RESUMEN
We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3CT minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3CLT, used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3CLT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle.
RESUMEN
To improve the production of foot-and-mouth disease (FMD) molecular vaccines, we sought to understand the effects of the FMD virus (FMDV) 2B viroporin in an experimental, plasmid-based, virus-like particle (VLP) vaccine. Inclusion of the FMDV viroporin 2B into the human Adenovirus 5 vectored FMD vaccine enhanced transgene expression despite independent 2B expression negatively affecting cell viability. Evaluating both wildtype 2B and mutants with disrupted viroporin activity, we confirmed that viroporin activity is detrimental to overall transgene expression when expressed independently. However, the incorporation of 2B into an FMD molecular vaccine construct containing a wildtype FMDV 3C protease, a viral encoded protease responsible for processing structural proteins, resulted in enhancement of transgene expression, validating previous observations. This benefit to transgene expression was negated when using the FMDV 3CL127P mutant, which has reduced processing of host cellular proteins, a reversion resulting from 2B viroporin activity. Inclusion of 2B into VLP production constructs also adversely impacted antigen extraction, a possible side effect of 2B-dependent rearrangement of cellular membranes. These results demonstrate that inclusion of 2B enhanced transgene expression when a wildtype 3C protease is present but was detrimental to transgene expression with the 3CL127P mutant. This has implications for future molecular FMD vaccine constructs, which may utilize mutant FMDV 3C proteases.
RESUMEN
Senecavirus A (SVA) is a member of the family Picornaviridae and enzootic in domestic swine. SVA can induce vesicular lesions that are clinically indistinguishable from Foot-and-mouth disease, a major cause of global trade barriers and agricultural productivity losses worldwide. The LF-BK αVß6 cell line is a porcine-derived cell line transformed to stably express an αVß6 bovine integrin and primarily used for enhanced propagation of Foot-and-mouth disease virus (FMDV). Due to the high biosecurity requirements for working with FMDV, SVA has been considered as a surrogate virus to test and evaluate new technologies and countermeasures. Herein we conducted a series of comparative evaluation in vitro studies between SVA and FMDV using the LF-BK αVß6 cell line. These include utilization of LF-BK αVß6 cells for field virus isolation, production of high virus titers, and evaluating serological reactivity and virus susceptibility to porcine type I interferons. These four methodologies utilizing LF-BK αVß6 cells were applicable to research with SVA and results support the current use of SVA as a surrogate for FMDV.
Asunto(s)
Virus de la Fiebre Aftosa , Fiebre Aftosa , Interferón Tipo I , Picornaviridae , Enfermedades de los Porcinos , Animales , Bovinos , Línea Celular , Integrinas , PorcinosRESUMEN
This report covers the methodology for generation of stable heterohybridoma clones producing Foot-and-mouth disease virus (FMDV) reactive porcine monoclonal antibodies (mAbs). Swine received five inoculations of an inactivated O1 Manisa FMDV vaccine prior to the harvest of splenocytes. Due to the lack of a species-specific hybridoma fusion partner, the Sp2/0 murine myeloma cell line was utilized for the formation of porcine-murine heterohybridoma clones. Twenty-nine FMDV-reactive parental clones were generated. Following sub-cloning and monitoring of reactivity over 20 serial passages, eleven subclones derived from unique parental origins were characterized and are reported herein. This methodology demonstrated the production of porcine mAbs by fusion of porcine splenocytes from immunized pigs with murine myeloma cells to generate heterohybridomas. The porcine immune response may differ from the murine immune response in relation to recognized epitopes. Therefore, application of this methodology may provide valuable resources for swine immunology and enhance the understanding of the mechanisms for antibody based protection from diseases in swine.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunas Virales/farmacología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Linfocitos B/inmunología , Línea Celular , Clonación Molecular , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Hibridomas , Inmunización , Ratones , Bazo/inmunología , Sus scrofa , Vacunas Virales/inmunologíaRESUMEN
To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.
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Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunación/métodos , Vacunas Virales/administración & dosificación , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Enfermedades de los Bovinos/virología , Línea Celular , Fiebre Aftosa/inmunología , Células HeLa , Humanos , Serogrupo , Vacunación/veterinaria , Vacunas de ADN , Vacunas Sintéticas , Vacunas Virales/inmunología , Viremia/prevención & controlRESUMEN
The production of experimental molecular vaccines against foot-and-mouth disease virus utilizes the viral encoded 3C protease for processing of the P1 polyprotein. Expression of wild type 3C protease is detrimental to host cells. The molecular vaccine constructs containing the 3C protease L127P mutant significantly reduce adverse effects associated with protease expression while retaining the ability to process and assemble virus-like particles. In published 3C protease crystal structures, the L127 residue is contained within the B2 ß-strand as part of the A2-B2 ß-sheet. To provide insight into the mechanism by which the L127P mutant alters the properties of the 3C protease, we performed scanning proline mutagenesis of residues 123-128 of the B2 ß-strand and monitored expression and P1 processing. Simultaneously, we utilized random mutagenesis of the full 3C sequence to identify additional mutations presenting a phenotype similar to the L127P mutation. Six of the tested mutants enhanced expression over wild type, and the I22P, T100P and V124P mutations surpassed the L127P mutation in certain cell lines. These data areinterpreted in conjunction with published 3C protease crystal structures to provide insight into the mechanism by which these mutations enhance expression.
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Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Virus de la Fiebre Aftosa/enzimología , Fiebre Aftosa/virología , Péptidos/genética , Proteínas Virales/química , Proteínas Virales/genética , Proteasas Virales 3C , Animales , Cisteína Endopeptidasas/metabolismo , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Regulación Viral de la Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Mutagénesis , Péptidos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Prolina/genética , Prolina/metabolismo , Conformación Proteica en Lámina beta , Procesamiento Postranscripcional del ARN , Proteínas Virales/metabolismoRESUMEN
Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.
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Anticuerpos Antivirales/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Vacunación/veterinaria , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Animales , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Femenino , Glicoproteínas/genética , Glicoproteínas/inmunología , Porcinos , Nicotiana/genética , Nicotiana/metabolismo , Vacunas de Subunidad/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
We investigated the serotype- and topotype versatility of a replication-deficient human adenovirus serotype 5 vectored foot-and-mouth disease (FMD) vaccine platform (AdtFMD). Sixteen AdtFMD recombinant subunit monovalent vaccines targeting twelve distinct FMD virus (FMDV) serotype/topotypes in FMD Regional Pools I-VII were constructed. The AdtA24 serotype conditionally licensed vaccine served as the basis for vaccine design and target dose for cattle clinical trials. Several vaccines contained an additional RGD motif genetic insertion in the adenovector fiber knob, and/or a full-length 2B gene insertion in the FMDV P1 gene cassette. In 13 of the 22 efficacy studies conducted, naïve control and AdtFMD vaccinated cattle were challenged intradermolingually at 2â¯weeks post-vaccination using a FMDV strain homologous to the AdtFMD vaccine strain. Each of the 16 AdtFMD vaccines were immunogenic based on the presence of homologous neutralizing antibodies in the serum of approximately 90% of total vaccinates (nâ¯=â¯375) on the day of challenge. Importantly, for 75% of vaccines tested, the effective dose that conferred 100% protection against clinical FMD was identical to or in some cases lower than, the minimum protective dose for the conditionally licensed AdtA24 vaccine formulated with ENABL® adjuvant. Results also confirmed the capability of the AdtFMD vaccine platform to differentiate infected from vaccinated animals (DIVA) across the five FMDV serotypes evaluated. Collectively, this comprehensive set of FMD cattle vaccine dose ranging studies highlights the serotype- and topotype versatility of the AdtFMD vaccine platform for further development, licensure, and application in FMD outbreak control and disease eradication efforts.
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Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Vacunación/veterinaria , Vacunas Virales/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Bovinos , Relación Dosis-Respuesta a Droga , Virus de la Fiebre Aftosa , Vectores Genéticos , Serogrupo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/uso terapéutico , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/uso terapéutico , Vacunas Virales/uso terapéuticoRESUMEN
Validated procedures for decontamination of laboratory surfaces and equipment are essential to biosafety and biorisk programs at high-containment laboratories. Each high-containment laboratory contains a unique combination of surfaces, procedures, and biological agents that require decontamination methods tailored to specific facility practices. The Plum Island Animal Disease Center (PIADC) is a high-containment laboratory operating multiple biosafety level (BSL)-3, ABSL-3, and BSL-3 Ag spaces. The PIADC facility requires the use of federally issued smart cards, called personal identity verification (PIV) cards, to access information technology (IT) networks both outside and within the high-containment laboratory. Because PIV cards may require transit from the BSL-3 to office spaces, a validated procedure for disinfecting PIV card surfaces prior to removal from the laboratory is critical to ensure biosafety and biosecurity. Two high-risk select agents used in the PIADC high-containment laboratory are foot-and-mouth disease virus (FMDV) and swine vesicular disease virus (SVDV). We evaluated disinfection of PIV cards intentionally spotted with FMDV and SVDV using a modified quantitative carrier test and the liquid chemical disinfectant Virkon® S. Our experimental design modeled a worst-case scenario of PIV card contamination and disinfection by combining high concentrations of virus dried with an organic soil load and use of aged Virkon® S prepared in hard water. Results showed that FMDV and SVDV dried on PIV card surfaces were completely inactivated after immersion for 30 and 60 seconds, respectively, in a 5-day-old solution of 1% Virkon® S. Therefore, this study provided internal validation of PIADC biosafety protocols by demonstrating the efficacy of Virkon® S to inactivate viruses on contaminated smart cards at short contact times.
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Contención de Riesgos Biológicos/métodos , Descontaminación/métodos , Desinfectantes/farmacología , Peróxidos/farmacología , Ácidos Sulfúricos/farmacología , Animales , Línea Celular , Enterovirus Humano B/efectos de los fármacos , Virus de la Fiebre Aftosa/efectos de los fármacos , Laboratorios , PorcinosRESUMEN
BACKGROUND: A direct contact transmission challenge model was used to simulate natural foot-and-mouth disease virus (FMDV) spread from FMDV A24/Cruzeiro/BRA/55 infected 'seeder' steers to naïve or vaccinated steers previously immunized with a replication-deficient human adenovirus-vectored FMDV A24/Cruzeiro/BRA/55 capsid-based subunit vaccine (AdtA24). In two independent vaccine efficacy trials, AdtA24 was administered once intramuscularly in the neck 7 days prior to contact with FMDV A24/Cruzeiro/BRA/55-infected seeder steers. RESULTS: In Efficacy Study 1, we evaluated three doses of AdtA24 to estimate the 50%/90% bovine protective dose (BPD50/90) for prevention of clinical FMD. In vaccinated, contact-challenged steers, the BPD50/90 was 3.1 × 1010 / 5.5 × 1010 AdtA24 particles formulated without adjuvant. In Efficacy Study 2, steers vaccinated with 5 × 1010 AdtA24 particles, exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers, did not develop clinical FMD or transmit FMDV to other vaccinated or naïve, non-vaccinated steers. In contrast, naïve, non-vaccinated steers that were subsequently exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers developed clinical FMD and transmitted FMDV by contact to additional naïve, non-vaccinated steers. The AdtA24 vaccine differentiated infected from vaccinated animals (DIVA) because no antibodies to FMDV nonstructural proteins were detected prior to FMDV exposure. CONCLUSIONS: A single dose of the AdtA24 non-adjuvanted vaccine conferred protection against clinical FMD at 7 days post-vaccination following direct contact transmission from FMDV-infected, naïve, non-vaccinated steers. The AdtA24 vaccine was effective in preventing FMDV transmission from homologous challenged, contact-exposed, AdtA24-vaccinated, protected steers to co-mingled, susceptible steers, suggesting that the vaccine may be beneficial in reducing both the magnitude and duration of a FMDV outbreak in a commercial cattle production setting.
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Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Vacunas Virales/inmunología , Adenovirus Humanos/genética , Animales , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/genética , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/virología , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/inmunología , Masculino , Serogrupo , Vacunación , Vacunas de Subunidad/inmunología , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Protective immunity to viral pathogens often includes production of neutralizing antibodies to virus capsid proteins. Many viruses produce capsid proteins by expressing a precursor polyprotein and related protease from a single open reading frame. The foot-and-mouth disease virus (FMDV) expresses a 3C protease (3Cpro) that cleaves a P1 polyprotein intermediate into individual capsid proteins, but the FMDV 3Cpro also degrades many host cell proteins and reduces the viability of host cells, including subunit vaccine production cells. To overcome the limitations of using the a wild-type 3Cpro in FMDV subunit vaccine expression systems, we altered the protease restriction sequences within a FMDV P1 polyprotein to enable production of FMDV capsid proteins by the Tobacco Etch Virus NIa protease (TEVpro). Separate TEVpro and modified FMDV P1 proteins were produced from a single open reading frame by an intervening FMDV 2A sequence. The modified FMDV P1 polyprotein was successfully processed by the TEVpro in both mammalian and bacterial cells. More broadly, this method of polyprotein production and processing may be adapted to other recombinant expression systems, especially plant-based expression.
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Proteínas de la Cápside/metabolismo , Endopeptidasas/metabolismo , Virus de la Fiebre Aftosa/genética , Endopeptidasas/genética , Virus de la Fiebre Aftosa/metabolismo , Células HEK293 , Humanos , Sistemas de Lectura Abierta , Transfección , Vacunas ViralesRESUMEN
A foot-and-mouth disease (FMD) recombinant subunit vaccine formulated with a lipid/polymer adjuvant was evaluated in two vaccine efficacy challenge studies in steers. The vaccine active ingredient is a replication-deficient human adenovirus serotype 5 vector encoding the FMD virus (FMDV) A24/Cruzeiro/BRA/55 capsid (AdtA24). In the first study, AdtA24 formulated in ENABL® adjuvant was compared to a fourfold higher dose of AdtA24 without adjuvant. Steers vaccinated with AdtA24â¯+â¯ENABL® adjuvant developed a significantly higher virus neutralizing test (VNT) antibody titer and an improved clinical response following FMDV A24/Cruzeiro/BRA/55 intradermal lingual challenge at 14â¯days post-vaccination (dpv) than steers vaccinated with the active ingredient alone. In the second study, vaccination with AdtA24 formulated in ENABL® at the same dose used in the first study, followed by FMDV A24/Cruzeiro/BRA/55 challenge on 7 or 14 dpv, prevented clinical FMD in all steers and conferred 90% protection against viremia. In addition, post-challenge FMDV titers in nasal samples from vaccinated steers compared to unvaccinated steers were significantly reduced. In both studies, none of the AdtA24 vaccinated steers developed antibodies to the FMDV non-structural proteins prior to challenge with FMDV, indicative of the capacity to differentiate infected from vaccinated animals (DIVA). These results demonstrate that administration of AdtA24 formulated in ENABL® adjuvant lowered the protective dose and prevented clinical FMD following exposure of vaccinated steers to virulent FMDV at 7 or 14 dpv.
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Adenovirus Humanos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Enfermedades de los Bovinos/prevención & control , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Potencia de la Vacuna , Vacunas Virales/inmunología , Adenovirus Humanos/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Bovinos , Virus de la Fiebre Aftosa/genética , Vectores Genéticos , Humanos , Serogrupo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Virales/administración & dosificación , Viremia/inmunologíaRESUMEN
The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production.
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Sustitución de Aminoácidos , Cisteína Endopeptidasas/inmunología , Virus de la Fiebre Aftosa/inmunología , Mutación Missense , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Proteasas Virales 3C , Animales , Cisteína Endopeptidasas/genética , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Virus de la Fiebre Aftosa/genética , Células HEK293 , Humanos , Proteínas Virales/genética , Vacunas Virales/genéticaRESUMEN
BACKGROUND: The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. RESULTS: We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. CONCLUSIONS: We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.
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Genes Reporteros/genética , Genes/genética , Vectores Genéticos/genética , Microscopía Fluorescente/métodos , Transfección/métodos , Transgenes/genética , Proteínas Virales/genética , Animales , Copépodos/enzimología , Luciferasas/metabolismo , Biosíntesis de Proteínas/genéticaRESUMEN
The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×10(8) to 2.1×10(11) particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R(2)=0.97) and viremia (R(2)=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R(2)=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.
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Adenoviridae , Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/inmunología , Bovinos , Enfermedades de los Bovinos/virología , Virus de la Fiebre Aftosa , Masculino , Pruebas de Neutralización , Serogrupo , Vacunas de Subunidad/inmunología , Proteínas no Estructurales Virales/inmunología , Esparcimiento de VirusRESUMEN
Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) - a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70% to 95% of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Brotes de Enfermedades , Genoma Viral , Virus de la Enfermedad Hemorrágica del Conejo/genética , Enfermedades de los Roedores/epidemiología , Animales , Antígenos Virales/genética , Secuencia de Bases , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/patología , Hemorragia/patología , Virus de la Enfermedad Hemorrágica del Conejo/aislamiento & purificación , Virus de la Enfermedad Hemorrágica del Conejo/patogenicidad , Indiana/epidemiología , Hígado/patología , Pulmón/patología , Datos de Secuencia Molecular , Necrosis/patología , Filogenia , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades de los Roedores/virología , Proteínas Estructurales Virales/genética , VirulenciaRESUMEN
African swine fever virus (ASFV) produces a fatal acute hemorrhagic fever in domesticated pigs that potentially is a worldwide economic threat. Using an expressed sequence tag (EST) library-based antisense method of random gene inactivation and a phenotypic screen for limitation of ASFV replication in cultured human cells, we identified six host genes whose cellular functions are required by ASFV. These included three loci, BAT3 (HLA-B-associated transcript 3), C1qTNF (C1q and tumor necrosis factor-related protein 6), and TOM40 (translocase of outer mitochondrial membrane 40), for which antisense expression from a tetracycline-regulated promoter resulted in reversible inhibition of ASFV production by >99%. The effects of antisense transcription of the BAT3 EST and also of expression in the sense orientation of this EST, which encodes amino acid residues 450 to 518 of the mature BAT3 protein, were investigated more extensively. Sense expression of the BAT3 peptide, which appears to reversibly interfere with BAT3 function by a dominant negative mechanism, resulted in decreased synthesis of viral DNA and proteins early after ASFV infection, altered transcription of apoptosis-related genes as determined by cDNA microarray analysis, and increased cellular sensitivity to staurosporine-induced apoptosis. Antisense transcription of BAT3 reduced ASFV production without affecting abundance of the virus macromolecules we assayed. Our results, which demonstrate the utility of EST-based functional screens for the detection of host genes exploited by pathogenic viruses, reveal a novel collection of cellular genes previously not known to be required for ASFV infection.
Asunto(s)
Virus de la Fiebre Porcina Africana/patogenicidad , Proteínas/genética , Replicación Viral , Virus de la Fiebre Porcina Africana/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorocebus aethiops , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Silenciador del Gen , Células HeLa , Humanos , Chaperonas Moleculares , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteínas/química , Proteínas/metabolismo , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
A number of viruses have evolved antiapoptotic mechanisms to promote infected-cell survival, either to ensure efficient productive viral replication or to promote long-term survival of virus-infected cells. Recent studies identified critical African swine fever virus genes involved in the complex regulation of ASFV-host interactions. Here we review the present knowledge of the recently identified ASFV genes with special attention to those which affect viral virulence, host range, and pathogenesis by regulating viral-induced apoptotic mechanisms.