Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs.

Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.

Intestinal Epithelial Cells Modulate the Production of Enterotoxins by Porcine Enterotoxigenic E. coli Strains.

Enterotoxigenic Escherichia coli (ETEC) strains are one of the most common etiological agents of diarrhea in both human and farm animals. In addition to encoding toxins that cause diarrhea, ETEC have evolved numerous strategies to interfere with host defenses. These strategies most likely depend on the sensing of host factors, such as molecules secreted by gut epithelial cells. The present study tested whether the exposure of ETEC to factors secreted by polarized IPEC-J2 cells resulted in transcriptional changes of ETEC-derived virulence factors. Following the addition of host-derived epithelial factors, genes encoding enterotoxins, secretion-system-associated proteins, and the key regulatory molecule cyclic AMP (cAMP) receptor protein (CRP) were substantially modulated, suggesting that ETEC recognize and respond to factors produced by gut epithelial cells. To determine whether these factors were heat sensitive, the IEC-conditioned medium was incubated at 56 °C for 30 min. In most ETEC strains, heat treatment of the IEC-conditioned medium resulted in a loss of transcriptional modulation. Taken together, these data suggest that secreted epithelial factors play a role in bacterial pathogenesis by modulating the transcription of genes encoding key ETEC virulence factors. Further research is warranted to identify these secreted epithelial factors and how ETEC sense these molecules to gain a competitive advantage in the early engagement of the gut epithelium.

Porcine Enterotoxigenic Escherichia coli Strains Differ in Their Capacity To Secrete Enterotoxins through Varying YghG Levels.

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens for humans and farm animals such as pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains, and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa, and STb. The strains differed greatly in their capacity to secrete LT, pSTa, and STb. Remarkably, in some strains, periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type II secretion system (T2SS) protein YghG plays a regulatory role in controlling LT secretion levels. These findings highlight YghG as an important mediator of the secretion of the heat-labile enterotoxin LT by porcine ETEC strains and provide better insights into ETEC enterotoxin secretion.IMPORTANCE Enterotoxigenic E. coli strains are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in the production and secretion capacities of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis.

DPP-4 inhibition has beneficial effects on the heart after myocardial infarction.

AIMS: Dipeptidyl peptidase-4 (DPP-4) inhibitors are reported to have protective effects on various cells but it is unclear how DPP-4 inhibitors have cardioprotective effects. Our aim was to study the mechanisms of cardioprotective effects by DPP-4 inhibition. METHODS AND RESULTS: C57BL/6 mice and DPP-4 knockout (DPP-4KO) mice were subjected to left coronary artery ligation to produce acute myocardial infarction (MI). C57BL/6 mice were then treated with vehicle or DPP-4 inhibitor. Left ventricular function, infarct size, the number of vessels, and myocardial ischemia were assessed at 5days after MI. The treatment with DPP-4 inhibitor significantly improved cardiac function and decreased the infarct size. DPP-4 inhibitor increased the ratio of endothelial cell numbers to a cardiomyocyte. The extent of myocardial ischemia and the number of TUNEL-positive cells in the border area were significantly decreased by DPP-4 inhibitor. Stromal cell-derived factor-1α (SDF-1α) level in myocardium was significantly increased by DPP-4 inhibitor. Those cardioprotective effects after MI were also recognized in DPP-4KO mice. DPP-4 protein was expressed on rat neonatal cardiomyocytes and DPP-4 inhibitor significantly reduced hypoxia-induced apoptosis in the cardiomyocytes. However, this effect was abolished by the pretreatment with a CXCR4 antagonist or a signal transducer and activator of transcription 3 (STAT3) inhibitor. The beneficial effects of DPP-4 inhibitor on heart failure after MI were abolished by cardiomyocyte-specific deletion of STAT3. CONCLUSIONS: DPP-4 inhibition may have direct protective effects on the post-MI heart by inducing an antiapoptotic effect and inhibiting a decrease in vessel number through the SDF-1α/CXCR4-mediated STAT3 signaling pathway.

Development of a reverse genetics system for epizootic hemorrhagic disease virus and evaluation of novel strains containing duplicative gene rearrangements.

Epizootic haemorrhagic disease is a non-contagious infectious viral disease of wild and domestic ruminants caused by epizootic hemorrhagic disease virus (EHDV). EHDV belongs to the genus Orbivirus within the family Reoviridae and is transmitted by insects of the genus Culicoides. The impact of epizootic haemorrhagic disease is underscored by its designation as a notifiable disease by the Office International des Epizooties. The EHDV genome consists of 10 linear dsRNA segments (Seg1-Seg10). Until now, no reverse genetics system (RGS) has been developed to generate replication-competent EHDV entirely from cloned cDNA, hampering detailed functional analyses of EHDV biology. Here, we report the generation of viable EHDV entirely from cloned cDNAs. A replication-competent EHDV-2 (Ibaraki BK13 strain) virus incorporating a marker mutation was rescued by transfection of BHK-21 cells with expression plasmids and in vitro synthesized RNA transcripts. Using this RGS, two additional modified EHDV-2 viruses were also generated: one that contained a duplex concatemeric Seg9 gene and another that contained a duplex concatemeric Seg10 gene. The modified EHDV-2 with a duplex Seg9 gene was genetically stable during serial passage in BHK-21 cells. In contrast, the modified EHDV-2 with a duplex Seg10 gene was unstable during serial passage, but displayed enhanced replication kinetics in vitro when compared with the WT virus. This RGS provides a new platform for the investigation of EHDV replication, pathogenesis and novel EHDV vaccines.

Identification of four novel group-specific bluetongue virus NS3 protein B-cell epitopes.

BACKGROUND: The non-structural protein 3 (NS3) of bluetongue virus (BTV) is the second smaller non-structural protein produced in host cells, playing an important role in BTV trafficking and release. RESULTS: In this study, we generated five BTV NS3-reactive monoclonal antibodies (mAbs), named 3D8, 2G9, 1B5, 4H8, and 2B12. A panel of overlapping NS3-derived peptides representing the entirety of the BTV15 NS3 protein was screened to identify linear peptide epitopes recognized by each mAb. Based on the initial screen, a series of progressively truncated peptides were produced to identify the minimal linear peptide sequence required to maintain mAb binding. We found that mAb 3D8 reacted with the motif (36)PPRYA(40), 2G9 reacted with the motif (82)AEAFRDDVRLRQIK(95), 1B5 reacted with the motif (205)YNDAVRMSF(213), 2B12 and 4H8 reacted with the motif (204)SYNDAVRMSF(213). Sequence alignments demonstrated that these linear epitopes are highly conserved among all BTV serotypes, consistent with the observation that each mAb was able to recognize cells infected with BTV1-24 serotypes tested and each identified B cell epitope was able to be recognized by BTV-infect sheep serum. CONCLUSION: This collection of mAbs along with defined linear epitopes may provide useful reagents for investigations of NS3 protein function and the development of BTV group-specific diagnostics.

Detection, discrimination and quantitation of 22 bluetongue virus serotypes using real-time RT-PCR with TaqMan MGB probes.

Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes.

DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1.

Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

Amplification of Protein Expression by Self-Amplifying mRNA Delivered in Lipid Nanoparticles Containing a ß-Aminoester Ionizable Lipid Correlates with Reduced Innate Immune Activation.

Self-amplifying mRNA (saRNA) is witnessing increased interest as a platform technology for protein replacement therapy, gene editing, immunotherapy, and vaccination. saRNA can replicate itself inside cells, leading to a higher and more sustained production of the desired protein at a lower dose. Controlling innate immune activation, however, is crucial to suppress unwanted inflammation upon delivery and self-replication of RNA in vivo. In this study, we report on a class of ß-aminoester lipids (ßAELs) synthesized through the Michael addition of an acrylate to diethanolamine, followed by esterification with fatty acids. These lipids possessed one or two ionizable amines, depending on the use of nonionic or amine-containing acrylates. We utilized ßAELs for encapsulating saRNA in lipid nanoparticles (LNPs) and evaluated their transfection efficiency in vitro and in vivo in mice, while comparing them to LNPs containing ALC-0315 as an ionizable lipid reference. Among the tested lipids, OC7, which comprises two unsaturated oleoyl alkyl chains and an ionizable azepanyl motif, emerged as a ßAEL with low cytotoxicity and immunogenicity relative to ALC-0315. Interestingly, saRNA delivered via the OC7 LNP exhibited a distinct in vivo transfection profile. Initially, intramuscular injection of OC7 LNP resulted in low protein expression shortly after administration, followed by a gradual increase over a period of up to 7 days. This pattern is indicative of successful self-amplification of saRNA. In contrast, saRNA delivered via ALC-0315 LNP demonstrated high protein translation initially, which gradually declined over time and lacked the amplification seen with OC7 LNP. We observed that, in contrast to saRNA OC7 LNP, saRNA ALC-0315 LNP induced potent innate immune activation by triggering cytoplasmic RIG-I-like receptors (RLRs), likely due to the highly efficient endosomal membrane rupturing properties of ALC-0315 LNP. Consequently, the massive production of type I interferons quickly hindered the amplification of the saRNA. Our findings highlight the critical role of the choice of ionizable lipid for saRNA formulation in LNPs, particularly in shaping the qualitative profile of protein expression. For applications where minimizing inflammation is desired, the use of ionizable lipids, such as the ßAEL reported in this study, that elicit a low type I interferon response in saRNA LNP is crucial.

Research on Temperature-Switched Dopamine Electrochemical Sensor Based on Thermosensitive Polymers and MWCNTs.

A temperature-controlled electrochemical sensor was constructed based on a composite membrane composed of temperature-sensitive polymer poly (N-isopropylacrylamide) (PNIPAM) and carboxylated multi-walled carbon nanotubes (MWCNTs-COOH). The sensor has good temperature sensitivity and reversibility in detecting Dopamine (DA). At low temperatures, the polymer is stretched to bury the electrically active sites of carbon nanocomposites. Dopamine cannot exchange electrons through the polymer, representing an "OFF" state. On the contrary, in a high-temperature environment, the polymer shrinks to expose electrically active sites and increases the background current. Dopamine can normally carry out redox reactions and generate response currents, indicating the "ON" state. In addition, the sensor has a wide detection range (from 0.5 µM to 150 µM) and low LOD (193 nM). This switch-type sensor provides new avenues for the application of thermosensitive polymers.

Heat-Stable Enterotoxins of Enterotoxigenic Escherichia coli and Their Impact on Host Immunity.

Enterotoxigenic Escherichia coli (ETEC) are an important diarrhea-causing pathogen and are regarded as a global threat for humans and farm animals. ETEC possess several virulence factors to infect its host, including colonization factors and enterotoxins. Production of heat-stable enterotoxins (STs) by most ETEC plays an essential role in triggering diarrhea and ETEC pathogenesis. In this review, we summarize the heat-stable enterotoxins of ETEC strains from different species as well as the molecular mechanisms used by these heat-stable enterotoxins to trigger diarrhea. As recently described, intestinal epithelial cells are important modulators of the intestinal immune system. Thus, we also discuss the impact of the heat-stable enterotoxins on this role of the intestinal epithelium and how these enterotoxins might affect intestinal immune cells. Finally, the latest developments in vaccination strategies to protect against infections with ST secreting ETEC strains are discussed. This review might inform and guide future research on heat-stable enterotoxins to further unravel their molecular pathogenesis, as well as to accelerate vaccine design.

Preparation and Characterization of a Monoclonal Antibody Against the Core Protein VP7 of the 25th Serotype of Bluetongue Virus.

Bluetongue virus (BTV) is a member of the genus Orbivirus, within the family Reoviridae. The VP7 protein of BTV is used for developing group-specific serological assays. To prepare monoclonal antibody (MAb) against VP7 of the 25th serotype BTV, the RNA S7 encoding VP7 was cloned into prokaryotic expression vectors pET-28a (+) and pGEX-6P-1 to generate recombinant plasmids. The recombinant protein VP7 was expressed in Escherichia coli BL21 (DE3), respectively. The results of SDS-PAGE revealed that the VP7 was expressed and the molecular mass of recombinant fusion protein pET-28a (+)/VP7 and pGEX-6P-1/VP7 was approximately 44 kDa and 64 kDa, respectively. The Western blot analysis indicated that the recombinant VP7 possessed good immunoreactivity. After purification, pET-28a (+)/VP7 was used to immunize BALB/c mice, while pGEX-6P-1/VP7 was used to screen for well-to-well MAb-secreting hybridomas. The hybridoma cell line 3H7 against recombinant VP7 that secreted MAbs was obtained. The isotype of 3H7 was identified as IgG1. The purification of recombinant VP7 protein and the monoclonal antibody will have potential applications on competitive ELISA format for BT-specific serum detection method.

Autophagy Activated by Bluetongue Virus Infection Plays a Positive Role in Its Replication.

Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. Despite extensive study in recent decades, the interplay between BTV and host cells is not clearly understood. Autophagy as a cellular adaptive response plays a part in many viral infections. In our study, we found that BTV1 infection triggers the complete autophagic process in host cells, as demonstrated by the appearance of obvious double-membrane autophagosome-like vesicles, GFP-LC3 dots accumulation, the conversion of LC3-I to LC3-II and increased levels of autophagic flux in BSR cells (baby hamster kidney cell clones) and primary lamb lingual epithelial cells upon BTV1 infection. Moreover, the results of a UV-inactivated BTV1 infection assay suggested that the induction of autophagy was dependent on BTV1 replication. Therefore, we investigated the role of autophagy in BTV1 replication. The inhibition of autophagy by pharmacological inhibitors (3-MA, CQ) and RNA interference (siBeclin1) significantly decreased viral protein synthesis and virus yields. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted viral protein expression and the production of infectious BTV1. These findings lead us to conclude that autophagy is activated by BTV1 and contributes to its replication, and provide novel insights into BTV-host interactions.