RESUMEN
African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.
Asunto(s)
Adenoviridae/genética , Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/inmunología , Antígenos Virales/inmunología , Porcinos/inmunología , Virus de la Fiebre Porcina Africana/inmunología , Animales , Antígenos Virales/genética , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Vectores GenéticosRESUMEN
A reverse vaccinology system, Vaxign, was used to identify and select a subset of five African Swine Fever (ASF) antigens that were successfully purified from human embryonic kidney 293 (HEK) cells and produced in Modified vaccinia virus Ankara (MVA) viral vectors. Three HEK-purified antigens [B646L (p72), E183L (p54), and O61R (p12)], and three MVA-vectored antigens [B646L, EP153R, and EP402R (CD2v)] were evaluated using a prime-boost immunization regimen swine safety and immunogenicity study. Antibody responses were detected in pigs following prime-boost immunization four weeks apart with the HEK-293-purified p72, p54, and p12 antigens. Notably, sera from the vaccinees were positive by immunofluorescence on ASFV (Georgia 2007/1)-infected primary macrophages. Although MVA-vectored p72, CD2v, and EP153R failed to induce antibody responses, interferon-gamma (IFN-γ+) spot forming cell responses against all three antigens were detected one week post-boost. The highest IFN-γ+ spot forming cell responses were detected against p72 in pigs primed with MVA-p72 and boosted with the recombinant p72. Antigen-specific (p12, p72, CD2v, and EP153R) T-cell proliferative responses were also detected post-boost. Collectively, these results are the first demonstration that ASFV subunit antigens purified from mammalian cells or expressed in MVA vectors are safe and can induce ASFV-specific antibody and T-cell responses following a prime-boost immunization regimen in swine.
Asunto(s)
Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/prevención & control , Antígenos Virales/inmunología , Vacunas Virales/inmunología , Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/genética , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Vectores Genéticos , Genoma Viral , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Masculino , Porcinos , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus Vaccinia/genética , Vacunas Virales/genéticaRESUMEN
The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.
Asunto(s)
Virus de la Fiebre Porcina Africana/inmunología , Antígenos Virales/inmunología , Inmunidad Celular , Inmunogenicidad Vacunal , Vacunas Virales/inmunología , Adenoviridae/genética , Animales , Antígenos Virales/química , Vectores Genéticos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Porcinos , Linfocitos T Citotóxicos/inmunología , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/inmunología , Vacunas Virales/efectos adversos , VirulenciaRESUMEN
Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni(2+)-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance.
Asunto(s)
Adyuvantes Inmunológicos , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Iones , Metales , Nanopartículas , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Virus de la Fiebre Aftosa/genética , Orden Génico , Genoma Viral , Lipoproteínas/inmunología , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas Virales/genéticaRESUMEN
African swine fever virus (ASFV) is an arbovirus which is vectored by soft ticks of the Ornithodoros spp. and in the sylvatic cycle infects wart hogs and bush pigs. ASFV infection of domestic swine causes a high mortality disease. On the other hand, ASFV infection of the tick can result in a high-titered and persistent infection depending upon the ASFV isolate and the tick combination. Recently, morphological, classical virology (titration) and recombinant ASFV have been used to study the cellular, molecular and genetic interactions that occur between ASFV and its host tick.
Asunto(s)
Virus de la Fiebre Porcina Africana/aislamiento & purificación , Ornithodoros/virología , Animales , Interacciones Huésped-PatógenoRESUMEN
Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.
Asunto(s)
Virus de la Fiebre Aftosa/patogenicidad , Fiebre Aftosa/genética , Pirofosfatasas/genética , ARN sin Sentido/genética , Animales , Bovinos , Línea Celular , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Humanos , ARN Viral/metabolismoRESUMEN
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.