Chimpanzee Adenovirus Vaccine Carriers
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Example 1
Isolation, Cloning, Sequencing and Characterization of ChAds Chimpanzee Adenovirus Isolation
[0159]Stool specimens were collected in viral transport medium (VTM; Microtest M4-R Multi-Microbe Transport Medium, Remel Inc.) then frozen or frozen directly at −70° C. at NIRC (New Iberia Research Center 4401 W. Admiral Doyle Drive New Iberia, La. 70560). The specimens were kept frozen at <−70° C. until they were processed for inoculation into cell cultures. At that time, the specimens were thawed and then vortexed in excess of chilled viral transport medium. After the specimens had dissociated into suspensions, they were centrifuged for 10 min at 1500-1800 rpm. The supernatants were filtered through 0.8 and 0.2 μm syringe filters in series and then the filtered material was inoculated into cell cultures (200-250 μL into shell vials and 250-300 μL into tube cultures). Each processed specimen was inoculated into tube cultures and shell vial cultures seeded with 293 cells or A549 cells.
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Example 2
ChAd Shuttle Vector and Expression Vector Construction and Rescue Vector Construction and Rescue
[0173]Genomic viral DNA was cloned into a standard plasmid vector by homologous recombination with an appropriate shuttle vector containing viral DNA sequences derived from both left and right end of viral genome (FIG. 2). As described more fully below, the sequence homology observed between viruses classified in the same serotype subgroup was exploited to develop group-specific shuttle vectors. Genomic viral DNA of Chimp adenovirus classified into subgroup D and E resulted to be sufficiently homologous to allow the construction of a common shuttle vector in order to clone viruses belonging to both subgroups.
Construction of a Subgroup D / E Shuttle Vector
[0174]The ChAd6 viral genome was fully sequenced (SEQ ID NO: 2) and the information obtained was used to construct a shuttle vector to facilitate cloning by homologous recombination of subgroup D and E chimpanzee adenovirus.
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Example 3
Neutralization Studies
[0184]Neutralization assays were carried out in order to evaluate the prevalence in human sera of neutralizing antibodies against the chimpanzee adenoviruses disclosed herein. The assay evaluated the effects of serum preincubation on the ability of chimp adenoviruses carrying the gene for secreted alkaline phosphatase (SEAP) to transduce human 293 cells. The neutralization titer is defined as the dilution of serum giving a 50% reduction of the SEAP activity observed in the positive control with the virus alone.
[0185]From 2×106 to 1.5×107 physical particles of CV33-SEAP, CV32-SEAP and ChAd3-SEAP vector were diluted in 100 μl of complete medium and added to an equal volume of human or chimp serum diluted in complete medium. Each serum samples was tested at various dilutions (five 4-fold increments starting from 1 / 18 dilution through 1:4608). Samples were pre-incubated for one hour at 37° C. and then added to 293 cells seeded into 96-well plates (3×104 cel
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