Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs.

BACKGROUND: African swine fever (ASF) is a highly fatal disease in domestic pigs caused by ASF virus (ASFV), for which there is currently no commercial vaccine available. The genome of ASFV encodes more than 150 proteins, some of which have been included in subunit vaccines but only induce limited protection against ASFV challenge. METHODS: To enhance immune responses induced by ASFV proteins, we expressed and purified three fusion proteins with each consisting of bacterial lipoprotein OprI, 2 different ASFV proteins/epitopes and a universal CD4+ T cell epitope, namely OprI-p30-modified p54-TT, OprI-p72 epitopes-truncated pE248R-TT, and OprI-truncated CD2v-truncated pEP153R-TT. The immunostimulatory activity of these recombinant proteins was first assessed on dendritic cells. Then, humoral and cellular immunity induced by these three OprI-fused proteins cocktail formulated with ISA206 adjuvant (O-Ags-T formulation) were assessed in pigs. RESULTS: The OprI-fused proteins activated dendritic cells with elevated secretion of proinflammatory cytokines. Furthermore, the O-Ags-T formulation elicited a high level of antigen-specific IgG responses and interferon-γ-secreting CD4+ and CD8+ T cells after stimulation in vitro. Importantly, the sera and peripheral blood mononuclear cells from pigs vaccinated with the O-Ags-T formulation respectively reduced ASFV infection in vitro by 82.8% and 92.6%. CONCLUSIONS: Our results suggest that the OprI-fused proteins cocktail formulated with ISA206 adjuvant induces robust ASFV-specific humoral and cellular immune responses in pigs. Our study provides valuable information for the further development of subunit vaccines against ASF.

Antigenic and immunogenic properties of recombinant proteins consisting of two immunodominant African swine fever virus proteins fused with bacterial lipoprotein OprI.

BACKGROUND: African swine fever (ASF) is a highly fatal swine disease, which threatens the global pig industry. There is no commercially available vaccine against ASF and effective subunit vaccines would represent a real breakthrough. METHODS: In this study, we expressed and purified two recombinant fusion proteins, OPM (OprI-p30-modified p54) and OPMT (OprI-p30-modified p54-T cell epitope), which combine the bacterial lipoprotein OprI with ASF virus proteins p30 and p54. Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The activation of dendritic cells (DCs) by these proteins was first assessed. Then, humoral and cellular immunity induced by the proteins were evaluated in mice. RESULTS: Both OPM and OPMT activated DCs with elevated expression of relevant surface molecules and proinflammatory cytokines. Furthermore, OPMT elicited the highest levels of antigen-specific IgG responses, cytokines including interleukin-2, interferon-γ, and tumor necrosis factor-α, and proliferation of lymphocytes. Importantly, the sera from mice vaccinated with OPM or OPMT neutralized more than 86% of ASF virus in vitro. CONCLUSIONS: Our results suggest that OPMT has good immunostimulatory activities and immunogenicity in mice, and might be an appropriate candidate to elicit immune responses in swine. Our study provides valuable information on further development of a subunit vaccine against ASF.

Development of an Indirect Chemiluminescence Immunoassay Using a Multiepitope Recombinant Protein To Specifically Detect Antibodies against Foot-and-Mouth Disease Virus Serotype O in Swine.

Foot-and-mouth disease virus (FMDV) has led to serious losses in animal husbandry worldwide. Seromonitoring of FMDV postvaccination is important for the control and eradication of foot-and-mouth disease (FMD) in regions and countries where vaccination is widespread. However, many commercial kits present high false-positive rates. In this study, a multiepitope-based indirect chemiluminescence immunoassay (ME-CLIA) was developed for specifically detecting antibodies against FMDV serotype O in swine sera. The developed method presented high diagnostic sensitivity and excellent diagnostic specificity, and it could detect a broad spectrum of antibodies against FMDV serotype O. The diagnostic performance, accuracy rate, and analytical sensitivity of ME-CLIA were compared with those of three commercial kits. The immune protection value of multiple-epitope recombinant vaccine detected using ME-CLIA was preliminarily determined by observation of clinical symptoms postimmunization challenge, the results of which indicated that the ME-CLIA can be employed as a matching detection method for evaluating multiple-epitope recombinant vaccine. The percent positive values of ME-CLIA determined using swine vaccinated with inactivated vaccine were significantly positively correlated with the titers of liquid-phase-blocking enzyme-linked immunosorbent assay (ELISA) (LBPE) (r = 0.8361; P < 0.0001). These results indicated that ME-CLIA is suitable for detection of antibodies against FMDV serotype O in swine and for potency evaluation of multiple-epitope and inactivated vaccines.

Development of a competitive chemiluminescence immunoassay using a monoclonal antibody recognizing 3B of foot-and-mouth disease virus for the rapid detection of antibodies induced by FMDV infection.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating animal disease. Anti-non-structural protein (NSP) antibody detection is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease. METHODS: In this study, a competitive chemiluminescence immunoassay (3B-cCLIA) was developed for the rapid detection of antibodies against NSPs in different species of livestock animals using the monoclonal antibody (mAb) 9E2 as a competitive antibody that recognizes NSP 3B. RESULTS: The cut-off value (50%), diagnostic sensitivity (Dsn) (97.20%, 95.71%, and 96.15%) and diagnostic specificity (Dsp) (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known-background sera from swine, cattle and sheep, respectively. The accuracy rate of the 3B-cCLIA was further validated and subsequently compared with that of two commercial diagnostic kits. The early diagnostic results showed that antibodies recognizing NSPs developed later (approximately 1-2 days) than antibodies recognizing structural proteins. Furthermore, anti-NSP antibody presence in animals vaccinated multiple times (false positives), especially cattle and sheep, was confirmed, and the false-positive rate increased with the number of vaccinations. CONCLUSIONS: These results indicate that the 3B-cCLIA is suitable for the rapid detection of antibodies against FMDV NSP 3B in a wide range of species.

Development of an indirect ELISA to specifically detect antibodies against African swine fever virus: bioinformatics approaches.

BACKGROUND: African swine fever (ASF), characterized by acute, severe, and fast-spreading, is a highly lethal swine infectious disease caused by the African swine fever virus (ASFV), which has caused substantial economic losses to the pig industry worldwide in the past 100 years. METHODS: This study started with bioinformatics methods and verified the epitope fusion protein method's reliability that does not rely on traditional epitope identification. Meanwhile, it will also express and purify the constructed genes through prokaryotic expression and establish antibody detection methods. RESULTS: The results indicated that the protein had good reactivity and did not cross-react with other swine diseases. The receiver-operating characteristic analysis was performed to verify the determination. The area under the receiver-operating characteristic curve was 0.9991 (95% confidence interval 0.9973 to 1.001). CONCLUSIONS: It was proved that the recombinant protein is feasible as a diagnostic antigen to distinguish ASFV and provides a new idea for ASFV antibody detection.

Development of alveolar-capillary-exchange (ACE) chip and its application for assessment of PM2.5-induced toxicity.

Although standard two-dimensional (2D) cell culture is an effective tool for cell studies, monolayer cultivation can yield imperfect or misleading information about numerous biological functions. In this study, we developed an alveolar-capillary exchange (ACE) chip aiming to simulate the cellular microenvironment at the alveolar-capillary interface. The ACE chip was designed with two chambers for culturing alveolar epithelial cells and vascular endothelial cells separately, which are separated by a microporous polycarbonate film that allows for the exchange of soluble biomolecules. Using this model, we further tested the toxic effects of fine particulate matter (PM2.5), a form of airborne pollutant known to induce adverse effects on human respiratory system. These effects are largely associated with the ability of PM2.5 to penetrate the alveoli, where it negatively affects the pulmonary function. Our results indicate that alveolar epithelial cells cultured in the ACE chip in solo and coculture with vascular endothelial cells underwent oxidative injury-induced apoptosis mediated via the PEAK-eIF2α signaling pathway of endoplasmic reticulum stress. The use of ACE chip in an alveolar epithelial cell-vascular endothelial cell coculture model revealed cellular vulnerability to PM2.5. Therefore, this chip provides a feasible surrogate approach in vitro for investigating and simulating the cellular microenvironment responses associated with ACE in vivo.

Demographics and medical disorders associated with smoking: a population-based study.

BACKGROUND: Few studies have investigated factors associated with smoking behaviors. In this population-based study, we investigated demographics and medical comorbid diseases to establish a prediction model for smoking behaviors by using the National Health Interview Survey (NHIS) and National Health Insurance Research Database (NHIRD). METHODS: We enrolled individuals aged ≥40 years who had participated in the NHIS in 2001, 2005, and 2009. We identified the smoking behaviors of the study participants in the NHIS. Smoking behaviors were divided into ever smokers (current smokers and ex-smokers) and nonsmokers (never smokers).We defined medical comorbid disorders of the study participants by using medical claim data from the NHIRD. We used multivariable logistic regression models to calculate the adjusted odds ratio and 95% confidence interval for variables associated with smoking. The significant variables in the multivariable model were included in the receiver operating characteristic curves (ROC) to predict the sensitivity and specificity of the model. RESULTS: In total, 26,375 participants (12,779 men and 13,596 women) were included in the analysis. The prevalence of smoking was 39.29%. The mean ages of the 16,012 nonsmokers were higher than those of the 10,363 smokers (57.86 ± 12.92 years vs. 53.59 ± 10.82 years). Men outnumbered women among smokers (68.18% vs. 31.82%). Male sex, young age and middle age, being insured categories, residence in suburban areas, and chronic obstructive pulmonary disease (COPD) were independent factors associated with smoking. The area under the ROC curve of these significant factors to predict smoking behaviors was 71.63%. CONCLUSION: Sex, age, insured categories, residence in suburban areas, and COPD were associated with smoking in people.

Reporter gene knock-in into Marc-145 cells using CRISPR/Cas9-mediated homologous recombination.

OBJECTIVES: Marc-145 cells (monkey embryonic kidney epithelial cells) play a critical role in the biotechnology industry as certain virus host cells. To investigate the expression of enhanced green fluorescent protein (eGFP) gene as a foreign gene in Marc-145 cells, which we developed an approach of foreign gene site-specific knock-in into Marc-145 cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and putatively explored appropriate genomic recombination sites in Marc-145 cells. RESULTS: Our study demonstrated that the specific homologous recombination (HR) site between the Rac GTPase activating protein 1 (RACGAP1) and the acid-sensing ion channel subunit 1 (ASIC1) genes of the 11th chromosome could be used as the target site of Cas9 for the generation of target gene knock-in into Marc-145 cells, by the insertion of the eGFP cassette into the specific HR site and subsequent expression. CONCLUSIONS: Junction PCR, sequencing, Southern blot and fluorescence assay determined eGFP gene-specific knock-in HR site between the RACGAP1 and ASIC1 genes of the 11th chromosome, which was identified by the genomic safe harbours in Marc-145 cells. Our study encouraged a broader range of applications, such as Marc-145 cells development and engineering for virus adaption and yield increase in the vaccine biotechnology industry.

Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis through the endosomal pathway in Drosophila.

Mutations of the retromer component Vps35 and endosomal kinase LRRK2 are linked to autosomal dominant forms of familial Parkinson's disease (PD). However, the physiological and pathological roles of Vps35 and LRRK2 in neuronal functions are poorly understood. Here, we demonstrated that the loss of Drosophila Vps35 (dVps35) affects synaptic vesicle recycling, dopaminergic synaptic release and sleep behavior associated with dopaminergic activity, which is rescued by the expression of wild-type dVps35 but not the PD-associated mutant dVps35 D647N. Drosophila LRRK2 dLRRK together with Rab5 and Rab11 is also implicated in synaptic vesicle recycling, and the manipulation of these activities improves the Vps35 synaptic phenotypes. These findings indicate that defects of synaptic vesicle recycling in which two late-onset PD genes, Vps35 and LRRK2, are involved could be key aspects of PD etiology.

Transcriptome analysis of responses to bluetongue virus infection in Aedes albopictus cells.

BACKGROUND: Bluetongue virus (BTV) causes a disease among wild and domesticated ruminants which is not contagious, but which is transmitted by biting midges of the Culicoides species. BTV can induce an intense cytopathic effect (CPE) in mammalian cells after infection, although Culicoides- or mosquito-derived cell cultures cause non-lytic infection with BTV without CPE. However, little is known about the transcriptome changes in Aedes albopictus cells infected with BTV. METHODS: Transcriptome sequencing was used to identify the expression pattern of mRNA transcripts in A. albopictus cells infected with BTV, given the absence of the Culicoides genome sequence. Bioinformatics analyses were performed to examine the biological functions of the differentially expressed genes. Subsequently, quantitative reverse transcription-polymerase chain reaction was utilized to validate the sequencing data. RESULTS: In total, 51,850,205 raw reads were generated from the BTV infection group and 51,852,293 from the control group. A total of 5769 unigenes were common to both groups; only 779 unigenes existed exclusively in the infection group and 607 in the control group. In total, 380 differentially expressed genes were identified, 362 of which were up-regulated and 18 of which were down-regulated. Bioinformatics analyses revealed that the differentially expressed genes mainly participated in endocytosis, FoxO, MAPK, dorso-ventral axis formation, insulin resistance, Hippo, and JAK-STAT signaling pathways. CONCLUSION: This study represents the first attempt to investigate transcriptome-wide dysregulation in A. albopictus cells infected with BTV. The understanding of BTV pathogenesis and virus-vector interaction will be improved by global transcriptome profiling.

Antiviral activity of porcine interferon omega 7 against foot-and-mouth disease virus in vitro.

Foot-and-mouth disease (FMD) is a disease of worldwide economic importance, and vaccines play an important role in preventing FMDV outbreaks. However, new control strategies are still needed since FMDV outbreaks still occur in some disease-free countries. Currently, interferon (IFN)-based strategies have been demonstrated to be an efficient biotherapeutic option against FMDV; however, interferon omega (IFN-ω) has not yet been assessed in this capacity. Thus, this study evaluated the antiviral activity of porcine IFN omega 7 (PoIFN-ω7) against FMDV. After the PoIFN-ω7 was expressed and purified, cell proliferation assays and quantitative real-time reverse transciption-polymerase chain reaction were used to evaluate the effective anti-cytopathic concentration of PoIFN-ω7 and its effectiveness pre- and post-infection with FMDV in swine kidney cells (IBRS-2). Results showed the rHis-PoIFN-ω7 fusion protein was considerably expressed using Escherichia coli BL21 (DE3) strain, and the recombinant protein exhibited significant in vitro protection against FMDV, including two strains belonging to type O and A FMDV, respectively. In addition, PoIFN-ω7 upregulated the transcription of Mx1, ISG15, OAS1, and PKR genes. These findings indicated that IFN-ω has the potential for serving as a useful therapeutic agent to prevent FMDV or other viral outbreaks in pigs.

Artificially designed hepatitis B virus core particles composed of multiple epitopes of type A and O foot-and-mouth disease virus as a bivalent vaccine candidate.

Recently, many countries, including China, have experienced a series of type A and O foot-and-mouth disease virus (FMDV) epidemics, causing serious economic losses. Although concerns about the safety of inactivated FMD vaccines have been raised, the development of a safe and effective subunit vaccine is necessary. We constructed two chimeric virus-like particles (VLPs; rHBc/AO and rHBc/AOT VLPs) displaying tandem repeats of B cell epitopes (VP1 residue 134-161 and 200-213) derived from type A and O FMDV and one T cell epitope (3 A residue 21-35) using the truncated hepatitis B virus core (HBc) carrier. Our results indicate that the chimeric HBc can self-assemble into VLPs with these FMDV epitopes displayed on the surface. Immunization with the chimeric VLPs induced specific IgG and neutralization antibodies against type A and O FMDV in mice. Compared with the commercial type A/O FMDV bivalent inactivated vaccine, rHBc/AO and rHBc/AOT VLPs significantly stimulated the production of Th1 type cytokines (IFN-γ and IL-2), whereas Th2 cytokine production (IL-4 and IL-10) was decreased. Compared with rHBc/AO, rHBc/AOT induced increased Th2 cytokine and specific IgG production. These results demonstrate that the VLPs constructed in the current study induced both humoral and cellular immune responses and may represent potential bivalent VLP vaccines targeting both FMDV type A and O strains.

Inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects cloven-hoof animals including cattle, swine, sheep, goats, and lots of wild species. Effectively control measures are urged needed. Here, we showed that homoharringtonine treatment exhibited a strong inhibitory effect against two different strains of FMDVs (O/MYA98/BY/2010 and A/GD/MM/2013) in swine kidney (IBRS-2) cells. Further experiments demonstrated that homoharringtonine did not affect virus attachment or entry. Using time-of-addition assays, we found that the antiviral activity of homoharringtonine occurred primarily during the early stage of infection. These results demonstrated that homoharringtonine might be an effective anti-FMDV drug. Further studies are required to explore the antiviral activity of homoharringtonine against FMDV replication in vivo.

Antiviral effect of amiloride on replication of foot and mouth disease virus in cell culture.

Recently, amiloride was shown to potently suppress Coxsackievirus B3 (CVB3) replication. In the current study, we investigated whether amiloride could also exhibit antiviral activity against foot-and-mouth disease virus (FMDV), which belongs to the same family (Picornaviridae) as CVB3. We found that amiloride exerted antiviral activity in a dose-dependent manner against two strains of FMDV in IBRS-2 cells, with slight cytotoxicity at 1000 µM. Besides, amiloride did not inhibit the attachment and entry of FMDV in IBRS-2 cells, but prevented early viral replication. These data implied that amiloride could be a promising candidate for further research as a potential antiviral drug against FMDV infection.

A novel type I interferon, interferon alphaomega, shows antiviral activity against foot-and-mouth disease virus in vitro.

Recently, a novel type I interferon alphaomega (IFN-αω), also known as IFN-µ, was identified. However, the biological activity of IFN-αω remain poorly understood. In this study, the porcine IFN-αω (PoIFN-αω) was expressed, purified, and its antiviral activities assessed by its ability to inhibit the cytopathic effect caused by FMDV on IBRS-2 cells. In addition, q-PCR was used to evaluate the expression of IFN-stimulated genes induced by PoIFN-αω. It was found that PoIFN-αω exerted effective antiviral activity against FMDV pre- and post-infection. Additionally, PoIFN-αω induced the transcription of IFN-stimulated genes, including Mx1, ISG15, OAS1, and PKR genes. Our study reported a new indication of PoIFN-αω as an effective anti-FMDV agent for the first time.

Identification of three linear B cell epitopes against non-structural protein 3ABC of FMDV using monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) has led to serious losses in the farming industry worldwide, particularly in cattle and swine. In developing countries, the control and eradication of FMD rely upon vaccination, in which the inactivated vaccine is predominant. In the preparation of inactivated vaccine, a series of purification methods were used to remove non-structural proteins (NSPs). It is necessary to develop a quantitative detection method of residual NSP and confirm a threshold value for the evaluation of the vaccine. Meanwhile, it is also important to develop a sensitive and rapid diagnostic method to distinguish infected animals from vaccinated animals (DIVA). In this study, three monoclonal antibodies (MAbs) against NSP 3ABC, designated 2G5, 9E2, and 1E10, were used. Subsequently, a series of overlapping peptides were expressed using a prokaryotic expression system to determine the minimal epitopes identified by the MAbs. Three linear B cell epitopes (BCEs), "92EYIEKA97" "23EGPYAGPLE31" and "209EPHH212", were identified by MAbs 2G5, 9E2, and 1E10, respectively. Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes. The epitope "92EYIEKA97" is located in 3A, which is deleted in some natural deletion mutants that result in a change in virus tropism. MAb 9E2 that identified the epitope "23EGPYAGPLE31" reacted with 3B1 and 3B2, but did not react with 3B3. In combination with sequence alignment analysis, the epitope "23EGPYAGPLE31" is highly conserved among different FMDV isolates. Preliminary screening using the known positive and negative sera indicated the MAb 9E2 has the potential for the development of a diagnostic method for DIVA. The residual NSP in inactivated vaccines can be detected using 9E2-HRP, which indicated the MAb 9E2 is able to evaluate inactivated vaccines. The four-amino acid epitope is the first reported to date that is recognized by 1E10. These results provide valuable insight into the diagnosis of DIVA and the NSP residual evaluation in inactivated vaccines.

In Vitro and in Vivo Antiviral Activity of Mizoribine Against Foot-And-Mouth Disease Virus.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which has significant economic consequences in affected countries. As the currently available vaccines against FMD provide no protection until 4-7 days post-vaccination, the only alternative method to control the spread of FMD virus (FMDV) during outbreaks is the application of antiviral agents. Hence, it is important to identify effective antiviral agents against FMDV infection. In this study, we found that mizoribine has potent antiviral activity against FMDV replication in IBRS-2 cells. A time-of-drug-addition assay demonstrated that mizoribine functions at the early stage of replication. Moreover, mizoribine also showed antiviral effect on FMDV in vivo. In summary, these results revealed that mizoribine could be a potential antiviral drug against FMDV.

Type I Interferons: Distinct Biological Activities and Current Applications for Viral Infection.

The interferons (IFNs) are a primary defense against pathogens because of the strong antiviral activities they induce. IFNs can be classified into three groups: type I, type II and type III, according to their genetic, structural, and functional characteristics and their receptors on the cell surface. The type I IFNs are the largest group and include IFN-α, IFN-ß, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN-τ and IFN-ζ. The use of IFNs for the treatment of viral infectious diseases on their antiviral activity may become an important therapeutic option, for example, IFN-α is well known for the successful treatment of hepatitis B and C virus infections, and interest is increasing in the antiviral efficacy of other novel IFN classes and their potential applications. Therefore, in this review, we summarize the recent progress in the study of the biological activities of all the type I IFN classes and their potential applications in the treatment of infections with immunodeficiency virus, hepatitis viruses, and influenza viruses.

Untangling the Tauopathy for Alzheimer's disease and parkinsonism.

Tau is a microtubule-associated protein that mainly localizes to the axon to stabilize axonal microtubule structure and neuronal connectivity. Tau pathology is one of the most common proteinopathies that associates with age-dependent neurodegenerative diseases including Alzheimer's disease (AD), and various Parkinsonism. Tau protein undergoes a plethora of intra-molecular modifications and some altered forms promote the production of toxic oligomeric tau and paired helical filaments, and through which further assemble into neurofibrillary tangles, also known as tauopathy. In this review, we will discuss the recent advances of the tauopathy research, primarily focusing on its association with the early axonal manifestation of axonal transport defect, axonal mitochondrial stress, autophagic vesicle accumulation and the proceeding of axon destruction, and the pathogenic Tau spreading across the synapse. Two alternative strategies either by targeting tau protein itself or by improving the age-related physiological decline are currently racing to find the hopeful treatment for tauopathy. Undoubtedly, more studies are needed to combat this devastating condition that has already affected millions of people in our aging population.