Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.

Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.

In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both alphav integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

[Isolation and study of recombinant strains of Salmonella typhimurium SL 7207, producing HBcAG and HBcAG-HBs]. / Poluchenie i issledovanie rekombinantnykh shtammov Salmonella typhimurium SL 7207, produtsiruiushchikh HBcAg i HBcAg-HBs.

Recombinant strains producing hepatitis B virus (HBcAg) core protein and chimeric core protein exposing on its surface the major immunogenic epitope of HBsAg (HBcAg-HBs) were constructed on the base of attenuated S. typhimurium SL 7202 strain. The resultant Salmonella strains produced proteins which were capable of self-assembly into virus-like particles and showed antigenic properties of both core and surface hepatitis B proteins. A single rectal immunization with recombinant S. typhimurium induced humoral and cellular immune response to HBcAg and HBsAg. Specific anti-HBcAg were detected in animal sera and intestinal tissues, which indicated the formation of specific mucosal immunity.

[Induction of immune mediators in human cultured mononuclear cells by Marburg virus]. / Induktsiia virusom Marburg mediatorov immuniteta v kul'ture mononuklearov cheloveka.

Infection of human mononuclear cells with Marburg virus results in production of inflammatory cytokines tumor necrosis factor and interleukin-1. These cytokines induce expression of adhesion receptors VCAM and ICAM by human endothelial cells. Mitogen-induced proliferation of human mononuclears is not suppressed 1 h after infection of these cells with Marburg virus in a dose of 0.01 PFU/cell. The capacity of cells to proliferate in response to mitogens significantly decreased 4 h after infection.

[Effects of tumor necrosis factor antiserum of the course of Marburg hemorrhagic fever]. / Vliianie syvorotki protiv faktora nekroza opukholi na techenie gemorragicheskoi likhoradki marburg.

The paper provides data on the effect of tumor necrosis factor (TNF) antiserum on the course of experimental Marburg hemorrhagic fever. On day 3 of the challenge of guinea pigs with the Popp strain of Marburg virus, the infection event was confirmed by PCR. On days 4-7, the infected animals intramuscularly received a TNF-alpha antiserum. The treated animals showed a 50% survival while all control animals died. It may be assumed that a course of death due to Marburg hemorrhagic fever in shocks brought about by inflammatory cytokines. Therefore, attempts may be made to treat this infection by using the drugs that are TNF antagonists.

[Immunologic indicators in vaccinated mice with Machupo virus infection]. / Immunologicheskie pokazateli u vaktsinirovannykh myshei pri myshei pri zarazhenii virusom Machupo.

The parameters of nonspecific immunity (interferon, interleukin-1, tumor necrosis factor, and natural killers) changed in immune BALB/c mice after challenge with Machupo virus in doses of 1000 and 5000 PFU. After challenge with 1000 PFU the activity of the above parameters increased during the first three days and no cases of animal death occurred. After challenge with 5000 PFU the maximal values of interferon, interleukin-1, tumor necrosis factor, and natural killers were observed on days 5-7, the animals dying at the height of these values. Hence, the formation of specific humoral and cell-mediated immune response in BALB/c mice immunized with inactivated Machupo virus does not protect the animals from infection with the homologous virus in a dose of 5000 PFU. High mediator activity of nonspecific immunity factors on days 5-7 after infection augments the disease course and leads to earlier death of vaccinated animals in comparison with the same dose of the virus in nonimmune mice.

[Experimental study of possible treatment of Marburg hemorrhagic fever with desferal, ribavirin, and homologous interferon]. / Eksperimental'noe izuchenie vozmozhnosti lecheniia gemorragicheskoi likhoradki Marburg desferalom, ribavirinom i gomologichnym interferonom.

The paper presents data on the effects of the drugs desferal, ribavirin and homologous (guinea pig) interferon on the course of experimental Marburg fever. Neither ribavirin nor interferon were effective. A moderate response to desferal was noted. It is suggested that tumor necrosis factor may contribute to development of Marburg hemorrhagic fever.

Inactivated Marburg virus elicits a nonprotective immune response in Rhesus monkeys.

In the present work the kinetics of some indices of immunity (tumor necrosis factor (TNF), interferon (IFN), natural killer cells (NK), lymphocyte proliferation activity, virus-specific antibodies, CD4/CD8 ratio) in response to Marburg virus infection in Macaca mulatta were studied. The different kinetics of immunological parameters for animals which survived Marburg virus infection and for animals which died after infection are shown. A comparison of the indices of IFN, TNF and spontaneous lymphocyte proliferation activity in Macaca mulatta infected with Marburg virus at different stages of life shows the relationship between the increase of these indices and the decrease in the animals' lifespan. Marburg virus immunosuppressive properties were corroborated by studying lymphocyte proliferation activity in response to antigenic stimulation in vitro and the CD4/CD8 index during experimental Marburg virus infection in Macaca mulatta. We conclude that the disease outcome depends on the dynamics of certain immunologic indices such as TNF and IFN.

[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.

[Mechanisms of protective immune response in models of Marburg fever in monkeys]. / Mekhanizmy protektivnogo immunogo otveta pri modelirovanii likhoradki Marburg na obez'ianakh.

The parameters of nonspecific immunity (interferon, tumor necrosis factor, natural killers), of spontaneous and mitogen-induced lymphocyte proliferation, of specific immunity (antibodies to Marburg virus, antigen-stimulated proliferation) were found to change in the course of experimental Marburg fever in immune and intact animals. The named factors were shown to influence the course of the disease; their role in the lethal outcome of the infection was demonstrated.

[The immunological indices of guinea pigs modelling Marburg hemorrhagic fever]. / Immunologicheskie pokazateli u morskikh svinok pri modelirovanii gemorragicheskoi likhoradki Marburg.

Presents data on changes in nonspecific immunity (interferon, tumor necrosis factor, natural killers), spontaneous and mitogen-induced lymphocyte proliferation in the course of development of experimental Marburg fever. Demonstrates the effects of the said factors and interleukin-2 on the course of the fever and their role in a lethal outcome of the disease.

[The immunity indices of animals immunized with the inactivated Marburg virus after infection with homologous virus]. / Nekotorye pokazateli immuniteta u zhivotnykh, immunizirovannykh virusom Marburg, posle zarazheniia gomologichnym virusom.

The data on changes in the parameters of specific and nonspecific immunity after challenge of the immunized animals with Marburg virus are presented. Mediators of the immune response: tumor necrosis factor, interferon, were shown to play different roles in the development of the disease and "protection" of animals. The survival rate of the immunized animals after the challenge with Marburg virus was determined by the intensity and level of the immune response after challenge rather than by the levels of postvaccination immunity at the time of the challenge.

[The immunity indices of BALB/c mice immunized with an inactivated antigen of the Machupo virus]. / Pokazateli immuniteta u myshei linii BALB/c, immunizirovannykh inaktivirovannym antigenom virusa Machupo.

The paper presents the data characterizing parameters of specific and nonspecific immunity in BALB/c mice immunized with gamma-ray-inactivated Machupo virus antigen or its formalinized antigen. The gamma-ray inactivated preparation was shown to be more immunogenic for BALB/c mice. A certain relationship between the time course of activity of nonspecific immunity factors in the immunized animals and the protective activity of the preparation under study was also noted. The decisive role of the T-cell part of the immune system was demonstrated in the resistance of this model animal to Machupo virus infection.

[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 immunogenic properties of Marburg virus proteins]. / Immunogennye svoistva belkov virusa Marburg.

Immunogenic and protective properties of VP40 and NP proteins of Marburg virus were studied. VP40 protein was shown to have insignificant immunogenicity and NP protein to be capable of protecting the animals from lethal infection by stimulation of cell-mediated immunity. No significant increase in the specific antibody level was found.