The half neutralization doses (ND50) of purified monovalent equine F(ab′)2 prepared by the VNRG, INRG or AHRG-immunized horses and polyvalent one against A/Ostrich/SZ/097/04 were 320, 1280, 1280 and 2560 in cultured MDCK cells, respectively. 10 μg polyvalent F(ab′)2 could completely protect mice infected with 100 half lethal doses (LD50) of A/Ostrich/SZ/097/04 in preventive settings. In therapeutic settings, even when injected three days post lethal infection, mice were still completely protected, although 200 μg of polyvalent F(ab′)2 was required. Conclusions Our work has provided experimental supports for testing the broad-spectrum protective efficacy of polyvalent equine immunoglobulin F(ab′)2 for the future large trials.

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.

The novel influenza A (H1N1) 2009 virus has emerged to cause the first pandemic of the twenty-first century. Disease outbreaks caused by the influenza A (H1N1) virus have prompted concerns about the potential for a pandemic and have driven the development of vaccines against this subtype of influenza A. In this study, we developed a monovalent influenza A (H1N1) split vaccine and evaluated its effects in BALB/c mice. Mice were immunized subcutaneously with 2 doses of the vaccine containing hemagglutinin (HA) alone or HA plus an aluminum hydroxide (Al(OH)(3)) adjuvant. Immunization with varying doses of HA (3.75, 7.5, 15, 30, 45 or 60 microg) was performed to induce the production of neutralizing antibodies. The vaccine elicited strong hemagglutination inhibition (HI) and microneutralization, and addition of the adjuvant augmented the antibody response. A preliminary safety evaluation showed that the vaccine was not toxic at large doses (0.5 ml containing 60 microg HA+600 microg Al(OH)(3) or 60 microg HA). Moreover, the vaccine was found to be safe at a dose of 120 microg HA+1200 microg Al(OH)(3) or 120 microg HA in 1.0 ml in rats. In conclusion, the present study provides support for the clinical evaluation of influenza A (H1N1) vaccination as a public health intervention to mitigate a possible pandemic. Additionally, our findings support the further evaluation of the vaccine used in this study in primates or humans.

This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium Strains (S. typhimurium) to express and deliver a L7/L12 and BLS fusion antigen of Brucella as a vaccination strategy to prevent Brucella infection in mice. S. typhimurium X4072 that contained a pTrc99A-BLS-L7/L12 plasmid, designated X4072bl, can deliver a L7/L12 and BLS fusion antigen expressed by the bacterium itself, while S. typhimurium X4550 that contained an asd-pVAX1-BLS-L7/L12 (asd-pBL) plasmid, designated X4550bl, can deliver the antigen to be expressed in target eukaryotic cells. When orally administered to BALB/c mice, both attenuated carrier strains were able to elicit mucosal and systemic immunity, which induced protection against B. abortus 544 infection in mice. The immunogenicity and protective efficacy of X4072bl and X4550bl were compared with a recombinant BLS-L7/L12 fusion protein vaccine (rBL) and a pVAX1-BLS-L7/L12 DNA vaccine (pBL) in this study. When rBL and pBL were intramuscularly injected into mice, both vaccines could also elicit comparable humoral and cellular immune responses, but not mucosal immunity, which therefore induced lower protection. Taken together, Salmonella-based subunit vaccines are a promising vaccine strategy in the prevention of Brucella infection.