RESUMO
White-nose syndrome is a fungal disease responsible for the rapid decline of North American bat populations. This study addressed a novel method for inactivating Pseudogymnoascus destructans, the causative agent of WNS, using ultraviolet A (UVA) or B (UVB) radiation in combination with methoxsalen, a photosensitizer from the furanocoumarin family of compounds. Fungal spore suspensions were diluted in micromolar concentrations of methoxsalen (50-500 µM), then exposed to fixed doses of UVA radiation (500-5000 mJ/cm2), followed by plating on germination media. These plates were examined for two to four weeks for evidence of spore germination or inactivation, along with resultant growth or inhibition of P. destructans colonies. Pretreatment of fungal spores with low doses of methoxsalen resulted in a UVA dose-dependent inactivation of the P. destructans spores. All doses of methoxsalen paired with 500 mJ/cm2 of UVA led to an approximate two-log10 (~99%) reduction in spore viability, and when paired with 1000 mJ/cm2, a four-log10 or greater (>99.99%) reduction in spore viability was observed. Additionally, actively growing P. destructans colonies treated directly with methoxsalen and either UVA or UVB radiation demonstrated UV dose-dependent inhibition and termination of colony growth. This novel approach of using a photosensitizer in combination with UV radiation to control fungal growth may have broad, practical application in the future.
Assuntos
Ascomicetos/efeitos da radiação , Quirópteros/microbiologia , Metoxaleno/administração & dosagem , Micoses/veterinária , Fármacos Fotossensibilizantes/administração & dosagem , Terapia Ultravioleta , Animais , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/patogenicidade , Micoses/radioterapia , Esporos Fúngicos/patogenicidade , Esporos Fúngicos/efeitos da radiação , SíndromeRESUMO
The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos , Células Musculares/citologia , Simplexvirus/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Distrofina/genética , Inativação Gênica , Genes Reporter , Terapia Genética/métodos , Genoma , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Lentivirus/metabolismo , Camundongos , Músculos/citologia , Neurônios , Regiões Promotoras Genéticas , Ratos , Transdução Genética , Células VeroRESUMO
The glycoprotein D of HSV-2 (gD2) is currently a leading candidate vaccine target for genital herpes vaccines as both cellular and humoral responses can be generated against it. However, little is known about how vaccine composition will affect T cell epitope selection. A panel of 15-mer peptides (with 11 amino acid overlap) spanning full-length gD2 was used to investigate the fine specificity of T cell responses to gD2 as well as the role of vaccine composition on epitope selection. Spleen cells from BALB/c mice (H-2(d)) immunized with gD2, formulated with or without AlPO(4) and/or IL-12, were stimulated in vitro with overlapping gD2 peptides. Cellular responses (lymphoproliferation and IFN-gamma expression) were mapped to four epitopes within the gD2 molecule: gD2(49-63), gD2(105-119), gD2(245-259), and gD2(333-347). CTL analysis of these four epitopes indicated that not all of them could serve as a CTL epitope. Mice immunized with gD2 expressed from a viral vector mounted CTL responses primarily to one epitope located in the extracellular domain of gD2 (gD2(245-259)). More importantly, mice immunized with gD2 co-administered with IL-12 mounted CTL responses to an additional epitope located at the transmembrane-cytoplasmic junction of gD2 (gD2(333-347)). The location of this novel epitope emphasizes the benefit of using full-length versions of glycoproteins when designing vaccine components.
Assuntos
Antígenos CD4/metabolismo , Membrana Celular/imunologia , Citoplasma/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito T/biossíntese , Herpesvirus Humano 2/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Epitopos de Linfócito T/genética , Feminino , Herpesvirus Humano 2/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/metabolismo , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
The type of immune response elicited against HSV-2 infection may be a factor in the frequency and severity of recurrent disease, with non-recurrent status being associated with a Th1-like response. As administration of glycoprotein D subunit formulated with an aluminum-based adjuvant induces predominantly Th2-like immune responses, we sought to assess the ability of IL-12 to redirect anti-HSV immunity towards a Th1 response. Co-administration of gD with IL-12 resulted in gD-specific antibody subclass switching from predominantly IgG1 observed in mice immunized with either gD or gD/AlPO4 to a more balanced combination of IgG1 and IgG2a, and enhanced virus neutralizing activity. Spleen cells from mice immunized with gD and IL-12, and restimulated in vitro with HSV-2, developed into effector cells capable of secreting IFN-gamma and lysing HSV-2 infected targets, while those obtained from gD or gD/ALPO4 immunized mice did not express lytic activity. In vitro studies determined that these CTLs were CD4+ and that the cytotoxicity was primarily perforin dependent. Vaginal challenge with HSV-2 demonstrated that IL-12 co-administration with gD resulted in increased efficacy of this vaccine as compared to administration of gD antigen alone. This acquired protection persisted up to 1 year. Finally, adsorbing gD and IL-12 to AlPO4 decreased the optimal dose of IL-12 required to enhance gD immunogenicity and shift responses towards a Th1-like profile.