Genetically stable poliovirus vectors activate dendritic cells and prime antitumor CD8 T cell immunity

Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses can provide a context optimal for generating antigen-specific CD8 T cells, as they have natural tropism for dendritic cells, preeminent inducers of CD8 T cell immunity; elicit Th1-promoting inflammation; and lack interference with innate or adaptive immunity. However, notorious genetic instability and underlying neuropathogenicity has hampered poliovirus-based vector applications. Here we devised a strategy based on the polio:rhinovirus chimera PVSRIPO, devoid of viral neuropathogenicity after intracerebral inoculation in human subjects, for stable expression of exogenous antigens. PVSRIPO vectors infect, activate, and induce epitope presentation in DCs in vitro; they recruit and activate DCs with Th1-dominant cytokine profiles at the injection site in vivo. They efficiently prime tumor antigen-specific CD8 T cells in vivo, induce CD8 T cell migration to the tumor site, delay tumor growth and enhance survival in murine tumor models

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration

BACKGROUND: Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. METHODS: We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. RESULTS: Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. CONCLUSION: These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis

Molecular Analysis of Virulent Determinants of Enterovirus 71

Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5′-NTR and 3′-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (GlyP710/GlnP710/ArgP710 and GluP729) on the DE and EF loop of VP1, one (LysP930) on the surface of protease 2A and four nucleotides on three positions (GP272, UP488 and AP700/UP700) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the AP700/UP700 was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR

Imaging Poliovirus Entry in Live Cells

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to–plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100–200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses

Limited Trafficking of a Neurotropic Virus Through Inefficient Retrograde Axonal Transport and the Type I Interferon Response

Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS). To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle–sciatic nerve–spinal cord–brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN) α/β receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN α/β receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses

Formulation technologies for oral vaccines

Many options now exist for constructing oral vaccines, which, in experimental systems, have shown themselves to be able to generate highly effective immunity against infectious diseases. Their suitability for implementation in clinical practice, however, for prevention of outbreaks particularly in LMIC, is not always guaranteed, because of factors such as cost, logistics, and cultural and environmental conditions. This brief overview provides a summary of the various approaches which can be adopted, and evaluates them from a pharmaceutical point of view, taking into account potential regulatory issues, expense, manufacturing complexity etc., all of which can determine whether a vaccine approach will be successful in the late stages of development. Attention is also drawn to problems arising from inadequate diet, which impacts on success in stimulating effective immunity, and identifies use of lipid‐based carriers as a way to counteract the problem of nutritional deficiencies in vaccination campaigns

Identification of Gemin5 as a Novel 7-Methylguanosine Cap-Binding Protein

A unique attribute of RNA molecules synthesized by RNA polymerase II is the presence of a 7-methylguanosine (m(7)G) cap structure added co-transcriptionally to the 5' end. Through its association with trans-acting effector proteins, the m(7)G cap participates in multiple aspects of RNA metabolism including localization, translation and decay. However, at present relatively few eukaryotic proteins have been identified as factors capable of direct association with m(7)G.Employing an unbiased proteomic approach, we identified gemin5, a component of the survival of motor neuron (SMN) complex, as a factor capable of direct and specific interaction with the m(7)G cap. Gemin5 was readily purified by cap-affinity chromatography in contrast to other SMN complex proteins. Investigating the underlying basis for this observation, we found that purified gemin5 associates with m(7)G-linked sepharose in the absence of detectable eIF4E, and specifically crosslinks to radiolabeled cap structure after UV irradiation. Deletion analysis revealed that an intact set of WD repeat domains located in the N-terminal half of gemin5 are required for cap-binding. Moreover, using structural modeling and site-directed mutagenesis, we identified two proximal aromatic residues located within the WD repeat region that significantly impact m(7)G association.This study rigorously identifies gemin5 as a novel cap-binding protein and describes an unprecedented role for WD repeat domains in m(7)G recognition. The findings presented here will facilitate understanding of gemin5's role in the metabolism of non-coding snRNAs and perhaps other RNA pol II transcripts

Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice

VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma.In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point.VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus

A Single Nucleotide in Stem Loop II of 5′-Untranslated Region Contributes to Virulence of Enterovirus 71 in Mice

BACKGROUND: Enterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored. PRINCIPAL FINDINGS: In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5'-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5'-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5'-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. CONCLUSIONS: These results presented the first reported virulence determinant in EV71 5'-UTR and first position discovered from unadapted isolates

Tissue Type-Specific Expression of the dsRNA-Binding Protein 76 and Genome-Wide Elucidation of Its Target mRNAs

Background: RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulatory functions for rapid adjustment to changing extra-or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins. Methodology: We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific subcellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered subcellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism. Significance: Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation. Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype