System Vaccinology for the Evaluation of Influenza Vaccine Safety by Multiplex Gene Detection of Novel Biomarkers in a Preclinical Study and Batch Release Test

Vaccines are beneficial and universal tools to prevent infectious disease. Thus, safety of vaccines is strictly evaluated in the preclinical phase of trials and every vaccine batch must be tested by the National Control Laboratories according to the guidelines published by each country. Despite many vaccine production platforms and methods, animal testing for safety evaluation is unchanged thus far. We recently developed a systems biological approach to vaccine safety evaluation where identification of specific biomarkers in a rat pre-clinical study evaluated the safety of vaccines for pandemic H5N1 influenza including Irf7, Lgals9, Lgalsbp3, Cxcl11, Timp1, Tap2, Psmb9, Psme1, Tapbp, C2, Csf1, Mx2, Zbp1, Ifrd1, Trafd1, Cxcl9, β2m, Npc1, Ngfr and Ifi47. The current study evaluated whether these 20 biomarkers could evaluate the safety, batch-to-batch and manufacturer-to-manufacturer consistency of seasonal trivalent influenza vaccine using a multiplex gene detection system. When we evaluated the influenza HA vaccine (HAv) from four different manufactures, the biomarker analysis correlated to findings from conventional animal use tests, such as abnormal toxicity test. In addition, sensitivity of toxicity detection and differences in HAvs were higher and more accurate than with conventional methods. Despite a slight decrease in body weight caused by HAv from manufacturer B that was not statistically significant, our results suggest that HAv from manufacturer B is significantly different than the other HAvs tested with regard to Lgals3bp, Tapbp, Lgals9, Irf7 and C2 gene expression in rat lungs. Using the biomarkers confirmed in this study, we predicted batch-to-batch consistency and safety of influenza vaccines within 2 days compared with the conventional safety test, which takes longer. These biomarkers will facilitate the future development of new influenza vaccines and provide an opportunity to develop in vitro methods of evaluating batch-to-batch consistency and vaccine safety as an alternative to animal testing.     

Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus

BackgroundJamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections.Methodology/Principal findingsThe antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2’-fluoro-2’-deoxycytidine (2’-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC₉₀) of FPV, RBV, and 2’-FdC were 41.0, 61.8, and 13.6 μM in Vero cells and 20.7, 25.8, and 8.8 μM in N2A cells, respectively. All mice infected with 1.0×10⁴ TCID₅₀ died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (−2 dpi–2 dpi) or on the day of infection (0 dpi–4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi–4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi).Conclusions/SignificanceAlthough the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.     

Identification of Fasciola species isolated from Egypt based on sequence analysis of genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers

Fascioliasis has a negative impact on the farming industry in both developed and developing countries, rather than a public health challenge. This study was performed to identify Fasciola sp. from different definitive hosts (buffalo, cattle, and sheep) based on the molecular parameters and spermatogenesis. Ninety-one adult flukes were collected from livers of slaughtered animals at abattoirs in different prefectures in Egypt. Microscopic examination of the analyzed flukes showed many normal spermatozoa in the seminal vesicles (spermic), suggesting that they have the ability of spermatogenesis. This study showed that no parthenogenic Fasciola species occurred in Egypt. Molecular analysis was performed utilizing genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Whereas 16 animals proved to have infection with a single Fasciola species, 2 were infected with both F. hepatica and F. gigantica. The results indicated that sheep were prone to F. hepatica (8 out of 10 animals) more than F. gigantica infection. Sequences of ITS1 and ITS2 ribosomal region indicated that the flukes were categorized into 3 groups F. hepatica-type (47), F. gigantica-type (42) and 2 flukes possessed sequences of both types indicating an existence of different alleles at the same loci. Unique overlapping of T/C bases were detected in both ITS1 (Position 96) and ITS2 (Position 416). Based on results of mitochondrial gene markers (NDI and COI), flukes were classified into F. hepatica-type and F. gigantica-type. Extensive intra-sequence polymorphism was detected at both markers. NDI and COI sequences of Egyptian strain of F. gigantica showed pronounced diversity compared with relevant sequences at database.     

The first detection of Babesia species DNA from Japanese black bears (Ursus thibetanus japonicus) in Japan

In this study, we tried to detect protozoan blood parasites from the liver or blood of 156 Japanese black bears (Ursus thibetanus japonicus) in Iwate Prefecture of Japan by polymerase chain reaction. Two amplicons (approximately 540 bp and 480 bp) were detected by amplification for V4 hyper-variable regions of the 18S rRNA gene. Approximately 540-bp products were obtained in 119 samples (76.3%) and were considered to be DNA of Hepatozoon ursi. Approximately 480-bp products were obtained in 22 samples (14.1%) and were considered to be DNA of Babesia species. The nucleotide sequences (1635 bp) of the 18S rRNA gene of Babesia sp. were very similar (99.3%) to those (AY190123, AY190124) of Babesia sp. detected previously from Ixodes ovatus. Phylogenetic analysis showed that Babesia sp. detected in this study closely related to Babesia sp. derived from raccoons in Japan and the U.S.A. This is the first report of Babesia species detected from Japanese black bears.     

HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3′ splice site 2 (A2) and 5′splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.     

Identification of Inter-Organ Vascular Network: Vessels Bridging between Organs

Development and homeostasis of organs and whole body is critically dependent on the circulatory system. In particular, the circulatory system, the railways shuttling oxygen and nutrients among various organs, is indispensible for inter-organ humoral communication. Since the modern view of the anatomy and mechanics of the circulatory system was established in 17^th century, it has been assumed that humoral factors are carried to and from organs via vascular branches of the central arteries and veins running along the body axis. Over the past few decades, major advances have been made in understanding molecular and cellular mechanisms underlying the vascularization of organs. However, very little is known about how each organ is linked by vasculature (i.e., inter-organ vascular networks). In fact, the exact anatomy of inter-organ vascular networks has remained obscure. Herein, we report the identification of four distinct vessels, V1^LP, V2^LP, V3^LP and V4^LP, that bridge between two organs, liver and pancreas in developing zebrafish. We found that these inter-organ vessels can be classified into two types: direct and indirect types. The direct type vessels are those that bridge between two organs via single distinct vessel, to which V1^LP and V2^LP vessels belong. The indirect type bridges between two organs via separate branches that emanate from a stem vessel, and V3^LP and V4^LP vessels belong to this type. Our finding of V1^LP, V2^LP, V3^LP and V4^LP vessels provides the proof of the existence of inter-organ vascular networks. These and other yet-to-be-discovered inter-organ vascular networks may facilitate the direct exchange of humoral factors that are necessary for the coordinated growth, differentiation and homeostasis of the connected organs. It is also possible that the inter-organ vessels serve as tracks for their connected organs to follow during their growth to establish their relative positions and size differences.     

Isoquinoline derivatives as potent CRTH2 antagonists: Design,synthesis and SAR

In this study, we describe the synthesis and structure–activity relationship (SAR) of a series of isoquinoline chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2) antagonists. TASP0376377 (15-20), one of the most potent compounds, showed a potent binding affinity (IC₅₀ = 19 nM) in addition to the excellent functional antagonist activity (IC₅₀ = 13 nM). Moreover, the efficacy of this compound in a chemotaxis assay (IC₅₀ = 23 nM) was in good agreement with its potency as a CRTH2 antagonist. In addition, 15-20 exhibited greater selectivity in binding to CRTH2 than to the DP1 prostanoid receptor (IC₅₀ >1 μM) or the enzymes COX-1 and COX-2 (IC₅₀ >10 μM).     

Bacillus thuringiensis Cry toxins bound specifically to various proteins via domain III, which had a galactose-binding domain-like fold

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.