Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses.
A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection approximately 10-100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity approximately 5-50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi-gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi-gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.
Department of Microbiology and Immunology, Indianapolis, Indiana 46202, USA.
Lakshmi
L
Sastry
Yi
Y
Xu
Terry
T
Johnson
Kunal
K
Desai
David
D
Rissing
Jonathan
J
Marsh
Kenneth
K
Cornetta
D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D015683:Gene Products, gag / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011786:Quality Control; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics
C028391:G protein, vesicular stomatitis virus; D015683:Gene Products, gag; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins