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Introduction. Administered nasally, spores of the Gram-positive bacterium Bacillus subtilis have been shown to be able to induce innate immunity sufficient to confer protection to influenza and respiratory syncytial virus.Hypothesis. Although members of the aerobiome, intranasal delivery of high numbers of live spores carries potential safety issues.Aim. To address the potential safety risk of using live spores, we assessed the safety of spores that had been completely inactivated using heat sterilization.Methodology. Using autoclaved, and therefore killed, spores of a generally recognized as safe-notified B. subtilis strain (DSM 32444), safety was assessed in vitro (biotype, genome and cell based cytoxicity) and in vivo, using intranasal administration in rodent models and lastly in human volunteers.Results. Using a 15-day, repeat-dose, regimen in a rodent model, no indication of toxicity was observed. In a registered human study (NCT05984004), a formulated preparation of inactivated DSM 32444 spores referred to as SPEROVID was developed, and tolerance in human volunteers was assessed following 7 days of nasal dosing (2-4 times/day).Conclusion. Our study demonstrated that in humans an intranasal dose of up to 3×108 killed spores was safe and well tolerated.
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Administración Intranasal , Bacillus subtilis , Esporas Bacterianas , Adulto , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Ratas , Adulto JovenRESUMEN
One problem encountered in the use of adenoviral vectors for gene therapy is their toxicity. Although many studies have analyzed this question in vivo, few researches have investigated adenovirus vector effects at the cellular level using a large-scale approach. In particular, no such data are available for helper-dependent adenovirus vectors (HD), which are promising adenovirus vectors for clinical applications since they are devoid of all viral genes and can host large transgene cassettes. The present study used gene chips to examine (Affymetrix HG-U95Av2 interrogating 12,626 unique human transcripts) the effect on liver cells of HD vectors versus that of DeltaE1/E3 adenovirus vector and wild type Adenovirus (Ad5). The effects of the DeltaE1/E3 adenovirus and of HD vectors were comparable, and significantly milder than that of Ad5. Interestingly the expression signatures of DeltaE1/E3 adenovirus and HD vectors were non-overlapping both at the single gene and the pathway level, suggesting specific and different interactions between the host cell and the two gene therapy vectors.
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Adenoviridae/fisiología , Regulación de la Expresión Génica , Vectores Genéticos , Transcripción Genética , Adenoviridae/genética , Línea Celular , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Virales/genética , Virus Helper , Hepatocitos/virología , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered lambda by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286-393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286-393) includes the principal receptor-binding motif ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda Pb (286-393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.
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Bacteriófago lambda/genética , Proteínas de la Cápside/genética , Cápside , Fibroblastos/metabolismo , Receptores Virales/metabolismo , Transducción Genética/métodos , Bacteriófago lambda/metabolismo , Proteínas de la Cápside/metabolismo , Línea Celular , Endosomas/metabolismo , Terapia Genética/métodos , Proteínas Fluorescentes Verdes , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/genéticaRESUMEN
Given the biological complexity of viral infections, the variability of the host response, and the safety concerns related to viral-mediated gene transfer, recent studies have made use of DNA mircoarrays to integrate multi-layered experimental approaches aimed at completely clarifying virus-host interactions. Particular attention has been given to those viruses that are implicated in clinical use and/or in life-threatening diseases. Examples of such use can be divided into three main categories, including: (i) the use of microarrays to study viral expression; (ii) the use of microarrays to analyze the host response to viral infection; and (iii) the use of microarrays to characterize the host response to viral vector-mediated transduction. Significant information on virus- and viral vector-host interactions can be obtained with the microarray approach, including the recognition of master pathways of virally-induced responses, the identification of new target genes for specific viruses, and indications on the molecular toxicity of specific gene transfer vectors currently used for gene therapy trials (in particular, adeno-associated viruses and adenovirus-derived vectors). We predict that the development of accessible repositories containing most of the DNA microarray data on viral infections will certainly help to elucidate the puzzling pictures of different viral infections. This will be crucially important for the correct handling of viral diseases and the intelligent amelioration of viral vectors for gene therapy.
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Terapia Genética , Vectores Genéticos , Virosis/terapia , Virus/genética , Animales , ADN Viral/análisis , Técnicas de Transferencia de Gen , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Virosis/virologíaRESUMEN
The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation. We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression. These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs.
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Adenovirus Humanos/metabolismo , Regulación de la Expresión Génica , Priones/metabolismo , Replicación Viral/fisiología , Infecciones por Adenoviridae/metabolismo , Adenovirus Humanos/genética , Línea Celular , Células Cultivadas , Células HeLa , Humanos , Proteínas PrPSc/metabolismo , Priones/genética , Eliminación de SecuenciaRESUMEN
The porcine endogenous retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID(50) and in vivo results in more RNA positive tissues and higher levels of proviral DNA using our human PERV-A receptor (HuPAR-2) transgenic mouse model [Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., Wilson, C.A., Patience, C., Salomon, D.R., 2006. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J. Virol. 80 (7), 3135-3146]. The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus-host context.
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Retrovirus Endógenos/fisiología , Retrovirus Endógenos/patogenicidad , Estructura Terciaria de Proteína/fisiología , Animales , Línea Celular , Productos del Gen gag/química , Humanos , Ratones , Ratones Transgénicos , Receptor PAR-2/genética , Receptores Virales/genética , Porcinos , Virulencia , Esparcimiento de VirusRESUMEN
The potential of porcine endogenous retrovirus (PERV) as a human pathogen, particularly as a public health risk, is a major concern for xenotransplantation. In vitroPERV transmission to human cells is well established. Evidence from human/pig hematopoietic chimeras in immunodeficient mice suggests PERV transmission from pig to human cells in vivo. However, recently Yang et al. demonstrated in such a model that PERV-C, a nonhuman-tropic class, could be transmitted via pseudotyping by xenotropic murine leukemia virus (X-MLV). We developed a mouse pig islet xenotransplant model, where pig and human cells are located in physically separate compartments, to directly assess PERV transmission from a functional pig xenograft. X-MLV efficiently pseudotypes all three classes of PERV, including PERV-A and -B that are known to productively infect human cell lines and PERV-C that is normally not infectious for human cells. Pseudotyping also extends PERV's natural tropism to nonpermissive, nonhuman primate cells. X-MLV is activated locally by the surgical procedure involved in the tissue transplants. Thus, the presence and activation of endogenous X-MLV in immunodeficient mice limits the clinical significance of previous reports of in vivo PERV transmission from pig tissues to human cells.
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ADN Viral/aislamiento & purificación , Retrovirus Endógenos/genética , Trasplante de Islotes Pancreáticos/inmunología , Islotes Pancreáticos/virología , Virus de la Leucemia Murina/fisiología , Infecciones por Retroviridae/transmisión , Trasplante Heterólogo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Retrovirus Endógenos/aislamiento & purificación , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Datos de Secuencia Molecular , Infecciones por Retroviridae/virología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Porcinos/virología , Quimera por Trasplante/virología , Integración ViralRESUMEN
Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of alpha3beta1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin alpha3beta1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-alpha3 and anti-beta1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with alpha3beta1, which involved multiple additional contact sites.