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1.
J Virol ; 90(11): 5343-5352, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27009956

RESUMO

UNLABELLED: Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. IMPORTANCE: Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, both in vitro and in vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.


Assuntos
Neoplasias Experimentais/terapia , Neoplasias Experimentais/virologia , Neoplasias/terapia , Vírus da Doença de Newcastle/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , DNA Intergênico/genética , Expressão Gênica , Terapia Genética , Humanos , Injeções Intravenosas , Camundongos , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus Oncolíticos/crescimento & desenvolvimento , Recombinação Genética , Genética Reversa/métodos , Replicação Viral/genética
2.
Biologicals ; 41(4): 247-53, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23665302

RESUMO

The cold-adapted (ca) live attenuated influenza vaccine (LAIV) strains are manufactured in embryonated hens' eggs. Recently, a clonal isolate from Madin Darby Canine Kidney (MDCK) cells was derived and characterized to assess its utility as a potential cell substrate for the manufacturing of LAIV [1]. Since MDCK cells are a transformed continuous cell line [2], and low levels of residual cellular components (DNA and protein) are found in the intermediates and final filled vaccine, we sought to characterize the uptake and clearance of MDCK DNA from tissues in order to assess theoretical risks associated with manufacturing LAIV in MDCK cell culture. In order to address this concern, MDCK DNA uptake and clearance studies were performed in Sprague Dawley rats. DNA extracted from MDCK Master Cell Bank (MCB) cells was administered via an intranasal (IN) or intramuscular (IM) route. Tissue distribution and clearance of MDCK DNA were then examined in fourteen selected tissue types at selected time points post-administration using a quantitative PCR assay specific for canine (SINE) DNA. Results from these studies demonstrate that the uptake and clearance of MDCK DNA from tissues vary depending on the route of administration. When DNA was administered intranasally, as compared to intramuscularly, detectable DNA levels were lower at all time points. Thus, the intranasal route of vaccine administration appears to reduce potential risk associated with residual host cell DNA that may be present in cell culture produced final vaccine products.


Assuntos
DNA/farmacocinética , Administração Intranasal , Animais , Galinhas , DNA/efeitos adversos , DNA/química , DNA/isolamento & purificação , DNA/farmacologia , Cães , Vacinas contra Influenza/isolamento & purificação , Vacinas contra Influenza/farmacologia , Injeções Intramusculares , Células Madin Darby de Rim Canino , Ratos , Vacinas Atenuadas/isolamento & purificação , Vacinas Atenuadas/farmacologia
3.
Vaccine ; 31(26): 2822-7, 2013 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-23602668

RESUMO

MEDI-534 is the first live vectored RSV vaccine candidate to be evaluated in seronegative children. It consists of the bovine parainfluenza virus type 3 (PIV3) genome with substituted human PIV3 F and HN glycoproteins engineered to express RSV F protein. A Phase 1 study of 49 healthy RSV and PIV3 seronegative children 6 to <24 months of age demonstrated an acceptable safety profile at the following doses: 10(4), 10(5) and 10(6)TCID50. After 3 doses of MEDI-534 at 10(6)TCID50, administered at 0, 2 and 4 month intervals, 100% of subjects seroresponded to PIV3, whereas only 50% seroresponded to RSV. To investigate the discordance in seroresponse rates, the RSV F transgene and its flanking non-coding nucleotides were sequenced from shed virus recovered from the nasal washes of 24 MEDI-534-vaccinated children. Eleven out of 24 samples contained no nucleotide changes in the analyzed region. The other 13 samples contained mixtures of variant subpopulations. Fifty-five percent exhibited changes in the transcription termination poly A gene sequences of the upstream bPIV3N gene while 21% had variant subpopulations in the RSV F open reading frame that resulted in pre-mature stop codons. Both types of changes are expected to reduce RSV F expression. Evaluation of the administered vaccine by dual immunofluorescence staining showed ~2.5% variants with low or no RSV F expression while single nucleotide primer extension detected ~1% variation at nucleotide 2045 that resulted in a pre-mature translational termination at codon 85. An association between shedding of variants and lower RSV F serological response was observed but it was not possible to establish a definitive clinical significance due to the small number of subjects in this study.


Assuntos
Vírus da Parainfluenza 3 Humana/genética , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/genética , Vacinas Virais/genética , Animais , Anticorpos Antivirais/sangue , Bovinos , Estudos de Coortes , Humanos , Lactente , Vírus da Parainfluenza 3 Bovina/genética , Vírus da Parainfluenza 3 Bovina/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Análise de Sequência de DNA , Transgenes , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Eliminação de Partículas Virais
4.
Biologicals ; 41(3): 201-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23313102

RESUMO

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.


Assuntos
DNA/análise , Eletroforese Capilar/métodos , Vacinas Virais/biossíntese , Animais , Linhagem Celular , Chlorocebus aethiops , DNA/genética , Fluorescência , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/genética , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Vacinas contra Vírus Sincicial Respiratório/biossíntese , Vacinas contra Vírus Sincicial Respiratório/genética , Células Vero , Vacinas Virais/genética
5.
PDA J Pharm Sci Technol ; 66(5): 445-52, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23035028

RESUMO

Sensitive and timely detection of bioburden in presterile filtration product in aseptic processing of biologics is a critical parameter for microbial control and assurance of final product sterility. An application of automated flow cytometry system was developed for rapid microbial assessment and in-process control in vaccine virus production. In order to minimize the background signal caused by the components of the chicken egg substrate sample matrix, a sample processing method to clear somatic cell debris was included. The sample processing and the automated analysis take approximately 5 to 7 min per test sample and the method provides objective results in real time, enabling uninterrupted processing. The flow cytometry method was compared with the standard aerobic plate count method using tryptic soy agar in a parallel study of 1566 independent production-scale samples. The method was further characterized by spike recovery of five model bacterial organisms in representative sample matrix. In comparison to the culture method, the flow cytometry method was shown to be 96.2% sensitive and 98.2% specific for the detection of bioburden at a level of sensitivity suitable for the process stage requirement with the advantage of a nearly instantaneous time to result. LAY ABSTRACT: In-process bioburden control in the manufacturing of biopharmaceuticals is essential for final product sterility and integrity. In manufacturing contexts where an in-process hold time is infeasible or in cases where uninterrupted processing is desired, conventional culture-based bioburden detection methods cannot be used, as they require significant time to results that may not fit within the time constraints. In this case study we demonstrate the use of flow cytometry as an alternative rapid method that provides real-time results to enable uninterrupted processing.


Assuntos
Filtração , Citometria de Fluxo , Produtos Biológicos , Meios de Cultura , Análise de Alimentos , Embalagem de Produtos , Vacinas
6.
PLoS One ; 7(12): e52327, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23284988

RESUMO

Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log(2) lower) or superior (>1 log(2) higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.


Assuntos
Anticorpos Neutralizantes/análise , Vírus da Influenza A/imunologia , Influenza Humana/virologia , Animais , Células CACO-2 , Linhagem Celular , Cães , Humanos
7.
Antiviral Res ; 72(1): 1-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16730076

RESUMO

Endoplasmic reticulum (ER) alpha-glucosidase inhibitors block the trimming of N-linked glycosylation and thus prevent the production of several viruses. The present study investigates the antiviral effects of the alpha-glucosidase and alpha-mannosidase inhibitors (castanospermine, 1-deoxynojirimycin, bromoconduritol, deoxymannojirimycin and swainsonine) on human parainfluenza virus type 3 (HPIV3). The alpha-glucosidase inhibitors (castanospermine, 1-deoxynojirimycin) in recombinant expression systems reduced the surface and intracellular expression of both HPIV3 F and HN proteins. On the other hand, alpha-mannosidase inhibitors prevented processing of the oligosaccharides on HPIV3 glycoproteins into the complex form. Consequently, alpha-glycosidase inhibitors (castanospermine and 1-deoxynojirimycin) significantly inhibited viral fusion activity. We demonstrated that the alpha-glucosidase inhibitors (castanospermine and 1-deoxynojirimycin) reduced the infectivity of newly released viral particles. We postulate that alpha-glucosidase inhibitors can prevent the first steps of HPIV3 envelope glycoprotein processing and that the inhibition of glucose trimming has antiviral effects.


Assuntos
Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glicosilação/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , alfa-Glucosidases/farmacologia , alfa-Manosidase/farmacologia , Animais , Linhagem Celular , Inibidores de Glicosídeo Hidrolases , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Vírus da Parainfluenza 3 Humana/química , Vírus da Parainfluenza 3 Humana/enzimologia , Vírus da Parainfluenza 3 Humana/fisiologia , Proteínas Recombinantes/biossíntese , Transfecção , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Replicação Viral , alfa-Manosidase/antagonistas & inibidores
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