RESUMO
Differences in fusion activity between measles virus (MV) attenuated, oncolytic strain MV(NSe) and pathogenic MV(wt323) are reflected in amino acid 94 of the fusion (F) proteins. A valine 94 in F(NSe) (naturally) or F(wt323) (introduced) correlated with enhanced cell-cell fusion activity during transient glycoprotein expression or recombinant MV infections irrespective of the strains' targeted receptors, whereas the reverse effect was found for methionine 94. Enhanced fusogenicity was determined by weaker glycoprotein interaction and correlated positively with cytotoxicity in both virus strains. Amino acid 94 of F can be used to tailor fusogenicity and cytotoxicity of recombinant MV, while the cellular receptor triggering fusion is not decisive.
Assuntos
Substituição de Aminoácidos , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Proteínas Virais de Fusão/genética , Internalização do Vírus , Animais , Fusão Celular , Sobrevivência Celular , Chlorocebus aethiops , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Células Vero , Proteínas Virais de Fusão/metabolismoRESUMO
Abstract Oncolytic viruses such as measles virus (MV) represent a new class of therapeutic agents that might help to overcome current limitations in cancer therapy. Although MV-based virotherapeutics already have entered clinical testing for various tumor entities, the preclinical safety of MV virotherapeutics so far has not been elucidated for particular regimens with high medical need, such as (1) direct injection into hepatic tumor sites, (2) employing high doses ibidem, and (3) concurrent usage of arming with cytotoxic genes required to further enhance oncolytic efficiency. Here, we assessed the safety and pharmacokinetics of suicide gene-armed vector MV-SCD when administered intrahepatically in two animal models, IFNAR(tm)-CD46(Ge) (interferon-α receptor deficient and CD46 MV receptor knock-in) transgenic mice and rhesus macaques (Macaca mulatta). Clinically, singular direct intrahepatic applications of MV-SCD were found to be well tolerated. Quantitative RT-PCR demonstrated the transient presence of viral RNA in various organs, whereas no shedding of infectious virus particles was observed at any time point. Histological analyses of organs did not exhibit adverse effects attributable to the test article. Blood parameters including liver enzymes revealed no deviations from normal. In both species an antiviral humoral immune response was mounted shortly after virus administration. Surprisingly, daily repeated systemic applications of MV-SCD under concomitant prodrug administration resulted in side effects in IFNAR(tm)-CD46(Ge) mice, but were less pronounced than in a 5-fluorouracil standard therapy control cohort. Taken together, these data indicate that "single shot" direct intrahepatic injections of MV-SCD in conjunction with systemic prodrug administration are safe and could be used in future virotherapeutic treatments of liver cancers.
Assuntos
Genes Transgênicos Suicidas/genética , Neoplasias Hepáticas/terapia , Vírus do Sarampo/genética , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Citocinas/metabolismo , Feminino , Fluoruracila/uso terapêutico , Técnicas de Introdução de Genes , Terapia Genética , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Macaca mulatta/metabolismo , Masculino , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , RNA Viral/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Distribuição TecidualRESUMO
BACKGROUND: Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx. METHODS AND FINDINGS: Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled. CONCLUSION: Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes.