RESUMEN
Vesicular stomatitis virus (VSV) expressing the Ebola virus (EBOV) glycoprotein (GP) in place of the VSV glycoprotein G (VSV/EBOV-GP) is a promising EBOV vaccine candidate which has already entered clinical phase 3 studies. Although this chimeric virus was tolerated overall by volunteers, it still caused viremia and adverse effects such as fever and arthritis, suggesting that it might not be sufficiently attenuated. In this study, the VSV/EBOV-GP vector was further modified in order to achieve attenuation while maintaining immunogenicity. All recombinant VSV constructs were propagated on VSV G protein expressing helper cells and used to immunize guinea pigs via the intramuscular route. The humoral immune response was analysed by EBOV-GP-specific fluorescence-linked immunosorbent assay, plaque reduction neutralization test and in vitro virus-spreading inhibition test that employed recombinant VSV/EBOV-GP expressing either green fluorescent protein or secreted Nano luciferase. Most modified vector constructs induced lower levels of protective antibodies than the parental VSV/EBOV-GP or a recombinant modified vaccinia virus Ankara vector encoding full-length EBOV-GP. However, the VSV/EBOV-GP(F88A) mutant was at least as immunogenic as the parental vaccine virus although it was highly propagation-restricted. This finding suggests that VSV-vectored vaccines need not be propagation-competent to induce a robust humoral immune response. However, VSV/EBOV-GP(F88A) rapidly reverted to a fully propagation-competent virus indicating that a single-point mutation is not sufficient to maintain the propagation-restricted phenotype.
Asunto(s)
Ebolavirus/inmunología , Glicoproteínas/inmunología , Inmunogenicidad Vacunal , Vesiculovirus/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales , Ebolavirus/genética , Vectores Genéticos , Glicoproteínas/genética , Cobayas , Inmunidad Humoral , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Vacunación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant.IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.
Asunto(s)
Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/aislamiento & purificación , Glicoproteínas/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Virus Vaccinia/genética , Virión/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Virus del Ébola/genética , Ebolavirus/genética , Ebolavirus/inmunología , Ebolavirus/fisiología , Glicoproteínas/genética , Humanos , Inmunoglobulina G/sangre , Ratones , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Virión/fisiologíaRESUMEN
UNLABELLED: Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-ß in murine and human cells in the presence of an intact E3L gene. IFN-ß induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo. IMPORTANCE: Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.
Asunto(s)
Inmunidad Innata , ARN Bicatenario/inmunología , Virus Vaccinia/inmunología , eIF-2 Quinasa/inmunología , Animales , Línea Celular , Citocinas/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Bicatenario/metabolismo , Virus Vaccinia/genética , eIF-2 Quinasa/metabolismoRESUMEN
Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Virus Vaccinia/metabolismo , Virus Vaccinia/patogenicidad , Vaccinia/enzimología , Proteínas Virales/metabolismo , Animales , Línea Celular , Activación Enzimática , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Vaccinia/metabolismo , Vaccinia/virología , Virus Vaccinia/genética , Proteínas Virales/genética , VirulenciaRESUMEN
The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.
Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Humanos , Ratones , Animales , Ligando de CD40/genética , Neoplasias/tratamiento farmacológico , Linfocitos T CD8-positivos , Anticuerpos Antineoplásicos , Virus Vaccinia/genéticaRESUMEN
Poxviruses such as the causative agent of smallpox have developed multiple strategies to suppress immune responses, including the suppression of DC activation. Since poxviruses are large DNA viruses, we hypothesized that their detection by DCs may involve the endosomal DNA recognition receptor TLR9. Indeed, we have shown here that DC recognition of ectromelia virus (ECTV), the causative agent of mousepox, completely depended on TLR9. The importance of TLR9 was highlighted by the fact that mice lacking TLR9 showed drastically increased susceptibility to infection with ECTV. In contrast, we found that the strongly attenuated poxvirus modified vaccinia virus Ankara (MVA) activated DCs by both TLR9-dependent and -independent pathways. We therefore tested whether we could use the broader induction of immune responses by MVA to protect mice from a lethal infection with ECTV. Indeed, MVA given at the same time as a lethal dose of ECTV protected mice from death. Importantly, MVA also rescued TLR9-deficient mice if administered 2 full days after an otherwise lethal infection with ECTV. Therefore, these data suggest an essential role for TLR9 in the defense against poxviruses. In addition, postexposure application of MVA may protect against lethal poxvirus infection.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por Poxviridae , Receptor Toll-Like 9/inmunología , Vacunación , Animales , Células Dendríticas/citología , Virus de la Ectromelia/inmunología , Virus de la Ectromelia/patogenicidad , Humanos , Inmunidad/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Poxviridae/inmunología , Infecciones por Poxviridae/mortalidad , Infecciones por Poxviridae/prevención & control , Tasa de Supervivencia , Receptor Toll-Like 9/genética , Virus Vaccinia/inmunologíaRESUMEN
Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Expresión Génica , Vectores Genéticos/genética , Regiones Promotoras Genéticas , Virus Vaccinia/genética , Vaccinia/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Secuencia de Bases , Antígenos CD8/genética , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/virología , Línea Celular , Células Cultivadas , Embrión de Pollo , Cricetinae , Femenino , Genes Inmediatos-Precoces , Vectores Genéticos/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ovalbúmina/genética , Ovalbúmina/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vaccinia/virología , Virus Vaccinia/química , Virus Vaccinia/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions.
Asunto(s)
Eliminación de Gen , Genoma Viral , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Animales , Línea Celular , Embrión de Pollo , Cromosomas Artificiales Bacterianos/genética , Efecto Citopatogénico Viral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Fenotipo , Conejos , Recombinación Genética , Vaccinia/etiología , Vaccinia/virología , Virus Vaccinia/fisiología , Virulencia/genética , Cultivo de Virus , Replicación ViralRESUMEN
T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.
Asunto(s)
Apoptosis/fisiología , Virus de la Enfermedad de Borna/fisiología , Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/fisiología , Neuronas/inmunología , Traslado Adoptivo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/patología , Enfermedad de Borna/metabolismo , Enfermedad de Borna/patología , Enfermedad de Borna/virología , Encéfalo/metabolismo , Encéfalo/patología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Citotoxicidad Inmunológica , Eosinófilos/citología , Eosinófilos/metabolismo , Eosinófilos/patología , Femenino , Citometría de Flujo , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/patología , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Inflamación , Linfocitos/citología , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Perforina/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Carga ViralRESUMEN
Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.
Asunto(s)
Antígenos Virales/inmunología , Virus de la Enfermedad de Borna/inmunología , Linfocitos T CD8-positivos/inmunología , Traslado Adoptivo , Animales , Antígenos Virales/genética , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Adrenocortical carcinoma (ACC) is a highly malignant tumor with poor response to classical antitumor therapy. Steroidogenic acute regulatory (StAR) protein is expressed in most human ACCs. The aim of this study was to induce antitumoral T cells directed against StAR in a murine tumor model. Because a suitable syngenic adrenocortical mouse tumor model is lacking, we established a clone of the mouse myeloma Sp2-0 tumor cell line stably expressing murine StAR (Sp2-mStAR). Using repeated im injections of plasmid DNA encoding mStAR followed by infection with a recombinant vaccinia virus (rVV) expressing mStAR, we induced a cytotoxic T-cell response as measured by enzyme-linked immunospot assay. To demonstrate antitumor activity of the vaccination procedure, mice were treated as follows: group A, mice immunized with plasmids and rVV encoding mStAR receiving Sp2-mStAR cells; control group B, mice immunized with the empty plasmid and the empty rVV receiving Sp2-mStAR cells; control group C, mice immunized with the empty plasmid and rVV encoding P450 side-chain cleavage enzyme receiving Sp2-mStAR cells; and control group D, mice immunized with plasmid and rVV encoding mStAR receiving parental Sp2-0 cells. A high proportion (89-100%) of the control groups B, C, and D developed subcutaneous tumors. In contrast, immunization specific for mStAR (group A) was highly protective against tumor growth (percentage of tumor-free animals, 67%; P < 0.001 vs. controls). In summary, these results show that T-cell tolerance toward mStAR can be broken, resulting in antitumoral immunity. Thus, StAR represents a candidate target antigen for immunotherapeutic strategies against ACC.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/terapia , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/terapia , Inmunoterapia , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/patología , Carcinoma Corticosuprarrenal/patología , Animales , División Celular , Línea Celular Tumoral , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Corticosterona/metabolismo , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Inmunidad , Inmunización , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/genética , Linfocitos T/inmunología , Vacunación , Virus Vaccinia/genéticaRESUMEN
Borna disease virus (BDV) can induce severe neurological disorder in Lewis rats and MRL mice. Antiviral CD8 T cells have been shown to be the mediators of disease in these animals. To define molecules involved in the disease process, we performed infection studies in MRL mice lacking either interferon-gamma, a functional Fas/FasL system, chemokine receptor CXCR3, or inducible NO synthase. We further used transgenic MRL mice expressing interferon-gamma-inducible, T cell-attracting chemokine CXCL10 in brain astrocytes. After intracerebral infection with BDV, wild-type and mutant mice developed CD8 T cell responses and neurological disease at similar frequency and with similar kinetics, suggesting that these factors are not required for initiation and maintenance of the immunopathological process. Similarly, the course of disease could not be altered by treating infected MRL mice or Lewis rats with the drug L-N(6)-(1-iminoethyl)-lysine (L-NIL) that specifically blocks the activity of the inducible NO synthase. We therefore have excluded a number of important factors that have been demonstrated to be crucial in the pathogenesis of a broad number of pathologic conditions. Thus, BDV-induced disease may not result from the action of a single dominant T cell-dependent effector molecule. Disease rather reflects a combined influence of several as yet undefined factors from CD8 T cells.
Asunto(s)
Enfermedad de Borna/inmunología , Enfermedad de Borna/fisiopatología , Virus de la Enfermedad de Borna/patogenicidad , Enfermedades del Sistema Nervioso/inmunología , Enfermedades del Sistema Nervioso/fisiopatología , Animales , Enfermedad de Borna/virología , Linfocitos T CD8-positivos/inmunología , Inducción Enzimática , Interferón gamma/genética , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Enfermedades del Sistema Nervioso/virología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Endogámicas Lew , Receptores CXCR3 , Receptores de Quimiocina/genética , Receptor fas/metabolismoRESUMEN
Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.
Asunto(s)
Vectores Genéticos/genética , Vectores Genéticos/inmunología , Regiones Promotoras Genéticas , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos/inmunología , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Línea Celular , Embrión de Pollo , Femenino , Expresión Génica , Orden Génico , Vectores Genéticos/administración & dosificación , Humanos , Inmunidad Celular , Inmunidad Humoral , Memoria Inmunológica , Ratones , Datos de Secuencia Molecular , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Modified vaccinia Ankara (MVA) was developed by serial passages on chicken embryo fibroblast cells. After passage 570, the virus was considered homogenous and genetically stable. Three MVA strains (MVA-572, MVA-I721 and MVA-BN) have been analyzed and shown to be 100% genetically identical; although significant differences in their phenotypes were illustrated. All MVA strains except MVA-BN replicated in human cells, or killed immune suppressed mice. Viruses isolated from dead animals were shown to represent variants present within MVA-572 or MVA-I721 used to inoculate the mice. These subpopulations were shown to encode mutations, or contain less than the six deletions associated with MVA and had significantly altered phenotypes compared to the parental MVA strains. MVA is a complex polyclonal mixture of viruses, the composition of which governs the phenotype.
Asunto(s)
Virus Vaccinia/genética , Virus Vaccinia/fisiología , Replicación Viral , Animales , Embrión de Pollo , ADN Viral/análisis , Femenino , Genoma Viral , Células HeLa , Humanos , Ratones , Ratones Noqueados , Ovario/virología , Fenotipo , Pase Seriado , Cultivo de VirusRESUMEN
Borna disease virus (BDV) is a highly neurotropic, noncytolytic virus. Experimentally infected B10.BR mice remain healthy unless specific antiviral T cells that infiltrate the infected brain are triggered by immunization. In contrast, infected MRL mice spontaneously mount an antiviral T-cell response that can result in meningoencephalitis and neurological disease. The antiviral T cells may, alternatively, eliminate the virus without inducing disease if they are present in sufficient numbers before the virus replicates to high titers. Since the immune response of H-2(k) mice is directed mainly against the epitope TELEISSI located in the viral nucleoprotein N, we generated BDV mutants that feature TQLEISSI in place of TELEISSI. We show that adoptive transfer of BDV N-specific CD8 T cells induced neurological disease in B10.BR mice persistently infected with wild-type BDV but not with the mutant virus expressing TQLEISSI. Surprisingly, the mutant virus replicated less well in adult MRL wild-type mice than in mutant mice lacking mature CD8 T cells. Furthermore, when MRL mice were infected with the TQLEISSI-expressing BDV mutant as newborns, neurological disease was observed, although at a lower rate and with slower kinetics than in mice infected with wild-type virus. These results confirm that TELEISSI is the major CD8 T-cell epitope in H-2(k) mice and suggest that unidentified minor epitopes are present in the BDV proteome which are recognized rather efficiently by antiviral T cells if the dominant epitope is absent.
Asunto(s)
Enfermedad de Borna/terapia , Virus de la Enfermedad de Borna/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T , Epítopos Inmunodominantes , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Virus de la Enfermedad de Borna/patogenicidad , Linfocitos T CD8-positivos/trasplante , Inmunidad , Ratones , Ratones Endogámicos , Enfermedades del Sistema Nervioso/etiología , Nucleoproteínas/inmunologíaRESUMEN
Chorioallantois vaccinia virus Ankara (CVA) is the parental virus of modified vaccinia virus Ankara (MVA), which was derived from CVA by more than 570 passages in chicken embryo fibroblasts (CEF). MVA became severely host-cell-restricted to avian cells and has strongly diminished virulence in mammalian hosts, while maintaining good immunogenicity. We determined the complete coding sequence of the parental CVA and mapped the exact positions of the six major deletions that emerged in the MVA genome. All six major deletions occurred in regions of the CVA genome where one or more truncated or fragmented open reading frames (ORFs) pre-existed. The CVA genome contains 229 ORFs of which 51 are fragments of full-length orthopoxvirus (OPV) genes, including fragmented orthologues of C9L and M1L (encoding two well-conserved ankyrin-like proteins), A39R (encoding a semaphorin-like protein) and A55R (encoding a kelch-like protein). Phylogenetic analysis demonstrated that MVA was most closely related to CVA, followed by the vaccinia virus (VACV) strain DUKE, a patient-derived isolate of the Dryvax vaccine virus. Loss or mutation of genes outside the six major deletions are assumed to contribute to the restricted host range phenotype of MVA. In support of this notion, deletions, insertions and non-synonymous mutations were found in 122 of the 195 ORFs remaining in MVA when compared with their CVA counterparts. Thus, detailed knowledge of the CVA genomic sequence is a prerequisite to further dissect the genetic basis of the MVA host range phenotype as well as the particular immunological properties of MVA.
Asunto(s)
Genoma Viral , Virus Vaccinia/genética , Proteínas Virales/genética , Animales , Células Cultivadas , Embrión de Pollo , Eliminación de Gen , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Pase Seriado , TurquíaRESUMEN
Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2(k)-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-gamma) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-gamma-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-gamma-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-gamma-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-gamma plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-gamma may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.
Asunto(s)
Enfermedad de Borna/inmunología , Enfermedad de Borna/prevención & control , Virus de la Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/aislamiento & purificación , Linfocitos T CD8-positivos/inmunología , Interferón gamma/inmunología , Enfermedades del Sistema Nervioso/inmunología , Enfermedades del Sistema Nervioso/prevención & control , Vacunación , Vacunas Virales/administración & dosificación , Factores de Edad , Animales , Enfermedad de Borna/virología , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , Quimiocinas/inmunología , Pruebas Inmunológicas de Citotoxicidad , Eosinófilos/patología , Vectores Genéticos , Inyecciones Intramusculares , Interferón gamma/deficiencia , Interleucina-13/inmunología , Ratones , Ratones Noqueados , Enfermedades del Sistema Nervioso/virología , Proteínas de la Nucleocápside/biosíntesis , Proteínas de la Nucleocápside/genética , Parapoxvirus/genética , Bazo/inmunología , Vacunas Sintéticas/administración & dosificación , Virus Vaccinia/genéticaRESUMEN
Borna disease virus (BDV) can persistently infect the central nervous system and induce CD8+ T-cell-mediated neurological disease in MRL mice. To determine whether specific immune priming would prevent disease, a prime-boost immunization protocol was established in which intramuscular injection of a recombinant parapoxvirus expressing BDV nucleoprotein (BDV-N) was followed by intraperitoneal infection with vaccinia virus expressing BDV-N. Immunized wild-type and perforin-deficient mice remained healthy after intracerebral infection with BDV and contained almost no virus in the brain at 5 weeks post-challenge. Immunization failed to induce resistance against BDV in mice lacking mature CD8+ T cells. Immunization of perforin-deficient mice with a poxvirus vector expressing mutant BDV-N lacking the known CD8+ T-cell epitope did not efficiently block multiplication of BDV in the brain and did not prevent neurological disease, indicating that vaccine-induced immunity to BDV in wild-type and perforin-deficient mice resulted from the action of CD8+ T cells.
Asunto(s)
Enfermedad de Borna/prevención & control , Virus de la Enfermedad de Borna , Vacunación , Vacunas Virales/administración & dosificación , Animales , Enfermedad de Borna/inmunología , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/inmunología , Encéfalo/virología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Vectores Genéticos , Inyecciones Intramusculares , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Mutación , Nucleoproteínas/biosíntesis , Nucleoproteínas/genética , Parapoxvirus/genética , Perforina , Proteínas Citotóxicas Formadoras de Poros , Vacunas Sintéticas/administración & dosificación , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
Borna disease virus (BDV) infection of the central nervous system (CNS) leads to severe neurological symptoms in susceptible MRL mice. The disease is mainly mediated by CD8+ T cells specific for the immunodominant epitope TELEISSI in the BDV nucleoprotein. In this study, TELEISSI/MHC class I tetramers were used to directly visualize antigen-specific CD8+ T cells. We found that on average approximately 30% of the ex vivo analyzed CD8+ T cells in the CNS of diseased mice were specific for TELEISSI. Unexpectedly, the frequency of tetramer-reactive brain-derived CD8+ T cells doubled following overnight culture in the absence of antigen. The majority of CD8+ T cells showed enhanced tetramer binding without up-regulation of T cell receptor surface expression. The frequency of IFN-gamma-secreting CD8+ T cells after antigen-specific stimulation was higher in overnight cultures than in freshly isolated BDV-specific brain lymphocytes, and enhanced tetramer binding correlated with elevated sensitivity to lower levels of peptide antigen in cytotoxicity assays. These results indicate that the functional avidity of virus-specific CD8+ T cells was down-modulated in vivo. Thus, quantification of tissue-infiltrating CD8+ T cells by the tetramer technique must be interpreted with caution as it may underestimate the real frequency of antigen-specific CD8+ T cells.
Asunto(s)
Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/inmunología , Linfocitos T CD8-positivos/inmunología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Animales , Enfermedad de Borna/diagnóstico , Enfermedad de Borna/patología , Linfocitos T CD8-positivos/metabolismo , Sistema Nervioso Central/patología , Regulación hacia Abajo , Antígenos H-2/inmunología , Interferón gamma/metabolismo , Ratones , Péptidos , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Dendritic cells (DCs) have been used successfully to induce CD8 T cells that control virus infections and growth of tumours. The efficacy of DC-mediated immunization for the control of neurotropic Borna disease virus (BDV) in mice was evaluated. Certain strains of mice only rarely develop spontaneous neurological disease, despite massive BDV replication in the brain. Resistance to disease is due to immunological ignorance toward BDV antigen in the central nervous system. Ignorance in mice can be broken by immunization with DCs coated with TELEISSI, a peptide derived from the N protein of BDV, which represents the immunodominant cytotoxic T lymphocyte epitope in H-2(k) mice. Immunization with TELEISSI-coated DCs further induced solid protective immunity against intravenous challenge with a recombinant vaccinia virus expressing BDV-N. Interestingly, however, this immunization scheme induced only moderate protection against intracerebral challenge with BDV, suggesting that immune memory raised against a shared antigen may be sufficient to control a peripherally replicating virus, but not a highly neurotropic virus that is able to avoid activation of T cells. This difference might be due to the lack of BDV-specific CD4 T cells and/or inefficient reactivation of DC-primed, BDV-specific CD8 T cells by the locally restricted BDV infection. Thus, a successful vaccine against persistent viruses with strong neurotropism should probably induce antiviral CD8 (as well as CD4) T-cell responses and should favour the accumulation of virus-specific memory T cells in cervical lymph nodes.