E1 Region Research Articles

410. Oncolytic Adenoviruses Armed with TNFa and IL-2 Induce Antitumor Immune Responses and Protection from Tumor Rechallenge

During the past few years, immune system-stimulating factors have emerged as promising means to treat malignant tumors. We aim to use cytokine-bearing oncolytic adenoviruses to improve and to extend the usage of adoptive T cell therapy, a treatment known to benefit a portion of melanoma and leukemia patients. Administered systemically or intratumorally the virus induces immune responses against the tumor by revealing tumor antigens, but immunostimulatory cytokines augment the effect further. We have shown that the most promising cytokines in this regard are interleukin (IL) -2 and Tumor Necrosis Factor alpha (TNFa). IL-2 is a common treatment for malignant melanoma and renal cell carcinoma, but systemic administration may lead to severe side effects. While IL-2 has a key role in recruiting and activating T cells, TNFa has prominent anti-immunosuppressive actions. Further, it directly promotes tumor cell death by apoptosis and necrosis. Armed oncolytic viruses accomplish local, long-lasting, high-level cytokine expression while systemic level remains low. Moreover, we have proven that adenoviruses enhance adoptive T cell therapy. In this study, we treated Syrian hamsters (Mesocricetus auratus) with Ad5/3-E2F-d24 virus bearing human IL-2, TNFa, or both in its E3 region. Hamster cell lines are semipermissive for 5/3-chimeric adenoviruses and produce active transgenes from these viruses. In addition, human IL-2 and TNFa are evidently active in hamsters. Hamster pancreatic cancer (HapT1) was implanted subcutaneously and treated once with TILs extracted from syngenic tumors, and with five viral injections. We saw synergy between unarmed virus and TILs, while the armed viruses turned out to be even more effective. When the cured animals were rechallenged with the same cancer cells, previous treatment with cytokine-armed viruses protected the animals against new tumors. Also, splenocytes derived from animals treated with cytokine-bearing viruses proliferated more actively ex vivo than the controls. To conclude, our data demonstrates the immunological benefit gained with viruses bearing IL-2 and TNFa, and further supports combining oncolytic viruses with adoptive cell transfer.

639. Oncolytic Adenovirus Expressing IFN Alpha Works Synergistically with Chemoradiation

Aside from curative resection, there is no curative treatment against pancreatic adenocarcinoma (PDAC) at present. Late diagnosis and high recurrence results in five-year survival of 6%. Notably, Phase II trials based on adjuvant therapy combining systemic IFN Alpha (IFN) and chemoradiation reported 30-50% increase in two-year survival and impressive 35% increase in five-year survival of PDAC patients. Despite promising results, trial drawbacks included high patient dropout due to IFN systemic toxicity and low IFN levels in tumors. Low intratumoral IFN hampered the full potential of the therapy while IFN is known to induce tumor apoptosis, chemoradio sensitization, and decreased tumor neo-vasculatization.Aiming to improve efficacy and tolerability of IFN therapy, we have developed an oncolytic adenovirus expressing human IFN (OAd-IFN). Vector has Ad5/3 fiber modification and overexpresses Adenoviral Death Protein respectively contributing to increased infectivity and oncolysis. Taking advantage of Cox-2 up-regulation in PDAC, the Cox-2 promoter was included in the upstream of Adenovirus E1 region, restricting vector replication to cancer cells. Human IFN-alpha gene was placed in the Adenovirus E3 region in the way that its expression is controlled by the adenovirus major late promoter. Therefore, IFN expression in this vector is replication dependent. To test the vector in an immunocompetent syngeneic hamster model of pancreatic cancer, OAd-IFN expressing hamster IFN was generated. Vector contains RGD fiber modification enhancing its infectivity in hamster cells.MTS and crystal violet assays demonstrated sensitization of PDAC cells to chemotherapy (5-FU, Cisplatin, and Gemcitabine), and radiation (4 and 8Gy) by OAd-IFN. Comparison between OAd-IFN and control vector not expressing IFN (OAd-LUC) indicated that IFN expressed by OAd-IFN is functional in combination therapy sensitizing PDAC cells to chemoradiation. Colony formation assay showed that combinations of OAd-IFN with chemotherapy, radiation, or chemoradiation are synergistic and exhibit superior killing effect compared to groups without OAd-IFN.In vivo studies using immunocompetent syngeneic hamster model of pancreatic cancer showed that combinations including OAd-IFN resulted in augmented tumor shrinkage and survival compared to groups treated with chemotherapy, radiation, chemoradiation, or OAd-LUC + radiation. Hexon staining and viral DNA quantification by qPCR show OAd-IFN effectively replicates and spreads in tumors.Our data suggests OAd-IFN synergistically improves chemoradioation in PDAC cells, and shows superior therapeutic effect when treating immunocompetent model of pancreatic cancer. Vector capacity to express high levels of IFN intratumorally and the strong synergism between OAd-IFN and radiation, chemotherapy, and chemoradiation indicates OAd-IFN will contribute to more olerable and effective IFN therapy. Strong synergism between OAd-IFN and chemoradiation combined with focal expression of IFN can help to reduce not only IFN toxicity, but also dose-dependent toxicity of chemoradiation. Considering systemic IFN injection combined with chemoradiation is one of the few therapies to effectively treat pancreatic cancer, usage of OAd-IFN in combination with chemoradiation has great potential to result in more effective and tolerable therapy agaisnt pancreatic cancer.

14. Engineering and Characterization of a Cloned Adenoviral-Library to Explore Natural Virus Diversity

Large double-stranded DNA (dsDNA) viruses such as adenovirus (Ad), herpesvirus, poxvirus and their recombinant counterparts were well explored in basic virology and biomedical research. Especially for the development of novel vaccines and gene therapies, Ad gained special attention and represents the most widely explored vector worldwide. Although ~70 types of human Ad and numerous nonhuman Ad (>200) have been identified so far a system for efficient Ad genome cloning and manipulation was lacking and therefore the majority of recombinant adenoviral vectors (AdVs) are based on a small fraction of Ad types. Here we report the generation and characterization of an engineered human adenoviral-library allowing exploration and system studies of the natural Ad diversity. Towards that end we first established that adenoviral genomes can be cloned and tagged in a high-throughput manner utilizing advanced homologous recombination techniques. Wild type Ads from clinical isolates including around half of the currently known adenovirus types that represent all seven human adenovirus species were propagated and direct high-throughput cloning (HTC) applied. The integrity of cloned Ad genomes was confirmed by DNA restriction enzyme pattern and virus reconstitution was conducted using optimized conditions. Most importantly next-generation sequencing (NGS) and phylogenetic analysis was performed. As a further step, half of all cloned adenoviruses representing each species were tagged with a 2A peptide-mediated multicistronic expression cassette providing a Turbo Green fluorescent protein as in vitro marker, a NanoLuc luciferase for in vivo studies and kanamycin/neomycin as selection marker. For HTC of tagged viruses the reporter cassette was inserted into the adenovirus E3 region using different orientations because the orientation of the transgene was essential for reconstitution efficiencies. After successful reconstitution these double reporters-labeled AdVs were evaluated in cultured cell lines and mouse models. In vitro characterization revealed distinct tropisms for tested viruses. Among the currently evaluated cell lines, specie B viruses demonstrated high transduction efficiencies in epithelial (Hela- and A549 cells) and endothelial cells, while Ad5 still displayed highest transduction rates in other human and murine cell types (hepatocytes, lymphocytes, neuroblastoma cells and myoblasts). For the further characterization of unknown receptor usage, we injected recombinant viruses into DSG2 or CD46 transgenic mice for in vivo bio-distribution analyses on genome level by quantitative PCR and on protein level by immunohistology analyses. We anticipate that our engineered adenoviral-library will provide a spacious novel view to the adenovirus field. As a broader perspective it will bring AdV and also other large dsDNA viruses from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology.

Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer.

Backgroundc-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1–E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter.ResultsWe now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression.ConclusionsOur data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0063-y) contains supplementary material, which is available to authorized users.

AChE and the amyloid precursor protein (APP) – Cross-talk in Alzheimer's disease

The amyloid precursor protein (APP) and acetylcholinesterase (AChE) are multi-faceted proteins with a wide range of vital functions, both crucially linked with the pathogenesis of Alzheimer's disease (AD). APP is the precursor of the Aβ peptide, the pathological agent in AD, while AChE is linked to its pathogenesis either by increasing cholinergic deficit or exacerbating Aβ fibril formation and toxicity. As such, both proteins are the main targets in AD therapeutics with AChE inhibitors being currently the only clinically available AD drugs. In our studies we have demonstrated an important inter-relation in functioning of these proteins. Both can be released from the cell membrane and we have shown that AChE shedding involves a metalloproteinase-mediated mechanism which, like the α-secretase dependent cleavage of APP, is stimulated by cholinergic agonists. Overexpression of the neuronal specific isoform APP695 in neuronal cells substantially decreased levels of the AChE mRNA, protein and catalytic activity accompanied by a similar decrease in mRNA levels of the AChE membrane anchor, PRiMA (proline rich membrane anchor). We further established that this regulation does not involve APP processing and its intracellular domain (AICD) but requires the E1 region of APP, specifically its copper-binding domain. On the contrary, siRNA knock-down of APP in cholinergic SN56 cells resulted in a significant upregulation of AChE mRNA levels. Hence APP may influence AChE physiology while released AChE may regulate amyloidogenesis through multiple mechanisms suggesting novel therapeutic targets.

Molecular determinants of alphavirus neuropathogenesis in mice.

Alphaviruses are enveloped viruses with a positive-stranded RNA genome, of the family Togaviridae. In mammals and birds they are mosquito-transmitted and are of veterinary and medical importance. They cause primarily two types of disease: encephalitis and polyarthritis. Here we review attempts to understand the molecular basis of encephalitis and virulence for the central nervous system (CNS) in mouse models. Sindbis virus (SINV) was the first virus to be studied in this way. Other viruses analysed are Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Eastern equine encephalitis virus and Western equine encephalitis virus. Neurovirulence was found to be associated with damage to neurons in the CNS. It mapped mainly to the E2 region of the genome, and to the nsP3 gene. Also, avirulent natural isolates of both SINV and SFV have been found to have more rapid cleavage of nonstructural proteins due to mutations in the nsP1-nsP2 cleavage site. Immune-mediated demyelination for avirulent SFV has been shown to be associated with infection of oligodendrocytes. For Chikungunya virus, an emerging alphavirus that uncommonly causes encephalitis, analysis of the molecular basis of CNS pathogenicity is beginning. Experiments on SINV and SFV have indicated that virulence may be related to the resistance of virulent virus to interferon action. Although the E2 protein may be involved in tropism for neurons and passage across the blood-brain barrier, the role of the nsP3 protein during infection of neurons is unknown. More information in these areas may help to further explain the neurovirulence of alphaviruses.

Genome analysis of four Old World monkey adenoviruses supports the proposed species classification of primate adenoviruses and reveals signs of possible homologous recombination.

Within the family Adenoviridae, presently Simian mastadenovirus A is the single species approved officially for monkey adenoviruses (AdVs), whilst the establishment of six further species (Simian mastadenovirus B to Simian mastadenovirus G) has been proposed in the last few years. We examined the genetic content and phylogenetic relationships of four Old World monkey (OWM) AdV types [namely simian AdV (SAdV)-8, -11, -16 and -19] for which it had been proposed that they should be classified into different AdV species: SAdV-11 to Human mastadenovirus G, and the other three viruses into three novel species. By full genome sequencing, we identified gene contents characteristic for the genus Mastadenovirus. Among the 36 ORFs, 2 genes of different lengths, predicted to encode the adenoviral cellular attachment protein (the fibre), were found. The E3 regions contained six genes, present in every OWM AdV, but lacked the E3 19K gene, which has seemingly appeared only in the ape (hominid) AdV lineages during evolution. For the first time in SAdVs, two other exons belonging to the gene of the so-called U exon protein were also predicted. Phylogenetic calculations, based on the fibre-1 and the major capsid protein, the hexon, implied that recombination events might have happened between different AdV species. Phylogeny inference, based on the viral DNA-dependent DNA polymerase and the penton base protein, further supported the species classification proposed earlier.

Lack of detection of human papillomavirus DNA in prostate carcinomas in patients from northeastern Brazil.

Prostate cancer is the second most common cancer among men in western populations, and despite its high mortality, its etiology remains unknown. Inflammatory processes are related to the etiology of various types of tumors, and prostate inflammation, in particular, has been associated with prostate cancer carcinogenesis and progression. Human papillomavirus (HPV) is associated with benign and malignant lesions in the anogenital tract of both females and males. The possible role of HPV in prostate carcinogenesis is a subject of great controversy. In this study, we aimed to examine the prevalence of HPV infections in prostate carcinomas of patients from northeastern Brazil. This study included 104 tissue samples from primary prostate carcinoma cases. HPV DNA was purified and then amplified using MY09/11 and GP5+/GP6+ degenerate primer sets that detect a wide range of HPV types, and with specific PCR primers sets for E6 and E7 HPV regions to detect HPV 16. None of the samples showed amplification products of HPV DNA for primer sets MY09/11 and GP5+/GP6+, or the specific primer set for the E6 and E7 HPV regions. HPV infection, thus, does not seem to be one of the causes of prostate cancer in the population studied.

A Glance at Recombination Hotspots in the Domestic Cat.

Recombination has essential roles in increasing genetic variability within a population and in ensuring successful meiotic events. The objective of this study is to (i) infer the population-scaled recombination rate (ρ), and (ii) identify and characterize regions of increased recombination rate for the domestic cat, Felis silvestris catus. SNPs (n = 701) were genotyped in twenty-two East Asian feral cats (random bred). The SNPs covered ten different chromosomal regions (A1, A2, B3, C2, D1, D2, D4, E2, F2, X) with an average region size of 850 Kb and an average SNP density of 70 SNPs/region. The Bayesian method in the program inferRho was used to infer regional population recombination rates and hotspots localities. The regions exhibited variable population recombination rates and four decisive recombination hotspots were identified on cat chromosome A2, D1, and E2 regions. As a description of the identified hotspots, no correlation was detected between the GC content and the locality of recombination spots, and the hotspots enclosed L2 LINE elements and MIR and tRNA-Lys SINE elements.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

ABSTRACTThe hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape.IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

Effects on the transcriptome upon deletion of a distal element cannot be predicted by the size of the H3K27Ac peak in human cells

Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. We used epigenomic information to identify H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC. Employing CRISPR/Cas9 nucleases, we deleted a CRC risk-associated H3K27Ac peak from HCT116 cells and observed large-scale changes in gene expression, resulting in decreased expression of many nearby genes. As a comparison, we showed that deletion of a robust H3K27Ac peak not associated with CRC had minimal effects on the transcriptome. Interestingly, although there is no H3K27Ac peak in HEK293 cells in the E7 region, deletion of this region in HEK293 cells decreased expression of several of the same genes that were downregulated in HCT116 cells, including the MYC oncogene. Accordingly, deletion of E7 causes changes in cell culture assays in HCT116 and HEK293 cells. In summary, we show that effects on the transcriptome upon deletion of a distal regulatory element cannot be predicted by the size or presence of an H3K27Ac peak.

Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome.

Presence of Human Papillomavirus DNA in Colorectal Cancer Tissues in Shiraz, Southwest Iran.

Colorectal cancer is one of the most common cancers worldwide. Viruses including human papillomavirus (HPV) have been reported to be associated with different cancers but any association with colorectal cancers remains controversial. To evaluate any association between HPV infection and adenocarcinoma of the colon and adenomatous polyps. Paraffin-embedded tissue specimens of 70 colorectal adenocarcinomas, 70 colorectal adenomatous polyps, and 70 colorectal normal tissues were subjected to DNA extraction. The quality of the extracted DNA was confirmed by amplification of a β-globin fragment using polymerase chain reaction (PCR). PCR using specific primers were performed to detect HPV DNA. Specific primers targeting the E6 region of the HPVs 16 and 18 were used for genotyping. HPV DNA was detected in 2 (2.85%) out of 70 adenocarcinoma colorectal tissues and 4 (5.71%) out of 70 adenomatous colorectal tissues. All normal colorectal tissues were negative for HPV DNA. HPV-16 was the most predominant genotype (5 sample) followed by HPV-18 (4 sample). Despite the above observations, statistical analyses indicated no significant differences in the frequencies of HPV positive subjects between the cancerous and normal samples. Although the differences observed in the frequencies of HPV positive cases in our study was not significant relative to those of control subjects, the fact of 6 positive samples among cancerous tissues, may still suggest a role of HPV in colorectal carcinogenesis. The study collectively indicated that some colorectal cancerous tissues are infected with high risk HPV genotype. The findings merit more investigation.

HIV infection and hepatitis C virus genotype 1a are associated with phylogenetic clustering among people with recently acquired hepatitis C virus infection

The aim of this study was to identify factors associated with phylogenetic clustering among people with recently acquired hepatitis C virus (HCV) infection. Participants with available sample at time of HCV detection were selected from three studies; the Australian Trial in Acute Hepatitis C, the Hepatitis C Incidence and Transmission Study — Prison and Community. HCV RNA was extracted and Core to E2 region of HCV sequenced. Clusters were identified from maximum likelihood trees with 1000 bootstrap replicates using 90% bootstrap and 5% genetic distance threshold. Among 225 participants with available Core-E2 sequence (ATAHC, n=113; HITS-p, n=90; and HITS-c, n=22), HCV genotype prevalence was: G1a: 38% (n=86), G1b: 5% (n=12), G2a: 1% (n=2), G2b: 5% (n=11), G3a: 48% (n=109), G6a: 1% (n=2) and G6l 1% (n=3). Of participants included in phylogenetic trees, 22% of participants were in a pair/cluster (G1a-35%, 30/85, mean maximum genetic distance=0.031; G3a-11%, 12/106, mean maximum genetic distance=0.021; other genotypes-21%, 6/28, mean maximum genetic distance=0.023). Among HCV/HIV co-infected participants, 50% (18/36) were in a pair/cluster, compared to 16% (30/183) with HCV mono-infection (P=<0.001). Factors independently associated with phylogenetic clustering were HIV co-infection [vs. HCV mono-infection; adjusted odds ratio (AOR) 4.24; 95%CI 1.91, 9.39], and HCV G1a infection (vs. other HCV genotypes; AOR 3.33, 95%CI 0.14, 0.61).HCV treatment and prevention strategies, including enhanced antiviral therapy, should be optimised. The impact of targeting of HCV treatment as prevention to populations with higher phylogenetic clustering, such as those with HIV co-infection, could be explored through mathematical modelling.

Covering the Cover

Covering the Cover

Identification and molecular characterization of border disease virus (BDV) from sheep in India.

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5'-UTR, N(pro) and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N(pro) and entire region coding structural proteins showed that the N(pro) (168), C (100 aa), E(rns) (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.

Attenuation of Replication-Competent Adenovirus Serotype 26 Vaccines by Vectorization.

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors.

Antibodies to an interfering epitope in hepatitis C virus E2 can mask vaccine-induced neutralizing activity.

Hepatitis C virus (HCV) neutralization occurring at the E2 region 412‐426 (EP‐I) could be enhanced when antibodies directed specifically to the E2 region 434‐446 (EP‐II) were removed from serum samples of persistently infected patients and vaccinated chimpanzees, a phenomenon of so‐called antibody interference. Here, we show that this type of interference can be observed in individuals after immunization with recombinant E1E2 proteins. One hundred twelve blinded serum samples from a phase I, placebo‐controlled, dose escalation trial using recombinant HCV E1E2 with MF59C.1 adjuvant in healthy HCV‐negative adults were tested in enzyme‐linked immunosorbent assay for binding reactivity to peptides representing the E2 regions 412‐426 (EP‐I) and 434‐446 (EP‐II). All samples were subsequently tested for neutralizing activity using cell‐culture HCV 1a(H77)/2a chimera, HCV pseudotype particles (HCVpp) H77, and HCVpp HCV‐1 after treatment to remove EP‐II‐specific antibodies or mock treatment with a control peptide. Among the 112 serum samples, we found 22 double positive (EP‐I and EP‐II), 6 EP‐II positive only, 14 EP‐I positive only, and 70 double negative. Depleting EP‐II antibodies from double‐positive serum samples increased 50% inhibitory dose (ID50) neutralizing antibody titers (up to 4.9‐fold) in up to 72% of samples (P ≤ 0.0005), contrasting with ID50 neutralization titer increases in 2 of 70 double‐negative samples (2.9%; P > 0.5). In addition, EP‐I‐specific antibody levels in serum samples showed a significant correlation with ID50 neutralization titers when EP‐II antibodies were removed (P < 0.0003). Conclusion: These data show that antibodies to the region 434‐446 are induced during immunization of individuals with recombinant E1E2 proteins, and that these antibodies can mask effective neutralizing activity from EP‐I‐specific antibodies. Elicitation of EP‐II‐specific antibodies with interfering capacity should be avoided in producing an effective cross‐neutralizing vaccine aimed at the HCV envelope proteins.(Hepatology 2015;62:1670–1682)

Functional Diversification of Motor Neuron-specific Isl1 Enhancers during Evolution.

Functional diversification of motor neurons has occurred in order to selectively control the movements of different body parts including head, trunk and limbs. Here we report that transcription of Isl1, a major gene necessary for motor neuron identity, is controlled by two enhancers, CREST1 (E1) and CREST2 (E2) that allow selective gene expression of Isl1 in motor neurons. Introduction of GFP reporters into the chick neural tube revealed that E1 is active in hindbrain motor neurons and spinal cord motor neurons, whereas E2 is active in the lateral motor column (LMC) of the spinal cord, which controls the limb muscles. Genome-wide ChIP-Seq analysis combined with reporter assays showed that Phox2 and the Isl1-Lhx3 complex bind to E1 and drive hindbrain and spinal cord-specific expression of Isl1, respectively. Interestingly, Lhx3 alone was sufficient to activate E1, and this may contribute to the initiation of Isl1 expression when progenitors have just developed into motor neurons. E2 was induced by onecut 1 (OC-1) factor that permits Isl1 expression in LMCm neurons. Interestingly, the core region of E1 has been conserved in evolution, even in the lamprey, a jawless vertebrate with primitive motor neurons. All E1 sequences from lamprey to mouse responded equally well to Phox2a and the Isl1-Lhx3 complex. Conversely, E2, the enhancer for limb-innervating motor neurons, was only found in tetrapod animals. This suggests that evolutionarily-conserved enhancers permit the diversification of motor neurons.

Enhanced Antitumor Efficacy and Reduced Toxicity of Docetaxel Loaded Estradiol Functionalized Stealth Polymeric Nanoparticles.

In spite of extensive research over the decades, breast cancer treatment is still a major challenge due to nonspecific distribution of the chemotherapeutics. This void can be filled by restricting the distribution of chemotherapeutics toward the cancerous cells. In the present report estradiol (E2) functionalization of docetaxel (DTX) loaded PLGA nanoparticles was supposed to have specific distribution of DTX to cancerous cells simultaneously avoiding the nonspecific distribution toward the normal cells. In line, E2-PEG-PLGA conjugate was synthesized and characterized by FTIR and NMR spectroscopy. Extensive optimization of different process variables resulted in the formation of spherical E2-PEG-PLGA NPs in the size range of 228.5 ± 11.8 nm and entrapment efficiency of 94.25 ± 2.49. Trehalose (5% w/v) resulted in the formation of a fluffy, easy to redisperse freeze-dried cake of nanoparticles. PXRD analysis revealed the amorphous nature of DTX encapsulated within the nanoparticles. X-ray photoelectron spectroscopy confirmed the presence of E2 over the surface of nanoparticles. In line with our hypothesis, cellular uptake studies on ER positive MCF-7 cells revealed relatively higher uptake and efficient localization into the nuclear region of E2-PEG-PLGA NPs in comparison with plain counterparts, while in the case of ER negative HeLa cells E2-PEG-PLGA NPs showed no difference in fluorescence pattern as compared to MCF-7 cells incubated with unmodified nanoformulation, indicating nonspecific delivery of DTX. Moreover, MTT assay revealed relatively higher cytotoxicity of E2-PEG-PLGA NPs in comparison with free DTX. Furthermore, in vivo pharmacokinetic studies revealed 9.36- and 4.79-fold enhancement in circulation half-life and AUC(0-∞), respectively, of E2-PEG-PLGA NPs in comparison with Taxotere. In vivo antitumor efficacy in DMBA induced rat model demonstrated significant reduction in tumor volume in comparison with the plain counterpart (PLGA-NPs) and a marketed formulation, Taxotere. Moreover, the safety of the estradiol functionalized PLGA NPs was confirmed by hepato- and nephrotoxicity studies.