A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism.

Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165,000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.

Estrogen-related receptor gamma regulates mitochondrial and synaptic genes and modulates vulnerability to synucleinopathy.

Many studies implicate mitochondrial dysfunction as a key contributor to cell loss in Parkinson disease (PD). Previous analyses of dopaminergic (DAergic) neurons from patients with Lewy-body pathology revealed a deficiency in nuclear-encoded genes for mitochondrial respiration, many of which are targets for the transcription factor estrogen-related receptor gamma (Esrrg/ERRγ). We demonstrate that deletion of ERRγ from DAergic neurons in adult mice was sufficient to cause a levodopa-responsive PD-like phenotype with reductions in mitochondrial gene expression and number, that partial deficiency of ERRγ hastens synuclein-mediated toxicity, and that ERRγ overexpression reduces inclusion load and delays synuclein-mediated cell loss. While ERRγ deletion did not fully recapitulate the transcriptional alterations observed in postmortem tissue, it caused reductions in genes involved in synaptic and mitochondrial function and autophagy. Altogether, these experiments suggest that ERRγ-deficient mice could provide a model for understanding the regulation of transcription in DAergic neurons and that amplifying ERRγ-mediated transcriptional programs should be considered as a strategy to promote DAergic maintenance in PD.

Cell vehicle targeting strategies.

Use of cells as therapeutic carriers has increased in the past few years and has developed as a distinct concept and delivery method. Cell-based vehicles are particularly attractive for delivery of biotherapeutic agents that are difficult to synthesize, have reduced half-lives, limited tissue penetrance or are rapidly inactivated upon direct in vivo introduction. Initial studies using cell-based approaches served to identify some of the key factors for the success of this type of therapeutic delivery. These factors include the efficiency of cell loading with a therapeutic payload, the means of cell loading and the nature of therapeutics that cells can carry. However, one important aspect of cell-based delivery yet to be fully investigated is the process of actual delivery of the cell payload in vivo. In this regard, the potential ability of cell carriers to provide site-specific or targeted delivery of therapeutics deserves special attention. The present review focuses on a variety of targeting approaches that may be utilized to improve cell-based therapeutic delivery strategies. The different aspects of targeting that can be applied to cell vehicles will be discussed, including physical methods for directing cell distribution, intrinsic cell-mediated homing mechanisms and the feasibility of engineering cells with novel targeting mechanisms. Development of cell targeting strategies will further advance cell vehicle applications, broaden the applicability of this delivery approach and potentiate therapeutic outcomes.

[Experimental study of the possibility of emergency prophylaxis of Bolivian hemorrhagic fever]. / Eksperimental'noe izuchenie vozmozhnosti ékstrennoi profilaktiki Boliviiskoi gemorragicheskoi likhoradki.

Describes changes in nonspecific immunity parameters (interferon, interleukin-1, tumor necrosis factor, and natural killers) in the course of experimental Bolivian fever (Machupo virus). Changes of these parameters were followed up after urgent prophylactic injections of Ridostin and specific gamma-globulin to infected animals. The possibility of treatment of experimental Machupo fever by intranasal administration of interferon inductor Ridostin has been demonstrated.

[The immunity indices in the infection of mice of different strains with the Machupo virus]. / Pokazateli immuniteta pri zarazhenii myshei razlichnykh linii virusom Machupo.

The pathomorphological patterns and the activity of serum interferon, interleukin-1, tumor necrosis factor, natural killers, and proliferative activity of lymphocytes were studied in BALB/c and C57B1/6 mice intracerebrally infected with Machupo virus. The BALB/c mice showed 100% lethality at 8-9 days after inoculation while C57B1/6 mice were found nonsusceptible to Machupo virus inoculation by this route. The pathomorphological findings at the peak of clinical manifestations in BALB/c mice revealed no organ whose functional deficiency could lead to the death of the animals. Investigations of nonspecific immunity parameters revealed a direct dependence between their high activity and susceptibility of the animals to Machupo virus infection. It is assumed that the endogenous shock due to the high activity of immune response mediators is the cause of death in Machupo virus infection.

[The ultrastructural changes in guinea pig organs during the serial passage of the Ebola virus]. / Ul'trastrukturnye izmeneniia v organakh morskikh svinok pri posledovatel'nom passirovanii virusa Ebola.

Morphological study of internal organs of guinea pigs inoculated with Ebola virus at 2-8 passages was carried out. In the course of these passages the number of infected cells and virus particles in the organs was shown to increase, and the destructive changes in organs became more pronounced. In the 1st-3rd passages Ebola virus replication was observed in macrophagal cells only but beginning from the 4th passage of virus reproduction was found also in hepatocytes, spongiocytes, and fibroblasts.

[Study of the immunobiological properties of parotitis viral strains]. / Izuchenie immunobiologichekikh svoistv shtammov virusa parotita.

The possibility of using different strains of parotitis virus (Enders, L-3, Jeryl-Leen) as antigens for enzyme immunoassay (EIA) to titer antibodies in human and animal blood sera is analyzed. Methods for preparation and purification of antigen on the basis of the said parotitis virus strains have been developed. Conditions of EIA were optimized. The sensitivity and specificity of EIA and hemagglutination inhibition test were compared.

[Study of certain indicators of immunity upon infecting CBA/Calac line mice with Lassa virus]. / Izuchenie nekotorykh pokazatelei immuniteta pri zarazhenii myshei linii CBA/Calac virusom lassa]

Some immunity parameters (interferon, tumor necrosis factor, interleukin 1, etc.) were studied in CBA/Calac mice infected with Lassa virus. The results permit a hypothesis that a pathologic inflammatory reaction is responsible for the death of animals in experimental Lassa fever. One of the components of this reaction is endogenous shock involving a manifest production of immune response mediators, such as interferon, interleukin 1. and tumor necrosis factor.

[Constructing the reference panel of sera with standard level of IgG-antibodies]. / Konstruirivanie standartnykh panelei syvorotok c normirovannym urovnem IgG-antitel.

Fundamentals of designing reference panels of sera for effective control of commercial test systems and immunoblotting, intended for detecting antiviral antibodies, have been developed. Reference low-titer panels of anti-IgG antibodies to HIV-a and hepatitis C virus have been designed. A reference panel contains diluted reactive sera with a standard level of IgG antibodies and native sera with undetectable level of antibodies to the major viral antigens from risk group subjects. The reactive sera of a panel contain the whole spectrum of antibodies to all principal viral antigens.

[Effect of lipid peroxidation on the insulin receptor in rat adipocytes]. / Vliianie perekisnogo okisleniia lipidov na retseptsiiu insulina adipotsitami krys.

Lipid peroxidation is studied for its effect on insulin receptors in isolated rat adipocytes. The results suggest that addition of two peroxidants (3 mM cumene hydroperoxide and 0.2 mM Fe2+) leads to malondialdehyde accumulation and binding inhibition through insulin receptors quantity and affinity decrease.

Targeting EGFR with metabolically biotinylated fiber-mosaic adenovirus.

Adenovirus (Ad)-based vectors are useful gene delivery vehicles for a variety of applications. Despite their attractive properties, many in vivo applications require modulation of the viral tropism. Targeting approaches applied to adenoviral vectors included genetic modification of the viral capsid, controlled expression of the transgene and combinatorial approaches that combine two or more targeting elements in single vectors. Most of these studies confirmed successful retargeting in cell cultures, however, in vivo gains of targeted adenoviral vectors have not been widely demonstrated. We have developed a combinatorial retargeting approach utilizing metabolically biotinylated Ad, where the biotin acceptor peptide was incorporated in one of the fibers in a dual fiber viral particle resulting in metabolically biotinylated fiber-mosaic Ad (mBfMAd). We have utilized this vector in complex with epidermal growth factor (EGF)-Streptavidin to retarget fiber-mosaic virus to EGF receptor (EGFR) expressing cells in vitro and confirmed an increased infectivity of the retargeting complex. Most importantly, the utility of this strategy was demonstrated in vivo in two distinct animal models. In both models tested, retargeted mBfMAd demonstrated an increased ratio of gene expression in target tissues compared to the liver expression profile. Thus, metabolically biotinylated fiber-mosaic virus in combination with appropriate adapters can be successfully exploited for adenoviral retargeting strategies.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy.

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

Phage display of adenovirus type 5 fiber knob as a tool for specific ligand selection and validation.

Adenovirus (Ad) vectors are most potent for use as gene delivery vehicles to infect human cells in vitro and in vivo with high efficiency. The main limitation in utilization of Ad as a gene transfer vector is the lack of specificity. Genetic modifications of Ad capsid proteins resulting in incorporation of foreign polypeptide ligand sequences can redirect the vector towards target cells. However, in many cases the incorporated ligands lose specificity or lead to conformational changes influencing virion integrity. In order to select target-specific ligands a priori structurally compatible with Ad, we propose a system for displaying polypeptide sequences in the context of the Ad fiber knob on the surfaces of filamentous bacteriophages. To establish this concept, we displayed the wild-type Ad serotype 5 knob and knobs containing c-Myc epitopes and six-histidine sequences in the pJuFo phage system. The knobs remained trimeric and bound the coxsackievirus-Ad receptor, and the phage knob-displayed ligands recognized and bound their cognates in the phage-displayed knob context. Further development of this system may be useful for candidate ligand fidelity and Ad structural compatibility validation prior to Ad modification.

Identification of antigenic sites on three hepatitis C virus proteins using phage-displayed peptide libraries.

A novel approach to screening phage-displayed peptide libraries has been used to identify hepatitis C virus (HCV) core, NS4 and NS5 sequences, which are antigenic in humans. Two random peptide libraries were used for screening using a mixture of HCV-positive sera or individual antibodies to core, NS3, NS4, and NS5 HCV proteins affinity-purified from this mixture. Sequencing of 56 selected phage clones resulted in 28 different peptide sequences and identification of seven antigenic regions, three in the core protein (19-26, 34-49, and 73-83), three in the NS4 (1681-1693, 1712-1718, and 1726-1736) and one in the NS5 protein (2251-2260). No NS3-specific peptides were identified. The immune response to core, NS4 and NS5 proteins includes a variety of linear determinants whereas epitopes on the investigated part of NS3 protein appear to be conformation-dependent.

Approaches to utilize mesenchymal progenitor cells as cellular vehicles.

Mammalian cells represent a novel vector approach for gene delivery that overcomes major drawbacks of viral and nonviral vectors and couples cell therapy with gene delivery. A variety of cell types have been tested in this regard, confirming that the ideal cellular vector system for ex vivo gene therapy has to comply with stringent criteria and is yet to be found. Several properties of mesenchymal progenitor cells (MPCs), such as easy access and simple isolation and propagation procedures, make these cells attractive candidates as cellular vehicles. In the current work, we evaluated the potential utility of MPCs as cellular vectors with the intent to use them in the cancer therapy context. When conventional adenoviral (Ad) vectors were used for MPC transduction, the highest transduction efficiency of MPCs was 40%. We demonstrated that Ad primary-binding receptors were poorly expressed on MPCs, while the secondary Ad receptors and integrins presented in sufficient amounts. By employing Ad vectors with incorporated integrin-binding motifs (Ad5lucRGD), MPC transduction was augmented tenfold, achieving efficient genetic loading of MPCs with reporter and anticancer genes. MPCs expressing thymidine kinase were able to exert a bystander killing effect on the cancer cell line SKOV3ip1 in vitro. In addition, we found that MPCs were able to support Ad replication, and thus can be used as cell vectors to deliver oncolytic viruses. Our results show that MPCs can foster expression of suicide genes or support replication of adenoviruses as potential anticancer therapeutic payloads. These findings are consistent with the concept that MPCs possess key properties that ensure their employment as cellular vehicles and can be used to deliver either therapeutic genes or viruses to tumor sites.

Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen.

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.

Ebola virus infection in guinea pigs: presumable role of granulomatous inflammation in pathogenesis.

An approach combining virology with light and electron microscopy was used to study the organs of guinea pigs during nine serial passages of Ebola virus, strain Zaire. It was observed that the wild type of Ebola virus causes severe granulomatous inflammation in the liver and reproduces in the cells of the mononuclear phagocyte system (MPS). Based on morphological characterization, two types of virus-cell interactions were demonstrated. The obtained data evidenced for heterogeneity of the population of wild type of Ebola virus. The virus accumulated in the liver of the infected animals, and the lesions became more pronounced with passage. Degenerative changes appeared, and their severity was increased with passage in the other organs as well. The set of target cells diversified and, as a result, not only the MPS cells, but also hepatocytes, spongiocytes, endotheliocytes and fibroblasts became involved in the reproduction of Ebola virus. The possible role of granulomatous inflammation in the development of the adaptive mechanism of Ebola virus to guinea pigs is discussed.