RESUMEN
Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401-4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).
Asunto(s)
Vacunas contra el Virus del Ébola/inmunología , Voluntarios Sanos , Fiebre Hemorrágica Ebola/prevención & control , Adenoviridae/genética , Adolescente , Adulto , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Portadores de Fármacos/administración & dosificación , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Vacunas contra el Virus del Ébola/administración & dosificación , Femenino , Humanos , Inmunoglobulina G/sangre , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Dolor/inducido químicamente , Dolor/epidemiología , Federación de Rusia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Vesiculovirus/genética , Voluntarios , Adulto JovenRESUMEN
Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to "nanoantibody-modified RPAN" binding.
RESUMEN
Anthrax is a particularly dangerous infectious disease that affects humans and livestock. It is characterized by intoxication, serosanguineous skin lesions, development of lymph nodes and internal organs, and may manifest itsself in either a cutaneous or septic form. The pathogenic agent is Bacillus anthracis, a grampositive, endospore-forming, rod-shaped aerobic bacterium. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. In this study, we designed three recombinant human adenovirus serotype-5-based vectors containing various modifications of the fourth domain of the B. anthracis protective antigen (PA). Three PA modifications were constructed: a secretable form (Ad-sPA), a non-secretable form (Ad-cPA), and a form with the protective antigen fused to the Fc fragment of immunoglobulin G2a (Ad-PA-Fc). All these forms exhibited protective properties against Bacillus anthracis. The highest level of protection was induced by the Ad-PA-Fc recombinant adenovirus. Our findings indicate that the introduction of the Fc antibody fragment into the protective antigen significantly improves the protective properties of the Ad-PA-Fc adenovirus against B. anthracis.
RESUMEN
Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg (SOD1*G93A)d11 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1(G93A) mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1(G93A) mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2-3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + ANG can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.
Asunto(s)
Terapia Genética , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/terapia , Neuronas Motoras/patología , Adenoviridae , Animales , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Ratones , Ratones Transgénicos , Enfermedad de la Neurona Motora/patología , Ribonucleasa Pancreática/biosíntesis , Ribonucleasa Pancreática/genética , Somatomedinas/genética , Médula Espinal/patología , Médula Espinal/cirugía , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
High frequency of epidemiological threats (H5N1 influenza, SARS, etc.) in modern world calls for the development of new flexible technologies for manufacturing efficacious vaccines and rapid reorientation of their production as appropriate. Genetic vaccination is one of such technologies aimed at prophylaxis of dangerous and socially significant infections. The technology is based on administration of one or several functionally active genes encoding for antigens of pathogens which induces formation of both cellular and humoral immunity against the respective microorganism. This property of genetic vaccines is used for the development of prophylactic schemes. New vaccines are currently being designed to prevent a variety of infections. The aim of the present review is to outline major trends in genetic vaccination leading to the improvement of its efficacy.
Asunto(s)
Control de Enfermedades Transmisibles/tendencias , Inmunización Pasiva , Inmunoterapia Activa , Subtipo H5N1 del Virus de la Influenza A , Gripe Humana , Síndrome Respiratorio Agudo Grave , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Disponibilidad Biológica , Control de Enfermedades Transmisibles/métodos , Predicción , Humanos , Inmunización Pasiva/métodos , Inmunización Pasiva/tendencias , Inmunoterapia Activa/métodos , Inmunoterapia Activa/tendencias , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Síndrome Respiratorio Agudo Grave/prevención & control , Síndrome Respiratorio Agudo Grave/virología , Vacunas de ADN/genética , Vacunas de ADN/farmacocinética , Vacunas de Virosoma/genética , Vacunas de Virosoma/farmacocinéticaRESUMEN
The present study is devoted to the feasibility of expressing the single-domain mini-antibody (nanoantibody) selected from the library of sequences of the variable domains of special single-stranded antibodies derived from an immunized camel, a gene of which was introduced into eukaryotic cells within a recombinant adenoviral vector. A vector bearing the gene of a single-domain nanoantibody was obtained using the AdEasy Adenoviral Vector System (Stratagene). This method of delivering the nanoantibody gene facilitates efficient expression of this gene and functional activity of the nanoantibody. The results obtained can be used to produce passive immunizing tools against pathogens or new-generation immunobiological antitoxic medication.
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Comparative antimicrobial activity of lactoferrins from various sources (native lactoferrin from Laprot, human hololactoferrin, recombinant human lactoferrin isolated from the cultural medium of permissive cell culture transfected using pseudoadenovirus nanostructure with the human lactoferrin gene, and native bovine lactoferrin) was studied to prove the possibility of their use for development of antimicrobial drugs. It was shown that all the substances were active against the Bacillus standard strains. The antibacterial activity was almost independent of the degree of saturation the lactoferrin molecules with Fe3+. The native human lactoferrin was more active than hololactoferrin against Candida when evaluated by the minimum inhibitory concentration (MIC). Fe(3+)-Non aturated recombinant human lactoferrin demonstrated the antimicrobial activity (by MIC) similar to that of the native human lactoferrin. The results showed that native and recombinant human lactoferrins might be used for the development of intravenous and intracavitary dosage forms, while the native bovine lactoferrin could be useful in development of oral drugs.
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Antibacterianos/farmacología , Antifúngicos/farmacología , Bacillus/efectos de los fármacos , Candida/efectos de los fármacos , Lactoferrina/farmacología , Proteínas Recombinantes/farmacología , Animales , Antibacterianos/biosíntesis , Antibacterianos/aislamiento & purificación , Antifúngicos/aislamiento & purificación , Bovinos , Humanos , Lactoferrina/biosíntesis , Lactoferrina/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
AIM: To design and study the properties of candidate vaccines against avian influenza based on recombinant adenoviral vectors expressing H5 hemagglutinin. MATERIALS AND METHODS: Recombinant adenoviral vectors were constructed as described in "Stratagene" (Ad Easy Adenoviral Vector System). For immunization of animals, recombinant candidate vaccines were administered intranasally twice. Titer of hemagglutinating antibodies were measured by hemaglutination inhibition assay. RESULTS: It was demonstrated that administration of vaccines to animals completely protects them from a lethal dose challenge with H5N2 influenza virus. Protective effect of vaccines remained for 6 months after immunization. Additionally, highly effective cross-protection of the immunized animals against heterologous strain of H5 influenza virus was demonstrated. CONCLUSION: Obtained results show good prospects for usage of recombinant adenoviral vectors as a basis for development of new generation effective vaccines against influenza.
Asunto(s)
Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Adenoviridae/genética , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Reacciones Cruzadas , Evaluación Preclínica de Medicamentos , Femenino , Ingeniería Genética , Vectores Genéticos/genética , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Esquemas de Inmunización , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Gripe Humana/inmunología , Ratones , Ratones Endogámicos BALB C , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The Ad5-Lf recombinant pseudoadenovirus nanostructure (RPAN) based on adenovirus of the 5th serotype and containing lactoferrin (Lf) gene was constructed. The goal of this work was to develop a system for efficient production of human lactoferrin (Lf) in human body. It was shown using the model of cisplatin (DDP)-induced toxicosis that human Ad5-based RPAN with human Lf gene expressing cassette in its genome provides high rate of expression of Lf gene in animal body. In vivo recombinant human Lf demonstrates detoxification effect against acute DDP-induced toxic reactions similar to that of the native Lf. RPAN does not stimulate growth of primary and metastatic nodes of experimental tumors. Moreover, it inhibits the growth of Lewis lung carcinoma (LLC), Ehrlich carcinoma (ELD), and S37 sarcoma in early periods after tumor transplantation. The obtained experimental data are indicative of the good prospects of further biologic and medical study of RPAN and development of RPAN-based genetic engineering medicine of the new generation.
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Proliferación Celular , Lactoferrina/biosíntesis , Nanoestructuras , Transducción Genética/métodos , Adenoviridae , Animales , Antineoplásicos/toxicidad , Línea Celular Tumoral , Cisplatino/toxicidad , Femenino , Ingeniería Genética , Humanos , Inactivación Metabólica , Lactoferrina/sangre , Lactoferrina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Influenza viruses are characterized by a high degree of antigenic variability, which causes the annual emergence of flu epidemics and irregularly timed pandemics caused by viruses with new antigenic and biological traits. Novel approaches to vaccination can help circumvent this problem. One of these new methods incorporates genetic vaccines based on adenoviral vectors. Recombinant adenoviral vectors which contain hemagglutinin-encoding genes from avian H5N1 and H5N2 (Ad-HA5-1 and Ad-HA5-2) influenza viruses were obtained using the AdEasy Adenoviral Vector System (Stratagene). Laboratory mice received a double intranasal vaccination with Ad-HA5-1 and Ad-HA5-2. This study demonstrates that immunization with recombinant adenoviruses bearing the Ð 5 influenza virus hemagglutinin gene induces a immune response which protects immunized mice from a lethal dose of the H5 influenza virus. Moreover, it also protects the host from a lethal dose of the H1 virus, which belongs to the same clade as H5, but does not confer protection from the subtype H3 influenza virus, which belongs to a different clade.
RESUMEN
The Ad5-Lf pseudoadenovirus nanostructure (RPAN) was produced using homologous recombination in E. coli cells. This construction provided efficient expression of the Lf gene in permissive cell culture with high production rate of recombinant protein similar to native human Lf in some physical, chemical, and biological properties. Single intravenous injection of the construction into mice and rats was effective for prolonged production and circulation of recombinant human Lf in blood of experimental animals without toxic effects. The produced construction is promising for providing prolonged production of recombinant human Lf in the human body.
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Adenoviridae , Lactoferrina/biosíntesis , Nanoestructuras , Proteínas Recombinantes/biosíntesis , Animales , Línea Celular , Expresión Génica , Humanos , Lactoferrina/genética , Masculino , Ratones , Ratas , Proteínas Recombinantes/genéticaRESUMEN
Aviadenovirus CELO is a promising vector for gene delivery to eukaryotic cells, including mammalian cells. In the present state of affairs, it gives a chance to apply recombinant adenoviruses CELO against animal pathogens. High expression level of transgene consisting of virus vector is necessary to reach protective humoral and T-cell immune answer. One of the approaches to enhance the expression level of transgene consisting of viral vectors is inclusion of addition 5'- and 3'- untranslated regulatory elements (PARS, IRES, WPRE) to the expression cassette. In this work, 5'-untranslated regulatory elements of major late transcription unit of CELO genome (a bipartite leader and gene hexon leader) were used for construction of the expression cassette consisting of the recombinant adenovirus CELO to provide high expression level of reporter gene of secreted alkaline phosphatase in nonpermissive cell system. It was shown in our experiments that the obtained recombinant adenovirus CELO was characterized by enhanced (2.4-3.5 times) expression level of reporter gene in transduced mammalian cells in vitro and in vivo.
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Fosfatasa Alcalina/biosíntesis , Adenovirus A Aviar/genética , Vectores Genéticos , Genoma Viral , Fosfatasa Alcalina/genética , Animales , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Elementos Reguladores de la Transcripción , TransgenesRESUMEN
The recombinant avian adenovirus CELO-gpRb expressing glycoprotein G of rabies virus (strain TS-80, ARRIW&M, Pokrov, Russia) was used for mice vaccination against rabies. Double intramuscular immunization by recombinant CELO-gpRb adenovirus in a dose 10(9) pfu per mouse caused the induction of virus neutralizing antibodies (VNA) synthesis in 78% of mice, while twice repeated intradermal injections of the recombinant adenovirus failed to induce the VNA production. The protection level in groups of vaccinated mice after intracerebral injection of CVS rabies virus in a dose of 100 MLD50 was equal to 45% at single intramuscular immunization and to 91% after twice repeated intramuscular immunization. The recombinant adenoviral vaccine against rabies, based on CELO viral genome, has a good perspective for domestic and wild animal vaccination, not only due to rather high protection level, but also because the production of adenoviral CELO vaccine in chicken embryos is of high technology and inexpensive.