Lentiviral Vectors

Background

Lentiviruses are a subset of retroviruses, which are enveloped single-stranded RNA viruses. Retroviruses are transmitted through direct exposure to bodily fluids, percutaneous exposures, or sexual contact. Lentiviruses can integrate into host chromosomes, like all retroviruses, to infect non-dividing cells to infect non-dividing cells, and have high mutation rates. These viruses cause multi-organ diseases characterized by long incubation periods, immune evasion, and persistent infections in their natural hosts. Acute infection with human lentiviruses can appear as non-specific “flu-like” and “mononucleosis-like” symptoms, including myalgia, diarrhea, nausea, vomiting, headache, weight loss, and neurological symptoms.

Types

Lentiviral vector systems can include viruses of non-human/non-primate origin (feline immunodeficiency virus, equine infectious anemia virus) as well as simian viruses (simian immunodeficiency virus) and human immunodeficiency viruses (HIV).

Pseudotyping

Replacement of the lentiviral envelope glycoprotein with a heterologous envelope, such as Vesicular stomatitis virus-g protein (VSV-g), provides a broad host range for the vector in terms of host and cell type transmissibility.

Vector Systems

Most of the lentiviral vectors presently in use are HIV-derived vectors. The cis- and trans-acting factors of lentiviruses are often on separate plasmid vectors, with packaging being provided in trans. Second-generation systems use two helper plasmids (one with gag, pol, rev, and tat; one with envelope protein) in addition to the transgene plasmid. Third-generation systems offer more safety features, including the use of three or more helper plasmids to separate the packaging and gene transfer functions in addition to the transgene plasmid. These systems are self-inactivating and do not include tat. Fourth generation systems further split gag and pol onto separate plasmids to reduce recombination, while adding back some HIV genes to enhance transduction efficiency and transgene expression. The IBC strongly encourages the use of third or fourth-generation systems where possible.

Recombination

Third and fourth-generation vectors have been designed to significantly diminish the possibility for recombination to occur resulting in a wild-type infectious virus. The potential for generation of replication-competent lentivirus (RCL) from HIV-1-based lentivirus vectors depends upon several factors, the most important of which are:
 

• the number of recombination events necessary to reassemble a replication competent virus genome
• the number of essential genes that have been deleted from the vector/packaging system

Earlier vector systems such as second-generation vectors (three-plasmid vector systems) may have a higher potential for generation of RCL.

Biosafety Level

All lentiviral work requires approval from the Institutional Biosafety Committee (IBC).  The biosafety level associated with lentiviral work is BSL-2 for non-human viruses, non-human pseudotyped viruses, or viruses that do not express transgenes with any known oncogenic potential or biological toxin; all work done in biosafety cabinet and use of safety lids when centrifugation occurs.

Enhanced BSL-2 is recommended for human viruses, amphotropic or VSV-g envelope pseudotyped viruses expressing transgenes with known oncogenic potential or a biological toxin. Enhanced BSL-2 containment means working with BSL-3 practices and procedures. Enhanced BSL-2 containment means working with BSL-3 practices and procedures, including all work in the biosafety cabinet, increased donning of PPE (2nd pair nitrile gloves, closed front disposable gown, mucous membrane protection), special attention to sharps use, and disinfection of all pipettes and other materials inside of a BSC.

Animal Biosafety Level

 All animal procedures require pre-approval by the OSU Institutional Animal Care and Use Committee (IACUC). Injections of rodents must be performed in a biosafety cabinet at ABSL-2 with appropriate PPE. ABSL-2 housing is required post-injection/exposure of animals. Animal bedding/cages are autoclaved from ABSL-2 animals.

For rodents that do not or will not contain any human cells or tissues, and the lentiviral vector used in the studies has been engineered to make the risk to animal care staff and researchers very minimal (replication incompetent) AHSP posting is required for the first 7 days, and then the posting may be removed. It is the responsibility of the researcher to review the AHSP and request a change in duration. The IBC may raise containment if deemed necessary.

Disinfection/Deactivation

10% bleach (there are other alternatives however it must be written in the IBC protocol and approved by the IBC for use)

Be safe today and remain a Buckeye tomorrow.