1. Viral Vector Training

2. Viral Vectors Can be used as gene delivery systems
Can also be used for human gene therapy
All work with viral vectors must be registered with the campus Institutional Biosafety Committee (IBC)
Prokaryotic or Eukaryotic viruses:
Recombinant prokaryotic viruses (bacteriophages) must be registered with IBC
Eukaryotic viruses present biohazard concerns, which is the focus of this training

3. Eukaryotic Viral Vectors (e.g., Adenovirus, Lentivirus)
Narrow or wide host range
Flexibility in the type of transgene that is delivered
Easily produced in the laboratory

4. Common Eukaryotic Viral Vectors
Adeno-associated Virus
Adenovirus
Retrovirus
Includes Lentivirus, MMLV, HIV or SIV replication, incompetent viruses
Herpes Virus
Vaccinia Virus

5. Production of Viral Vectors
Construction of recombinant vector with transgene(s) of interest
Transfection of plasmids (number of plasmids differ) into host cell (typically HEK293 cells) to package recombinant viral genome
Virus collected and used for infection of animal, cell, gene therapy, etc.
HEK293 cells are human cells (requiring BSL-2 practices), and require Hepatitis B vaccination or proof of immunity

6. Biosafety Concerns Require Risk Assessment
Risk Assessment considers the potential for the following risks which pose a hazard to laboratory staff which include:
Generation of Replication Competent Viruses (RCV)
Infection of unintended target cells
Insertional mutagenesis/oncogenic potential
Inappropriate expression of gene product
Germ-line transfer of genes
Rescue by other human pathogenic viruses

7. Risk Assessment Risk Assessments include:
Hazard Characteristics of Agent
Hazard Characteristics of Laboratory Procedures
Hazard Potential associated with work practices, safety equipment & facility safeguards
Determination of appropriate Biosafety Level (BSL) & any extra precautions
Risks for infection are DIMINISHED by the nature of the vector system (and its safety features)
OR;
EXACERBATED by the nature of the transgene insert encoded by the vector!.

8. Risk Assessment Summary
Biosafety Considerations
Higher Risk
Lower Risk
Vector Replication
Replication competent
Replication incompetent
Vector Design
Vector packaging functions on two plasmids
Viral genes present & expressed
Vector and packaging functions separated onto multiple (3+) plasmids
Viral genes deleted
Transgene
Oncogene, Toxin encoding, Tumor Suppressor
Non – oncogene, structural gene
Vector Generation
Large Scale
Laboratory Scale
Animal Host
Permissive host
Animal engrafted with human cells
Non-permissive host
Animal Manipulation
Vector administration (e.g., use of sharps during injection)
Housing and husbandry (no use of sharps)

9. Risks Associated with Viral Vectors:
Rescue of Replication Deficient Viruses
by Superinfection with Wild Viruses
Viral DNA
Gene of Interest
Virus
Wild
Virus
Cell’s DNA
Target Cell
Complementation
The genome from the wild virus provides the missing proteins needed for the viral vector to replicate. The superinfected cell functions similarly to a packaging line.

10. Risks Associated with Viral Vectors:
Rescue of Replication Deficient Viruses
by Superinfection with Wild Viruses
Virus
Wild
Virus
Viral DNA
Gene of Interest
Cell’s DNA
Target Cell
Unlikely to be HIV (due to gene deletions); theoretical risk is pseudo-typed VSV-g replication-competent virus bearing gene of interest and able to infect dividing and non-dividing cells.
Recombination
The genome from the wild virus randomly recombines with the viral vector, providing sufficient genetic material for the viral vector to replicate. The resulting rescued virus may possess pieces of the original insert gene. The viral genome is impossible to predict due to random recombination. The virus may exhibit altered virulence.

11. to increased cancer risk.
Risks Associated with Viral Vectors:
Insertional Mutagenesis
Viral DNA
Gene of Interest
Virus
Host Cell DNA
Target Cell
Proto-Oncogene
Oncogene
Random integration of viral genome may disrupt endogenous host genes. Of special concern is disruption of proto-oncogenes, which can lead
to increased cancer risk.

12. Viral Pseudotyping: A Double-Edged Sword
Tropism
The ability of a virus to infect a particular type of host cell
Psuedotyping
Altering the viral envelope protein to alter tropism, thus allowing the virus to infect cells it originally could not, typically VSV-G envelope is used

13. Viral Envelope Protein Receptor for Viral Envelope
Viral Pseudotyping: A Double-Edged Sword
Tropism
Host Range
Viral Envelope Protein
Receptor for Viral Envelope
Ecotropic
Mouse/Rat (narrow host range)
Gap70
mCAT-1
Amphotropic / Dualtropic
Mammals (wider host range)
4070A / 10A1
Ram-1 / GALV
Pantropic
All Animals
VSV-G
Phosphotidyl serine
Phosphotidyl inositol
GM3 ganglioside
Special care should be used when working with pantropic
or amphotropic viruses which can infect humans!

14. Adeno-Associated Virus (AAV)
Icosahedral, enveloped, ssDNA virus
Requires a helper virus to replicate
Typically Adenovirus, Herpesvirus or Vaccinia
Able to stably insert DNA into host chromosome, and remain latent in the absence of helper virus
Infectious to humans with no known disease association
May be transmitted by aerosol, droplet exposure to mucous membrane, injection and ingestion

15. AAV Vector Characteristics
Limited cloning capacity
Multi-plasmid packaging system
Ability to be produced in high titers
Ability to infect broad range of cells
Long term, stable expression from randomly integrated sequences
Replication in the presence of wild type (WT) AAV or helper virus
BSL-1 without helper virus, BSL-2 with helper virus or when working with human cells

16. Specific Risks for AAV Vectors
Insertional mutagenesis
Increased risk when using helper virus
Increased risk when gene of interest is an oncogene
Latent infection

17. Adenovirus Non-enveloped, icosahedral dsDNA
49 immunologically distinct types
Infectious through respiratory, mucous membranes, eye & gastrointestinal routes
Replication deficient strains can cause respiratory inflammation, corneal injury & conjunctival damage

18. Adenovirus Vector Characteristics
Vector capacity kb
Wide host range, including humans
Most used are replication deficient, by way of E1a and E1b deletion
Packaged using HEK293 cells
BSL2 recommended for in vitro and in vivo use

19. Specific Risks for Adenovirus Vectors
Formation of replication competent viruses
Increased risk when gene of interest is an oncogene or biotoxic material
Inflammation
Latentcy
Recombination with vector and natural Adenovirus

20. Retroviruses Enveloped, ssRNA virus
Able to inject into host DNA and become latent viruses
Host range determined by envelope proteins
Able to infect both proliferating & non-proliferating cells
Include ecotropic, amphotropic & pseudotyped viruses
BSL2 recommended for in vitro and in vivo use

21. Retroviral Vector Characteristics
Vector capacity: 8kb
Most common:
Lentivirus
MMLV
HIV/ SIV (replication incompetent forms)
Often psuedotyped with VSV-G
Multiple plasmid packaging systems
More plasmids = less risk (e.g. a 4 plasmid systems are better than 2 plasmid systems, less recombination risk)

22. 2 Plasmid System
2 plasmid systems present safety concerns due to the increased risk of recombination from homologous recombination resulting in a replication competent virus
Helper: all structural proteins needed to package new virus within the packaging cell line; packaging sequence deleted
Vector: gene of interest and packaging sequence – lacks structural genes needed to form a replicative competent virus in the host cell.
Construct efficient at delivery of the transgene but was shown to be replication competent via several recombination events in the packaging cell – double crossover event resulted in packaging sequence in vector plasmid to join helper plasmid (containing structural proteins).
Earlier generation vector systems (MMLV) used a 2 plasmid system:
Helper: all structural proteins needed to package new virus within the packaging cell line and the packaging sequence deleted

23. HIV – an upgrade in retroviral vectors
3 & 4 Plasmid Systems
Spread the genomes of the helper plasmid into multiple plasmids which would require multiple replication events to form a replicative competent virus More plasmids= less risk
HIV – an upgrade in retroviral vectors
Further separating the structural proteins of the helper plasmid.
HIV has many accessory genes required for replication – removal makes recombination event less likely

24. Specific Risks for Retroviral Vectors
Replication competent viruses
Recombination with WT viruses to form replication competent strains
Insertional Mutagenesis
Activation of endogenous sequences
Increased risk if the gene of interest is an oncogene
Latent infection

25. Herpes Virus Icosehedral, ds DNA virus
Two immunologically distinct types - HSV1 and HSV2
Vectors are typically replication deficient due to deletions in viral genome
Wide host range and cell tropism
Establishes latent infection indefinitely in post-mitotic neurons
Useful for nervous system applications

26. Specific Risks for Herpes Vectors
Insertional mutagenesis
Recombination that will result in a replication competent/ infectious particle
Viral infection resulting in illness for replication competent vectors
Latent infection

27. Vaccinia Virus dsDNA virus - member of poxviridae family
Wild type virus can replicate in enucleated cells
Vaccinia is a human pathogen, causing severe disease in immunocompromised and some healthy individuals
Virus is the component of the smallpox vaccination
Can cause infection through ingestion, parenteral injection, absorption through broken skin, droplet or aerosol exposure
Vaccination is available for laboratory workers
Replication competent strains available
Mutated with decreased pathogenicity

28. Vaccinia Vector Characteristics
Can hold large amount (30 kb) of foreign DNA, stably inserted into genome for efficient replication and expression in host cells
Can infect all mammalian cells
Most are replication competent
One variant, MVA, can grow only in avian cells and can remain in cytoplasm
Other variants mutated to prevent infection, targeted to specific cells within organism

29. Specific Risks for Vaccinia Vectors
Replication competent viruses
Potential for viral infection resulting in illness, especially in immuno-compromised subjects
A vaccination for vaccinia virus is available.
Occupational Health Services can provide additional information & counseling regarding its safety & protection for laboratory workers

30. Ways to Minimize Exposure
Engineering Controls:
Use of available technology and devices to isolate hazards from the worker
e.g., Biosafety cabinets (BSC), safer needle devices, puncture-resistant sharps containers
Administrative Controls:
Standard Operating Procedures, Exposure Control Plan, Biosafety Manual
e.g., Controls to monitor compliance, provide accessibility of control methods, investigate exposures to prevent future occurrences

31. Ways to Minimize Exposure
Work Practice Controls:
Manner in which task is performed to reduce exposure
e.g., Wash hands after removal of gloves; disposal of needles without recapping; no lab coats outside of lab
PPE (Personal Protective Equipment):
Specialized clothing or equipment used to protect workers from exposure
e.g., lab coats, gloves, face shields, eye protection, fluid resistant aprons, head and foot coverings

32. Engineering Controls
The following MUST be used when working with viral vectors:
Biological Safety Cabinet (Class II)
Chemical disinfectant traps with vacuum line HEPA filters
Sharps containers & “safe needle” devices
Centrifuge safety devices
Specimen transport containers
Replace glass with plastic

33. Engineering Controls Biosafety Cabinets (aka BSC, Tissue Culture Hood)
All work with viral vectors, infection of animals, handling infected animals, animal necropsy, cage changing, etc. MUST be performed inside a certified, Class II, biosafety cabinet
Click to view video on this topic

34. Working Inside a BSC
Allow cabinet to run for min before starting work
Check magnahelic gauge to be sure hood is functioning properly (compare with number on annual certification sticker)
Disinfect surfaces (including equipment)
Cover work surface with disinfectant-soaked towel
Place materials as far into cabinet as possible
Work Clean to Dirty

35. Working Inside a BSC (Cont’d)
Work “clean to dirty”
Use horizontal pipette trays and interior biohazard containers
Disinfect spills with appropriate disinfectant
Do not place items on the front grill or block the back grill
Prevent turbulence when working in the BSC, use slow and deliberate motions when moving hands out of cabinet
Work Clean to Dirty

36. The pressure readings on the sticker MUST match the gauge!
Certification
Required annually!
Contracted outside vendors certify biosafety cabinets annually
Filters are tested for leaks
Air flow is verified
Vibration, lighting, etc.
Also should be certified when moved or repaired
The pressure readings on the sticker MUST match the gauge!

37. The pressure readings on the sticker MUST match the gauge!
Repairs
Do NOT use the cabinet if it is malfunctioning (e.g.: noise, vibration, or the pressure gauge reads no pressure/ too much pressure)
Physical Plant does NOT perform repairs. Certified vendors must be contacted by your department
Some repairs will require decontamination of the cabinet
The pressure readings on the sticker MUST match the gauge!

38. More on BSCs
Absorbent Pad covering the grill. Nothing should be placed on or covering the grill
Items placed on the grill. Again, nothing should be placed on the grill

39. Filtered Vacuum Lines for Liquid Waste
Flask for liquid waste MUST have appropriate disinfectant
No hazardous chemicals to be used with vacuum flasks
Overflow flask is recommended
All vacuum lines MUST have HEPA filters

40. Centrifuge Safety Cups
Centrifuge safety cups or sealed rotors must be used when working with viral vectors They are to be loaded and unloaded in the biological safety cabinet

41. Work Practices
Decontaminate all waste (autoclave or chemical disinfectant)
No “sharps” (needles, glass Pasteur pipettes) may be used with these cultures unless approved by the Institutional Biosafety Committee
Use plastic aspiration pipets
Do not use “sharps” to harvest virus pellet
All sharps MUST be properly disposed in a sharps container
For experiments requiring needles- safer devices MUST be considered and are recommended

42. Work Practices (Cont’d)
Access to the laboratory should be limited or controlled
Viral vector work is NOT permitted on the open bench
A biosafety cabinet must be used for all manipulations including (but not limited to):
Pipetting
Harvesting infected cells
Loading and opening containers
Initial delivery of vector to animals
Handling of infected animals

43. Work Practice Controls
No eating, drinking, smoking, applying cosmetics, or handling contact lenses
No food or drink storage in the lab
Minimize production of droplets or aerosols
Transport specimens in secondary containment
Use mechanical pipetters
Decontaminate equipment after use
Use universal precautions:
Treat everything as if it is infectious!!

44. Work Practice Controls (Cont’d)
Biohazard labels must be placed to indicate each area where viral vectors are used / stored:
biosafety cabinets
Incubators
Centrifuge
Refrigerators
laboratory entrance doors
Waste containers

45. Animal Studies
Some animal systems are not permissive hosts and do not support replication competent viruses. These are safer systems, but all animals infected with viral vectors should be handled using ABSL-2 procedures
The initial delivery of vector is performed under ABSL-2 containment

46. Animal Studies (Cont’d)
All infected animals are to be manipulated in a certified BSC
Ventilated or filtered bonnet cages are required for housing
All cages must be changed in BSC
All carcasses and bedding must be autoclaved or chemically treated before disposal
Signage posted on room to indicate infected animals, and the vector of infection

47. Personal Protective Equipment
Use of the following personal protective equipment is required to reduce the potential for exposure:
Gloves
Lab Coats
Safety eyewear
Disposable gowns (animal work)
Other PPE as determined by the IBC

48. Disinfection & Waste Disposal
Most effective germicides for viral vectors are:
1% sodium hypochlorite (bleach)
2% glutaraldehyde
5% phenol
All waste generated MUST be autoclaved or chemically disinfected PRIOR TO disposal in regulated medical waste bins (red bag)

49. Autoclaving Autoclaves: Time, Pressure, Heat
Pressure vessels that use saturated steam under a pressure of approximately 15 psi to achieve a chamber temperature of a least 121°C (250°F) for a minimum of 30 minutes

50. Work Practices - Autoclaves
Use autoclave bags (regular plastic bags melt!)
Do not overload bags
Ensure bag is partially open to allow steam to penetrate the contents
Use appropriate secondary container for autoclaving and transporting the bag:
Plastic: Polypropylene pans preferred over:
Polyethylene
polystyrene
Stainless steel: durable & a good conductor of heat

51. Work Practices – Autoclaves (Cont’d)
Autoclave Indicators used to validate decontamination
Chemical indicators change color after being exposed to 121°C (250°F), but they have no time factor!
Tape indicators can ONLY be used to verify that the autoclave has reached normal operating temperatures for decontamination
Biological indicators are designed to demonstrate that an autoclave is capable of killing microorganisms
A load test using Geobacillus stearothermophilus should be performed monthly

52. Testing for Replication Competent Viruses (RCV)
Test producer cells and vector stocks periodically for the presence of RCV
If obtaining the viral vector from a commercial source, please check the manufacturer’s information as to the quality control concerning replication competent viruses
Information as to the methods and frequency for checking viral vectors for RCV should be included with the IBC application

53. Testing for Replication Competent Viruses (RCV) (Cont’d)
Adenoassociated Virus:
No helper virus: Not required
Helper virus used: Every viral preparation must be tested for the presence of adenovirus prior to in vitro or in vivo use
Heat inactivate viral preparations for 15 minutes at 56⁰C, test for RCV by plaque assay or cytopathic effect
Hehir, KM, Armentano, D, Cardoaz, LM, et al “Molecular characterization of replication-competent
variants of adenovirus vectors and genome modifications to prevent their occurrence”. J. Virol. 70:

54. Potential for Replication Competent Viruses
Adenovirus:
Replication competent viruses can be produced upon successive amplification. These viruses are produced when adenoviral DNA recombines with E1-containing DNA in HEK293 cells
The E1a assay can be used to check for RCV and must be done before in vitro or in vivo use. The vector stock should be tested at a limit of sensitivity of 1 in 106 virus particles compared to known positive control
Zhang WW, Kock, PE, Roth, JA “Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.” Biotechniques. 18:

55. Potential for Replication Competent Viruses
Retrovirus - Test every 6 months, for 1 infectious unit per mL
Retrovirus (ecotropic & amphotropic)
Amplification in permissive cell lines, and screening by appropriate assay (i.e. PG-4S+L- or marker rescue)
Forestell, SP, Nando, JS, Bohnlein, E and Rigg, RJ Improved detection of replication competent retrovirus. J Virol Methods. 60:
Wilson, CA, Ng TH, and Miller AE Evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors. Human Gene Therapy. 8(7):
Lentivirus
Serial transfer and by ELISA for p24 antigen
Marker rescue assay
Dull, T, Zufferey, R, Kelly M, mandel, RJ, Nguyen M, Trono D, Naldini L A third generation lentivirus vector with a conditional packaging system. J Virol. 72:
Murine Retrovirus - Marker rescue assay, PERT, PG3S+L- or infectivity RT-PCR assays

56. Potential for Replication Competent Viruses
Herpesvirus
Viral preparations should be tested every 6 months for RCV by plaque assay
These assays should be tested at a sensitivity level of 1 infectious unit per mL
For in vivo work, viral preparations should be tested before each use by plaque assay
Strathdee CA, McLeod, MR “A modular set of helper dependent simplex virus expression vectors.”
Mol Ther. 5:

57. Potential for Replication Competent Viruses
Vaccinia virus
Testing is not required since replicating viruses are used

58. Institutional Biosafety Committee Review of Viral Vector Protocols
The NIH rDNA guidelines indicate that the IBC is responsible for performing a risk assessment of rDNA work and will determine the appropriate biosafety level (BSL)
Major considerations to the BSL for viral vector work:
Potential human tropism of the vector
Potential pathogenic effects of expressed transgene

59. Institutional Biosafety Committee Review of Viral Vector Protocols
It is the responsibility of the protocol applicant to provide enough information to the IBC to justify WHY a particular vector should be used at BSL-2 and not BSL-3, particularly in the cases in which the transgene is potentially oncogenic or immunosuppressive to humans

60. Standard Operating Procedures
EOHSS has developed standard operating procedures for working with viral vectors, which includes the information in this training The signature page must be signed by all those working with the viruses in the lab AND the Principal Investigator
Adenoassociated Viruses Adenovirus Retroviruses Herpes Virus Vaccinia Virus

61. EOHSS Contact Information
Director:
Marta Figueroa, MS, CIH
Newark/ Scotch Plains Biosafety:
Jessica McCormick, Ph.D. RBP
Tamara McNair, MS
Brian Eggert, MPH
Piscataway/ New Brunswick Biosafety:
Tracy Pfromm, MPH
Camden/ Stratford Biosafety:
Tom Boyle, MS, RBP

62. We’re on the Web! ready.umdnj.edu http://emergency.umdnj.edu

64. References
Braun, A “Biosafety in Handling Gene Transfer Vectors.” Current Protocols in Human Genetics
CDC-BMBL, 5th ed.,
Dull, T, Zufferey, R, Kelly M, mandel, RJ, Nguyen M, Trono D, Naldini L A third generation lentivirus vector with a conditional packaging system. J Virol. 72:
Environmental Health and Safety. The University of Iowa, “ Adeno-Associated Virus and Adeno-Associated Viral Vectors”
Forestell, SP, Nando, JS, Bohnlein, E and Rigg, RJ Improved detection of replication competent retrovirus. J Virol Methods. 60:  
Hazardous and Radioactive Waste Disposal Standard Operating Procedure, Comparative Medicine Resources
Hehir, KM, Armentano, D, Cardoaz, LM, et al “Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence”. J. Virol. 70:
MSDS Health Canada

65. References
NCI-Fredrick Safetygram (ISM-193, April 2001):
Strathdee CA, McLeod, MR “A modular set of helper dependent simplex virus expression vectors.” Mol Ther. 5:
Stanford University, “Working with Viral Vectors,”
University of Texas Health Science Center at Houston “Guidelines for the Safe Handling of Adenoviral Vectors in Laboratory, Animal and Human Experiments”
Wilson, CA, Ng TH, and Miller AE Evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors. Human Gene Therapy. 8(7):
Young, L.S., Searle, P.F., Onion, D., and V. Mautner “Viral gene therapy strategies: from basic science to clinical application.” J. of Pathology. 208:  
Zhang WW, Kock, PE, Roth, JA “Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.” Biotechniques. 18:

66. Completed Training! Let’s take the Quiz!
Thank you for taking the time to complete the:
UMDNJ Viral Vector Online Training
Let’s take the Quiz!