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OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.
Assuntos
Colite , Doença de Crohn , Doenças Inflamatórias Intestinais , Nanopartículas , Camundongos , Animais , Doença de Crohn/genética , Doença de Crohn/complicações , Linfócitos T CD8-Positivos/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/prevenção & controle , Inflamação/complicações , Sulfato de Dextrana , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 22/genéticaRESUMO
In 2014 we observed a noticeable increase in the number of sudden deaths among green tree pythons (Morelia viridis). Pathological examination revealed the accumulation of mucoid material within the airways and lungs in association with enlargement of the entire lung. We performed a full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterize the pathogenesis of this mucinous pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung tissue samples from a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome whose sequence was <85% identical to that of nidoviruses previously identified in ball pythons and Indian pythons. We then employed reverse transcription-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cultures of Morelia viridis liver and brain cells, which we inoculated with homogenates of lung tissue from infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA in situ hybridization to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium, which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover.IMPORTANCE Novel nidoviruses associated with severe respiratory disease were fairly recently identified in ball pythons and Indian pythons. Herein we report on the isolation and identification of a further nidovirus from green tree pythons (Morelia viridis) with fatal pneumonia. We thoroughly characterized the pathological changes in the infected individuals and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death by inhibiting normal gas exchange in the lungs. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high rates of mortality in breeding collections, and most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.
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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.
Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/isolamento & purificação , Glicoproteínas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vaccinia virus/genética , Vírion/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Ebola/genética , Ebolavirus/genética , Ebolavirus/imunologia , Ebolavirus/fisiologia , Glicoproteínas/genética , Humanos , Imunoglobulina G/sangue , Camundongos , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Vírion/fisiologiaRESUMO
Rotaviruses (RVs) are important enteric pathogens of newborn humans and animals, causing diarrhea and in rare cases death, especially in very young individuals. Rotavirus vaccines presently used are modified live vaccines that lack complete biological safety. Previous work from our laboratory suggested that vaccines based on in situ produced, non-infectious rotavirus-like particles (RVLPs) are efficient while being entirely safe. However, using either vaccine, active mucosal immunization cannot induce protective immunity in newborns due to their immature immune system. We therefore hypothesized that offspring from vaccinated dams are passively immunized either by transfer of maternal antibodies during pregnancy or by taking up antibodies from milk. Using a codon optimized polycistronic gene expression cassette packaged into herpesvirus particles, the simultaneous expression of the RV capsid genes led to the intracellular formation of RVLPs in various cell lines. Vaccinated dams developed a strong RV specific IgG antibody response determined in sera and milk of both mother and pups. Moreover, sera of naïve pups nursed by vaccinated dams also had RV specific antibodies suggesting a lactogenic transfer of antibodies. Although full protection of pups was not achieved in this mouse model, our observations are important for the development of improved vaccines against RV in humans as well as in various animal species.
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Anticorpos Antivirais/imunologia , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Leite/imunologia , Vacinas contra Rotavirus/genética , Vacinas contra Rotavirus/imunologia , Rotavirus/imunologia , Vacinação , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Linhagem Celular Tumoral , Chlorocebus aethiops , Códon , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Gravidez , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/administração & dosagem , Transdução Genética , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Células Vero , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines.
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Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Herpesvirus Humano 1/imunologia , Infecções por Rotavirus/prevenção & controle , Rotavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Chlorocebus aethiops , Feminino , Vetores Genéticos , Células HEK293 , Herpesvirus Humano 1/genética , Humanos , Imunidade Humoral , Imunidade nas Mucosas , Imunização , Camundongos , Rotavirus/genética , Infecções por Rotavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Células Vero , Vírion/genética , Vírion/imunologiaRESUMO
Application of dextran sodium sulfate (DSS) is often used to induce experimental colitis. Current state of the art is to refrain from the use of analgesics due to their possible interaction with the model. However, the use of analgesics would be beneficial to reduce the overall constraint imposed on the animals. Here, we analyzed the effect of the analgesics Dafalgan (paracetamol), Tramal (tramadol) and Novalgin (metamizole) on DSS-induced colitis. To study the effect of those analgesics in colitis mouse models, acute and chronic colitis was induced in female C57BL6 mice by DSS administration in the drinking water. Analgesics were added to the drinking water on days four to seven (acute colitis) or on days six to nine of each DSS cycle (chronic colitis). Tramadol and paracetamol had minor effects on colitis severity. Tramadol reduced water uptake and activity levels slightly, while mice receiving paracetamol presented with a better overall appearance. Metamizole, however, significantly reduced water uptake, resulting in pronounced weight loss. In conclusion, our experiments show that tramadol and paracetamol are viable options for the use in DSS-induced colitis models. However, paracetamol seems to be slightly more favorable since it promoted the overall wellbeing of the animals upon DSS administration without interfering with typical readouts of colitis severity.
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Colite , Água Potável , Tramadol , Animais , Feminino , Camundongos , Tramadol/farmacologia , Dipirona/farmacologia , Acetaminofen/efeitos adversos , Água Potável/efeitos adversos , Sulfato de Dextrana/efeitos adversos , Camundongos Endogâmicos C57BL , Colite/induzido quimicamente , Colite/tratamento farmacológico , Analgésicos/efeitos adversos , Modelos Animais de DoençasRESUMO
BACKGROUND AND AIMS: Exacerbated immune activation, intestinal dysbiosis and a disrupted intestinal barrier are common features among inflammatory bowel disease [IBD] patients. The polyamine spermidine, which is naturally present in all living organisms, is an integral component of the human diet, and exerts beneficial effects in human diseases. Here, we investigated whether spermidine treatment ameliorates intestinal inflammation and offers therapeutic potential for IBD treatment. METHODS: We assessed the effect of oral spermidine administration on colitis severity in the T cell transfer colitis model in Rag2-/- mice by endoscopy, histology and analysis of markers of molecular inflammation. The effects on the intestinal microbiome were determined by 16S rDNA sequencing of mouse faeces. The impact on intestinal barrier integrity was evaluated in co-cultures of patient-derived macrophages with intestinal epithelial cells. RESULTS: Spermidine administration protected mice from intestinal inflammation in a dose-dependent manner. While T helper cell subsets remained unaffected, spermidine promoted anti-inflammatory macrophages and prevented the microbiome shift from Firmicutes and Bacteroides to Proteobacteria, maintaining a healthy gut microbiome. Consistent with spermidine as a potent activator of the anti-inflammatory molecule protein tyrosine phosphatase non-receptor type 2 [PTPN2], its colitis-protective effect was dependent on PTPN2 in intestinal epithelial cells and in myeloid cells. The loss of PTPN2 in epithelial and myeloid cells, but not in T cells, abrogated the barrier-protective, anti-inflammatory effect of spermidine and prevented the anti-inflammatory polarization of macrophages. CONCLUSION: Spermidine reduces intestinal inflammation by promoting anti-inflammatory macrophages, maintaining a healthy microbiome and preserving epithelial barrier integrity in a PTPN2-dependent manner.
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Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.
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Artrite Reumatoide , Osteoartrite , RNA Longo não Codificante , Humanos , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Osteoartrite/genética , Osteoartrite/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismoRESUMO
Plastic pollution is a major global challenge of our times, baring a potential threat for the environment and the human health. The increasing abundance of nanoplastic (NP) and microplastic (MP) particles in the human diet might negatively affect human health since they - particularly in patients suffering from inflammatory bowel disease (IBD) - might surpass the intestinal barrier. To investigate whether ingested plastic particles cross the intestinal epithelium and promote bowel inflammation, mice were supplemented with NP or MP polystyrene (PS) particles for 24 or 12 weeks before inducing acute or chronic dextran sodium sulfate (DSS) colitis with continuous plastic administration. Although ingested PS particles accumulated in the small intestine and organs distant from the gastrointestinal tract, PS ingestion did not affect intestinal health nor did it promote colitis severity. Although the lack of colitis-promoting effects of small PS particles might be a relief for IBD patients, potential accumulative effects of ingested plastic particles on the gastrointestinal health cannot be excluded.
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Colite , Doenças Inflamatórias Intestinais , Animais , Colite/induzido quimicamente , Humanos , Doenças Inflamatórias Intestinais/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Microplásticos , Plásticos , PoliestirenosRESUMO
Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.
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Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Dependovirus/fisiologia , Herpesvirus Humano 1/fisiologia , Transativadores/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Apoptose , Chlorocebus aethiops , Dano ao DNA , DNA Viral/metabolismo , Dependovirus/crescimento & desenvolvimento , Herpesvirus Humano 1/crescimento & desenvolvimento , Fosforilação , Deleção de Sequência , Células VeroRESUMO
BACKGROUND AND AIMS: A single nucleotide polymorphism in protein tyrosine phosphatase non-receptor type 22 [PTPN22] has been associated with the onset of autoimmune disorders, but protects from Crohn's disease. PTPN22 deficiency in mice promotes intestinal inflammation by modulating lymphocyte function. However, the impact of myeloid PTPN22 in colitis development remains unclear. The aim of this study was to investigate the role of PTPN2 in the IL-10 and the T cell transfer colitis models. METHODS: PTPN22-deficient mice were crossed with IL-10-/- and RAG2-/- mice. Naïve T cells were injected in RAG-/- mice to induce T-cell transfer colitis. Spontaneous colitis in IL-10-/- mice was monitored for up to 200 days. RESULTS: Here, we demonstrate that PTPN22 in non-lymphoid immune cells is required to protect against T cell transfer-mediated and IL-10 knock-out colitis. Analysis of the intestinal immune landscape demonstrated a marked reduction of granulocyte influx into the inflamed colon in PTPN22-deficient mice. On a molecular level, granulocytes were not only reduced by numbers, but also revealed a defective function. In particular, granulocyte activation and granulocyte-mediated bacteria killing was impaired upon loss of PTPN22, resulting in elevated bacterial burden and translocation beyond the intestinal epithelial barrier in PTPN22-deficient mice. Consistently, antibiotic-induced depletion of bacteria reverted the increased colitis susceptibility in PTPN22-deficient mice, whereas granulocyte depletion induced acolitis phenotype in wild-type mice similar to that observed in PTPN22-deficient mice. CONCLUSIONS: In conclusion, our data demonstrate that PTPN22 is essential for adequate granulocyte activation and antimicrobial defence to protect the inflamed intestine from bacterial invasion and exacerbated colitis.
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Doença de Crohn/genética , Predisposição Genética para Doença , Granulócitos/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Animais , Doença de Crohn/imunologia , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal , Inflamação , Camundongos , Camundongos Knockout , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Vedolizumab is a widely used and safe therapy in inflammatory bowel disease, particularly in ulcerative colitis (UC), making it a promising candidate for enhanced efficacy by combining it with additional immunomodulatory medications. In this study, we studied the impact of vedolizumab monotreatment vs vedolizumab coadministration with other immunomodulatory drugs on intestinal inflammation and intestinal immune cells in vivo. METHODS: Colon tissue from human patients with UC with active disease or in remission with or without vedolizumab treatment was stained by immunohistochemistry. We reconstituted NOD-SCID-SGM3 mice with human CD34+ cells and treated them with dextran sodium sulfate to induce acute colitis. Mice were treated with vedolizumab alone, or in combination with tacrolimus, ozanimid, or tofacitinib. RESULTS: Vedolizumab reduced the number of CD3+ T cells and CD68+ monocytes/macrophages in the colon of patients with UC with active disease. Vedolizumab moderately decreased immune cell numbers in acute dextran sodium sulfate-induced colitis. The combination of vedolizumab with tacrolimus further reduced the number of infiltrating CD3+ T cells and CD68+ monocytes/macrophages and was superior in ameliorating intestinal inflammation when compared to vedolizumab monotreatment. In contrast, cotreatment using vedolizumab with ozanimod or tofacitinib had no additive effect. CONCLUSIONS: Our data show that vedolizumab reduces the number of innate and adaptive immune cells in the mucosa of patients with UC. Further, the combination of vedolizumab with tacrolimus was more efficient to reduce immune cell numbers and to increase therapeutic efficacy than vedolizumab monotreatment. This finding indicates that combination treatment using these two drugs may be beneficial for patients who do not respond to vedolizumab monotherapy.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Colite Ulcerativa , Fármacos Gastrointestinais , Tacrolimo , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Dextranos , Fármacos Gastrointestinais/uso terapêutico , Humanos , Agentes de Imunomodulação , Inflamação/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Tacrolimo/uso terapêutico , Resultado do TratamentoRESUMO
HSV-1 amplicon vectors have been used as platforms for the generation of genetic vaccines against both DNA and RNA viruses. Mice vaccinated with such vectors encoding structural proteins from both foot-and-mouth disease virus and rotavirus were partially protected from challenge with wild-type virus (D'Antuono et al., Vaccine 28:7363-7372, 2010; Laimbacher et al., Mol Ther 20:1810-1820, 2012; Meier et al., Int J Mol Sci 18:431, 2017), indicating that HSV-1 amplicon vectors are attractive tools for the development of complex and safe genetic vaccines.This chapter describes the preparation and testing of HSV-1 amplicon vectors that encode individual or multiple viral structural proteins from a polycistronic transgene cassette. We further put particular emphasis on generating virus-like particles (VLPs) in vector-infected cells. Expression of viral genes is confirmed by Western blot and immune fluorescence analysis and generation of VLPs in vector-infected cells is demonstrated by electron microscopy. Furthermore, examples on how to analyze the immune response in a mouse model and possible challenge experiments are described.
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Vetores Genéticos , Herpesvirus Humano 1 , Transdução Genética , Vacinas Virais , Animais , Chlorocebus aethiops , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Camundongos , Células Vero , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.
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Herpesvirus Humano 1/crescimento & desenvolvimento , Proteínas Virais/biossíntese , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Membrana Celular/química , Núcleo Celular/química , Chlorocebus aethiops , Cricetinae , Citoplasma/química , Imunofluorescência , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Humano 1/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Membrana Nuclear/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Virais/genética , Proteína Vermelha FluorescenteRESUMO
Group C Rotavirus (RVC) has been associated globally with sporadic outbreaks of gastroenteritis in children and adults. RVC also infects animals, and interspecies transmission has been reported as well as its zoonotic potential. Considering its genetic diversity and the absence of effective vaccines, it is important and necessary to develop new generation vaccines against RVC for both humans and animals. The aim of the present study was to develop and characterize an HSV-1-based amplicon vector expressing a human RVC-VP6 protein and evaluate the humoral immune response induced after immunizing BALB/c mice. Local fecal samples positive for RVC were used for isolation and sequencing of the vp6 gene, which phylogenetically belongs to the I2 genotype. We show here that cells infected with the HSV[VP6C] amplicon vector efficiently express the VP6 protein, and induced specific anti-RVC antibodies in mice immunized with HSV[VP6C], in a prime-boost schedule. This work highlights that amplicon vectors are an attractive platform for the generation of safe genetic immunogens against RVC, without the addition of external adjuvants.
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Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Expressão Gênica , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Rotavirus/genética , Rotavirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Humanos , Imunidade Humoral , Masculino , Camundongos , Filogenia , Proteínas Recombinantes , Células VeroRESUMO
U94, the latency gene of human herpesvirus 6, was found to inhibit migration, invasion and proliferation of vascular endothelial cells (ECs). Because of its potent anti-migratory activity on ECs, we tested the capability of U94 to interfere with the individual steps of the metastatic cascade. We examined the U94 biological activity on the human breast cancer cell line MDA-MB 231, as a model of highly aggressive cancer cell. Here we show that the expression of U94 delivered by an HSV-1-based amplicon promoted down-modulation of Src and downstream molecules linked to cell motility and proliferation. Indeed, U94 expression strongly inhibited cell migration, invasiveness and clonogenicity. We investigated the effects of U94 in a three-dimensional rotary cell-culture system and observed the ability of U94 to modify tumor cell morphology by inducing a partial mesenchymal-to-epithelial transition. In fact, despite U94 did not induce any expression of the epithelial marker E-cadherin, it down-modulated different mesenchymal markers as ß-catenin, Vimentin, TWIST, Snail1, and MMP2. In vivo data on the tumorigenicity of MDA-MB 231 displayed the capability of U94 to control tumor growth, invasiveness and metastasis, as well as tumor-driven angiogenesis. The antitumor U94 activity was also confirmed on the human cervical cancer cell line HeLa. The ability of U94 to inhibit cell growth, invasion and metastasis opens the way to a promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.
Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Genes src , Herpesvirus Humano 6/fisiologia , Proteínas Virais/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Modelos Animais de Doenças , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Transdução de Sinais , Transfecção , Microambiente Tumoral/genética , Proteínas Virais/metabolismoRESUMO
Heterologous virus-vectored vaccines, particularly those based on canarypox virus vectors, have established a firm place in preventive veterinary medicine. However, herpesvirus-based vaccines have paved the way for DIVA vaccines (discrimination of infected against vaccinated animals), which are particularly desirable for highly contagious livestock diseases that are otherwise combatted by culling of infected animals.In this chapter, we describe the design, the preparation, and the testing of a polycistronic herpesvirus amplicon vaccine against rotaviruses with a particular emphasis on generating heterologous virus-like particles for immunization. After the design, the procedure consists of three steps, first, transient expression of the construct in cell cultures, second, expression and antibody response in a mouse model, and third, application of the system to the desired host species. As a whole, the present information will facilitate the design of novel vaccines of veterinary interest from the designing process until pre-licensing.
Assuntos
Doenças Transmissíveis/imunologia , Infecções por Rotavirus/prevenção & controle , Vacinação/métodos , Vacinas Virais/imunologia , Animais , Doenças Transmissíveis/genética , Doenças Transmissíveis/veterinária , Genes/genética , Vetores Genéticos , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Imunização/métodos , Imunização/veterinária , Camundongos , Rotavirus/imunologia , Rotavirus/patogenicidade , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/veterinária , Vacinação/veterinária , Vacinas Virais/genética , Vacinas Virais/uso terapêuticoRESUMO
HSV-1 amplicon vectors have been used as platforms for the generation of genetic vaccines against both DNA and RNA viruses. Mice vaccinated with such vectors encoding structural proteins from both foot-and-mouth disease virus and rotavirus were partially protected from challenge with wild-type virus (D'Antuono et al. Vaccine 28: 7363-7372, 2010; Laimbacher et al. Mol Ther 20: 1810-1820, 2012), indicating that HSV-1 amplicon vectors are attractive tools for the development of complex and safe genetic vaccines. This chapter describes the use of HSV-1 amplicon vectors that encode individual or multiple viral structural proteins from a polycistronic transgene cassette in mammalian cells. More precisely, amplicon vectors that encode multiple structural viral proteins support the in situ production of viruslike particles (VLPs) in vector-infected cells. The expression of the viral genes is confirmed by Western blot and immune fluorescence analysis, and the generation of VLPs in vector-infected cells is demonstrated by electron microscopy.
Assuntos
Herpesvirus Humano 1/genética , Biologia Molecular/métodos , Rotavirus/imunologia , Vacinas Virais/genética , Animais , Chlorocebus aethiops , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Febre Aftosa/terapia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Herpesvirus Humano 1/imunologia , Humanos , Camundongos , Rotavirus/genética , Rotavirus/patogenicidade , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/prevenção & controle , Infecções por Rotavirus/terapia , Células Vero , Proteínas Estruturais Virais/antagonistas & inibidores , Proteínas Estruturais Virais/imunologia , Vacinas Virais/uso terapêuticoRESUMO
The herpes simplex virus type 1 (HSV-1) tegument proteins pUL36 (VP1/2) and pUL37 are essential for viral egress. We previously defined a minimal domain in HSV-1 pUL36, residues 548-572, as important for binding pUL37. Here, we investigated the role of this region in binding to pUL37 and facilitating viral replication. We deleted residues 548-572 in frame in a virus containing a mRFP tag at the N-terminus of the capsid protein VP26 and an eGFP tag at the C-terminus of pUL37 (HSV-1pUL36∆548-572). This mutant virus was unable to generate plaques in Vero cells, indicating that deletion of this region of pUL36 blocks viral replication. Imaging of HSV-1pUL36∆548-572-infected Vero cells, in comparison to parental and resucant, revealed a block in secondary envelopment of cytoplasmic capsids. In addition, immunoblot analysis suggested that failure to bind pUL37 affected the stability of pUL36. This study provides further insight into the role of this essential interaction.
Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Liberação de Vírus , Animais , Chlorocebus aethiops , Análise Mutacional de DNA , Células Vero , Ensaio de Placa ViralRESUMO
Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only a very small percentage of the 152-kbp viral genome. Consequently, replication and packaging of amplicons depend on helper functions that are provided either by replication-defective mutants of HSV-1 or by replication-competent, but packaging-defective, HSV-1 genomes. Sets of cosmids that overlap and represent the entire HSV-1 genome can form, via homologous recombination, circular replication-competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. The resulting stocks of packaged amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of amplicon and cosmid or bacterial artificial chromosome (BAC) DNA into 2-2 cells by cationic liposome-mediated transfection and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid and BAC DNA and determining the titers of amplicon stocks.