Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses

Abstract

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.

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

“…This has been tackled for HIV based vectors by use of an attenuated replication competent HIV-1 virus with HIV envelope, 29 or another attenuated HIV-1 virus that was transiently pseudotyped with VSV-G for the first round of infection, and subsequently amplified on an HIV-1 susceptible cell line. 30,31 However, both these approaches rely on the amplification of RCL in a human cell that expresses the receptor for HIV-1 envelope.…”

“…Other groups have recently included HEK293-based cells in strategies to devise a RCL assay for HIV-1 vectors for similar reasons. 29,32 In addition, as they are the parent of the production cells, it is highly likely that any RCL that can replicate in the production cells will also replicate in the test cells. We considered using equine cells, but as we did not pursue this option for multiple reasons.…”

Prolonged Adherence of Human Immunodeficiency Virus-Derived Vector Particles to Hematopoietic Target Cells Leads to Secondary Transduction In Vitro and In Vivo

“…In an effort to exclude gain of replication competency underlying the observed vector marking in recipients, we performed prior p24 (HIV Gag) testing on all vector lots by standard methods (34). In addition, we tested recipient peripheral blood samples from all animals involved in this study in duplicate assay samples and on two separate occasions, early after transplantation and within 2 weeks of sacrifice.…”

“…Each vector batch used was tested prior to use. In addition, for each transplanted animal, serum was obtained early after SCT and at sacrifice for determination of p24 (Gag) protein to exclude the presence of replication-competent virus (34). Samples were run in duplicate with a commercial assay (Perkin-Elmer, Boston, MA) according to the manufacturer's instructions, with a lower limit of detection at 4.3 pg/ml.…”

Generation of lentivirus vectors using recombinant baculoviruses

“…Cells were cultured for 4 weeks and concentration of p24 in the supernatant was repeatedly measured. An increasing concentration of p24 would indicate an ongoing viral replication, 29 but no such increase was detected. Media collected from the transduced HeLa cells after 2.5 weeks were further used to transduce naive HeLa cells but no GFP expression was detected neither with fluorescent microscopy nor a flow cytometer.…”