CMB1003/L16 Viruses as Tools Flashcards
Give 2 useful properties of viruses that we can exploit.
Natural gene-delivery vehicle
Trigger appropriate immune response
Many have cytopathic effect
What does the term ‘attenuated’ mean?
Human viruses that have lost the properties to replicate efficiently in human cells
Give 2 powerful promoters/enhancers.
Human cytomegalovirus (HCMV)
Simian virus 40 (SV40)
Human immunodeficiency virus LTRs
How can viruses be used for gene expression?
A promoter region from a virus cloned into a DNA plasmid vector
Generate cDNA of protein wish to express from mRNA using PCR
How are viruses used to express genes? (4)
Isolate ORF of interest
Clone into expression vector
Isolate/purify plasmid DNA
Transfect target cells & detect gene expression
What is a VLP and why are they used as vectors?
A virus-like particle
Allow more efficient deliver than chemical/liposomal transfection
Give 2 advantages to adenoviral vectors.
Small genome, easy to work with & purify
Will infect many cell types, high level of gene expression
Give 2 disadvantages to adenoviral vectors.
Limited to ORFs <8kb
Immunogenic, means single shot
Transient expression
Describe adenovirus replication in the lab. (2)
Most of late phase ORF are removed
Replace viral protein ORFs with ORF/protein of interest
What is modified in adenoviral vectors before use? (3)
Missing E1 gene region (replication)
Missing all viral proteins
Require a ‘helper’ or ‘packaging’ cell-line
Give 2 advantages to lentiviral vectors.
Small genome, easy to work with
Will infect many cell types, high level of gene expression
Infect/express in non-dividing cells
Stable gene expression over time, gene integrates into genome of target cell
Give 2 disadvantages of lentiviral vectors.
Limited to ORFs <10kb
Safety concerned if transgene inserts near a proto-oncogene
Constitutive expression
Give 3 different types of vaccine.
Live
Killed whole virus
Subunit vaccines
Gene delivery
Describe the synthesis of attenuated vaccines. (4)
Pathogenic virus isolated and cultured on host cells
Virus incubated on cells from another host
Virus spontaneously mutates and grows on (monkey) cells
Virus can be used as a vaccine as it cannot grow on human cells
Give 3 advantages to attenuated vaccines.
Mimic wildtype infection
Strong humoral & cellular responses
Induces innate immunity
Fewer doses required for complete immunity
Give 3 disadvantages to attenuated vaccines.
Reversion can occur to wildtype
Cannot be used in immunosuppressed individuals
Required careful handling/storage (low temps)
Define gene therapy.
Replacement of a defective gene function by introducing a ‘normal’ gene
What is an oncolytic virus?
Virus which destroys tumour cells
What is an oncogenic virus?
Virus which promotes development of tumour cells
What can occur once a tumour cell lyses and releases oncolytic viruses (oncolytic virotherapy)? (3)
Destruction of tumour microenvironment
Infect more tumour cells
Systemic anti-tumour immune response
Give 3 barriers to oncolytic therapy.
Overcoming vector neutralisation
Enriching T cell response
Increasing speed
Enhancing tumour uptake
Give 3 advantages to phage therapy.
Specific to a bacteria spp.
Can evolve and adapt to resistance
Self-amplifying until all bacteria are dead
Give 3 disadvantages to phage therapy.
Lysis of Gram -ve bacteria, release of endotoxins (sepsis)
Theoretical potential to transfer DNA between bacteria (Ab resistance)
May need cocktail of phage
Individualised
What are lysins/endolysins?
Enzymes produced by some bacteriophages to cleave host cell wall during final stage of lytic cycle
What is phage therapy used for? (2)
Treat burn infections
Treatment of biofilms
