Introduction to Viral Vector SafetyAmanda Haley, Ph.D., RBP, MRSB

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OBJECTIVESAt the end of this course you should be able to:• Be able to understand the need to risk assess modifications of

viral vectors.• Identify viral vectors commonly used in research.• Understand the regulatory reIdentify the hazards associated

with using viral vectors.• quirements for working with viral vectors.

This course assumes a basic understanding of virology and viral infections.

INTRODUCTION• Viruses are highly efficient at transfecting their own nucleic acid

into a host cell.• Viral vectors have a greater chance of delivering genetic

information compared to traditional transfection methods.• Commercialization has increased the ease of obtaining vectors

for research.What is a viral vector?

A viral genome with deletions in some or all essential genes with, or without, the insertion of a transgene.

BASIC RISK ASSESSMENT FOR VIRAL VECTORS

• Understanding Biosafety Levels and Risk Groups• Cell tropism• Transgene risks• Vector safety features• Stability

BIOSAFETY LEVEL vs RISK GROUP

BIOSAFETY LEVEL• 4 Biosafety Levels.• Identify the protective measures

in a laboratory to protect workers and the environment.

• Defined by a combination of equipment, practices and lab design.

• Biosafety Levels are determined by risk assessment.

RISK GROUPS• 4 Risk Groups defined by the

National Institutes for Health (NIH).

• Describe the relative hazard posed by an agent.

• Primary basis of a biosafety risk assessment.

• Risk Group does not always match Biosafety Level.

RISK GROUPS• RISK GROUP 1 (RG1) – Agents not associated with disease in healthy adult

humans. Low or no risk to an individual or community.

• RISK GROUP 2 (RG2) – Agents associated with human disease which is rarely serious for which preventative or therapeutic options are often available. Moderate risk to an individual but low risk to the community.

• RISK GROUP 3 (RG3) – Agents associated with serious or lethal disease in humans for which preventative or therapeutic measures may be available. High risk to the individual but low risk to the community.

• RISK GROUP 4 (RG4) – Agents likely to cause serious or lethal disease in humans for which preventative or therapeutic measures are not usually available. High risk to the individual and the community.

BASIC RISK ASSESSMENT EXAMPLE

HIV (RG3)

10ml cultures of low concentration virus

Biosafety Level 2

• HIV is a Risk Group 3 organism.• How can an experiment change the Biosafety Level?

HIV (RG3)

10L cultures of high concentration virus

Biosafety Level 3

BASIC RISK ASSESSMENT EXAMPLE• So what changed?• The risk group of HIV is set at RG3 by NIH.• Scenario 1 – Volume and concentration of virus is low therefore

the risk to the worker is low. Biosafety Level 2 would be appropriate.

• Scenario 2 – Volume and concentration of virus is high therefore the risk to the worker is greater in the event of an incident. Biosafety Level 3 provides more protective control measures (PPE, engineering controls, practices and procedures).

RISK GROUPS AND VIRAL VECTORS• A viral vector has genes removed from the wild-type parent virus to

allow for insertion of a transgene and to increase safety.• Modifications generally make the vector unable to replicate.• Viral vectors may have the ability to revert to the original form if the

deleted genes are regained.• The Risk Group of the wild-type parent virus is important in

determining the Biosafety Level of the research however also consider the following:

• Cell tropism• Transgene• Potential for reversion• Vector Safety Features

CELL TROPISM• Vectors have a natural cell target that they

can infect.• Attachment is mediated by surface

structures.• Host cell ranges may be limited or broad.• Host cell targets may be altered by a

transgene.• Altering surface proteins to change cell

tropism is referred to as pseudotyping.

CELL TROPISM• Viral Vectors fall into 3 basic categories:• Ecotropic

• Able to infect only murine (mice and rat) cells.• Amphotrophic

• Able to infect mammalian cells including human cells.

• Pantropic• Able to infect cells of any species.

• Greater care should be taken when working with amphotropic and pantrophicviruses

TRANSGENE• Viral vectors may not be able to replicate however they can still

infect an individual and transfect the gene they are carrying!• The transgene is essential in determining risk.• Can your gene alter the host cell’s normal cell cycle?

• Growth Factors• Kinases• Transcription Factors• Oncogenes – genes which can cause cancer

• Use of any of these gene types should be carefully risk assessed to protect workers.

REPLICATION COMPETENCY• Viral vectors may be derived from a pathogenic wild-type parent

with deletions to reduce the risk of using them.• Most common deletion is of genes involved in replication.• The viral vector may be able to re-acquire these genes from:

• Plasmids• Helper virus• Packaging cell lines • Wild-type virus present in the host (this includes a worker exposed

to the vector).

REPLICATION COMPETENCY• Viral vectors are engineered to decrease the ability to become

replication competent by:• Increasing the number of recombination events needed to

become replication competent• Genes can be split between multiple constructs.

• Producing viral vectors in a single, simultaneous transfection of plasmids.

• Decreases the chance of acquiring genes from packaging cell lines through multiple transfections.

VIRAL STABILITY• Viral stability can be affected by whether they are:

• Enveloped – acquire a lipid envelope by exiting the cell through budding.

• Non-enveloped – do not have a lipid envelope and exit the cell through cell lysis.

• Alcohol is effective for inactivating enveloped viruses.• Bleach should be considered for use with non-enveloped virus.

COMMON VIRAL VECTORS USED IN

RESEARCH

RETROVIRUSES• Alpharetrovirus

• ALV, RSV• Betaretrovirus

• MMTV• Gammaretrovirus

• MLV, FeLV, SNV• Deltaretrovirus

• HTLV I and II

• Epsilonretrovirus• WDSV

• Lentivirus• HIV

• Spumavirus• HFV, SFV

RETROVIRIDAE - LENTIVIRUS• Lentiviral vectors are one of the most common viral vectors

used in research.• Lentiviral vectors are usually created using Human

Immunodeficiency Virus 1 (HIV) as the backbone.• Four generations of HIV vectors are available

• Generation 3 and 4 vectors have more safety features engineered in.• Extra safety measures should be considered when using Generation 1

and 2 vectors.• Knowing the generation of your vector is essential in

determining safety measures for research.

HAZARDS ASSOCIATED WITH LENTIVIRAL (HIV) VECTORS

• Amphotropic – can infect human cells.• High mutation rate.• High transduction efficiencies.• Capable of recombination.• Nature of the transgene present.• Risk of interaction with endogenous retroviruses in the host.• Seroconversion of workers following an exposure.

ADENOVIRUSES• Mastadenovirus

• Human adenovirus A-F• (approximately 50 identified

serotypes in humans)

HAZARDS ASSOCIATED WITH ADENOVIRAL VECTORS

• Amphotropic – can infect human cells.• Can be transmitted by aerosolization.• Recombination with wild-type strains.• Difficult to detect an exposure in workers.• Potential for contamination with a helper virus.• Viral recombination during production.

ADENO ASSOCIATED VIRUSES (AAV)• Parvoviridae

• Parvovirus – dogs, cats, mink• Erythrovirus – human• Dependovirus – human (needs

an AAV helper virus)

HAZARDS ASSOCIATED WITH ADENO ASSOCIATED VIRAL VECTORS

• Potential for insertional mutagenesis.• Deletions and rearrangements may occur during integration.• Multiple copies can integrate.• Reactivation.• Herpesvirus, adenovirus and poxvirus can act as helper viruses

for AAV.

REGULATORY REQUIREMENTS FOR

WORKING WITH VIRAL VECTORS

NIH GUIDELINES• All work with viral vectors and transgenes is subject to the NIH

Guidelines and review by the Institutional Biosafety Committee (IBC).

• No research with viral vectors can be conducted without approval from the IBC.

• The following slides detail the sections of the Guide that may be applicable to viral vector research.

NIH GUIDELINES – SECTION III-D-3• Experiments involving the use of infectious DNA or RNA viruses or

defective DNA or RNA viruses in the presence of helper virus in tissue culture systems.

• III-D-3-a: Experiments involving the use of infectious or defective Risk Group 2 viruses in the presence of helper virus may be conducted at BSL-2

• III-D-3-b: Experiments involving the use of infectious or defective Risk Group 3 viruses in the presence of helper virus may be conducted at BSL-3

• III-D-3-c: Experiments involving the use of infectious or defective Risk Group 4 viruses in the presence of helper virus may be conducted at BSL-4

NIH GUIDELINES – SECTION III-D-3• III-D-3-d: Experiments involving the use of infectious or

defective restricted poxviruses in the presence of a helper virus shall be determined on a case-by-case basis by NIH/OBA review. These experiments must be reviewed directly by NIH before work can begin. Grant review is not sufficient.

• III-D-3-e: Experiments involving the use of infectious or defective viruses in the presence of helper virus not covered in in Sections III-D-3-a through III-D-3-d may be conducted at BSL-1.

NIH GUIDELINES – SECTION III-E-1• Experiments involving the formation of Recombinant DNA

molecules containing no more than two-thirds of the genome of any eukaryotic virus.

CONCLUSIONSWork with viral vectors can be done safely if you:• Understand the hazards of the parent strain when working with

the vector strain.• Understand how your vector was generated.• Know the potential risks posed by your transgene, particularly if

it’s function is not fully understood.• Do not assume the Biosafety Level without conducting a full risk

assessment and obtaining approval from the IBC.• Report any potential or overt exposures to the Biosafety Officer.

RESOURCES• NIH Guidelines - https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.html• NIH Lentivirus Guidance Document - https://osp.od.nih.gov/wp-

content/uploads/Lenti_Containment_Guidance.pdf• Centers for Disease Control• American Biosafety Association• Wright State University• Dartmouth College• University of Cinncinati