Viral vectors (Dr. Kozak) Flashcards
Why do we use virus as vectors?
able to mount a robust immune response –> long lasting
What needs to be balanced when making a viral vector?
i) amount of immune repsonse it produces
ii) attenuated virulence (cannot be too strong or else have symtpoms on ppl)
What happened in 1950s in viral vectors?
first attenuated viruses for polio and influenza for vaccine use
What happened in 1985 in viral vectors?
introduce foerign gene HbsAg to vaccinia virus
What happened in 1990s in viral vectors?
Develop DNA plasmid as vector
What happened in 2000s in viral vectors?
heterologous prime boosting, replicons, virus like particles (safer)
Things to consider when desgining a viral vector
i) promoters for virus
ii) tropism and host receptor (entry mechanism)
iii) carrying capacity of foreign genes
iv) exposure in population
v) delivery to individuals
vi) attenuation
Difference between human adenovirus and poxvirus
i) Promoter: much more in Adv, only poxviral promoters in poxvirus
ii) Host receptor: Adv use Coxsackie adenovirus receptor, complex mech for poxvirus
iii) carrying capacity: much more (25kb) in poxvirus
iv) Exposure: high in Adv, low in poxvirus
Similarities between human adenovirus and poxvirus as viral vector
both have broad tropism
Why we need to attenuate the virus before using as vector?
i) prevent disease (especially in immunocompromised grp)
ii) deleting genes can increase carrying capacity
What genes are usually removed in virus for attenuation?
genes that help evade or counteract host immune response
Traditional methods of attenuation
do lots of passages of virus in cell lines or animals
–> try to lower immune response through lots of passages
con of using traditional method of attenuation
time consuming, inefficient
example of using traditional method of attenuation
vaccinia virus unable to replicate in mammalian cells after more than 500 passages
Better method of attenuation
selectively target key genes through deletion, recombination or generating replicon-like system
Things to consider when doing attenuation
need to look into possible side effects of deleting a gene
–> may reduce immunogenicity
Example of possible side effects of deleting a gene
E3L in vaccinia –> inhibits interferon response
–> when removed, induce interferon response
BUT, low B cell response, less antibodies
Ways to deliver viral vectors
IV, oral, intramuscular, nasal, aerolisized…
Why veterinay vaccines preferred over human vaccine?
i) lower safety concerns
ii) reduce disease spreading to human
iii) allow identification between vaccinated from infected animals
iv) easier route of administration (food, drinking water…)
Common route of admin for veterinary vaccines
i) admin in food or drinking water
ii) aerosol spray
iii) in ovo (injected in eggs)
What happened to HIV vaccine trial for Adv-5?
increased risk of getting HIV after vaccination
Why did this happen in HIV vaccine trial for Adv-5?
i) Pre-exposed to Adv-5
–> more target cells to infect + inflammatory environment to help
ii) uncircumcised (higher risk of HIV exposure)
iii) alteration to epithelial cell barrier with inflammatory response to vector (e.g. increase CCR5+ T cells)
What are solutions to pre-existing immunity?
i) use novel vectors
ii) use rare serotypes
iii) use animal viruses as vector
iv) modify route of immunity
E.g. of using animal virus as vectors to help with pre-existing immunity
Chimp adenovirus in AZ covid 19 vaccine
E.g. of using novel vector to help with pre-existing immunity
VSV
E.g. of rare serotypes to help with pre-existing immunity
HAdV, AAV
E.g. of virus more susceptible to pre-existing immunity
human adenovirus, herpesviruses
What does heterologous prime boosting mean?
combining two vaccines where on their own is not effective
Example of heterologous prime boosting
Thai HIV Vaccine trial
–> efficacy 33% after 3 years
Examples of adenovirus vectors used as vaccines
i) Adv-5 –> Covid
ii) Adv-26 –> Covid
iii) ChAdv –> Covid, Ebola
What did researchers do to lower the risk of pre-existing immunity in Adv-5?
use alternate route of delivery
What is ChAdV used in vaccine?
Covid, Ebola
What is Adv-5 used in vaccine?
Covid
What is Adv-26 used in vaccine?
Covid
Two ways to administer gene therapy
i) in vivo, by injecting viral vector through IV
ii) Ex vivo, by extracting patient’s cells, transform and grow in culture
–> then introduced back to patient
Things to consider when choosing vector for gene therapy
i) need long term gene expression
ii) tissue tropism
iii) carrying capacity
iv) potential for integration
v) toxicity
Describe the Jesse Gelsinger Story
- have disease in which she cannot break down ammonia in body
- died a couple of days after getting AdV vector with functional OTC gene
Why did Jesse Gelsinger pass away after receiving gene therapy?
massive inflammatory response to vector
–> organ failure
What went wrong in Jesse Gelsinger’s case?
i)informed consent not done properly
ii) didn’t tell family about experiments where animals died from therapy
iii) lead researcher had financial stake in therapy
iv) patient had too high ammonia levels
–> should be excluded
Why does our immune system also attack viral vector?
unable to distinguish between viral infection and vector for treatment
–> still trigger immune response
Where does adeno-associated viruses integrate into our genome?
locus AAVS1
Features of genome of adeno-associated viruses
i) flanked by 2 T-shaped inverted terminal repeats
ii) single stranded
Why is AAV a good vector?
i) only need one viral factor for replication (inverted genome repeats + promoter)
ii) ITR flanked genome can be cloned into plasmid + modified
iii) its lifecycle allow intro of novel genes into host cells
iv) use viral capsid to determine AAV tropism (helps gene therapy)
v) can replace viral genome with gene of interest
limitation of using AAV as viral vector
i) its capacity to carry genesis small (less than 5kb)
ii) re-administration might not be allowed as viral capsid generate humoral immunity
When does AAV integrate into host genome?
once enter nucleus, it uncoats and genetic material can integrate after turning into double stranded
When is AAV discovered?
1965-66
When is first FDA approval of using AAV for gene therapy?
2015
When is AAV vector first made for gene delivery?
1984
What immune response will AAV elicit once it enters out body?
i) humoral immunity from viral capsid (limit re-admin)
ii) activate innate immune response thru TLR2, 9
iii) weak T cell immunity
iv) antibodies against transgene
Strategies to overcome humoral immunity from viral capsid of AAV
i) do serological screening of patients
ii) remove neutralizing antibody epitopes
Strategies to overcome innate immune response from AAV infection
i) modify genome by reducing GC content
ii) include TLR silencing sequence
What vectors are used to treat cancer?
lentiviral vectors
Target of lentiviral vectors for cancer treatment
hematopoietic stem cells
What immune response is elicited thru lentiviral vectors?
tumor specific B and T cell response
How is lentiviral vectors used in cancer treatment?
i) lentivirus engineered to express T cell receptor genes
ii) transduced to hematopoietic stem cells
iii) induce tumor specific B and T cell reponse to direct immune system against tumor
An improvement made to adenoviral vectors
removing E1
–> inhibits viral replication
–> improve transgene carry capacity
Challenges for lentiviral vector to help with cancer
i) induce a robust durable response
ii) overcome tolerance to self-antigens
When are viruses for polio adn influenza attenuated and used as vaccine?
1950s
When are technologies like heterologous prime boosting, replicons and virus like particles developed?
2000s
When is the first time scientist introduced foreign genes (HbsAg) to vaccinia virus?
1985
When are DNA plasmids developed as vectors?
1990s
example of selectively targeting key genes for attenuation
deleting B15R gene in vaccinia
–> codes for soluble IL-1B receptor
increase vector specific CD8 T cell response
Why is adenovirus used as viral vector?
i) low pathogenicity (common to get infected)
ii) stable and safety (as it is DNA virus)
iii) don’t integrate into human genome
iv) able to generate strong immune response + infect diff. cell types
Ways that oncolytic virus help destroy tumor cells
i) direct lysing of tumor cells
ii) activate immune system against the tumor
How is oncolytic virus better than immunotherapies?
it can target tumor independent of tumor antigen expression patterns
What components of immune system does oncolytic virus induce?
i) NK cells thru secreting cytokines to activate them
–> kill tumor
ii) CD8+ T cells (cytokine or help with helper cells)
iii) activation antigen-presenting cells
Difference between antiviral and antitumor immunity
i) speed: quicker in antiviral
ii) neutralizing antibodies: increase in antiviral, decrease in antitumor
iii) T cells: increase antiviral T cells vs increase antitumor T cells
iv) signals: pro-inflammatory in antiviral, DAMP/PAMP in antitumor
features of innate immune system for antiviral immunity
increase complement production
features of innate immune system for antitumor immunity
increase in tumor antigen presentation
Balance is needed between what factors for oncolytic viruses?
i) immune response against the virus
–> will limit oncolytic efficiency
ii) activate immunity targets of tumor
What happens if immune response to oncolytic virus is too strong??
i) limit efficacy of oncolytic virus
ii) if too strong –> cytokine storm and toxicity
Methods to reduce the strength of immune response
attenuate the oncolytic virus
What is the first FDA-approved oncolytic virus?
T-vec
What is T-vec used for?
treating nodal lesions in patients with recurrent melanoma
–> those that cannot be removed with surgery
What is the oncolytic virus for T-vec?
Herpes simplex virus (HSV)
What are the gene edits done on HSV for T-vec?
i) delete y34.5 virulence gene
ii) delete US12
iii) express granulocyte macrophage colony stimulating factor (GM-CSF)
Why is y34.5 deleted from HSV?
it activates host phosphatase to dephosphorylate eIF-2a
–> continue translation when the host tries to stop it
What happens after y34.5 is deleted from HSV?
i) can stop translation
ii) promote transcription of viral gene US11
–> improve replication in tumor cells
Why is US12 deleted HSV?
it blocks peptides from antigen processing pathway
What happens after US12 gene is deleted?
tumor-associated peptides is presented by MHC-1
What happens after HSV is engineered to express GM-CSF?
chemokine promotes anti-tumor immune response
How to administer T-vec to patient?
directly into the tumor
–> location of tumor important
Effects on HSV seropositive ppl vs HSV seronegative
response rate similar
–> BUT seropositive ones have more severe response due to pre-existing immunity
What can we do to increase the efficacy of T-vec?
combine it with targeted immunotherapies
Factors to consider when choosing the optimal virus for cancer purposes
i) RNA virus vs DNA virus
ii) Enveloped vs non-enveloped
iii) Smaller virus vs larger
iv) viral tropism
v) possible modifications to the oncolytic virus
Difference between choosing RNA and DNA virus for OV
i) speed: RNA one kill tumor faster as they replicate in cytoplasm
ii) selective: DNA virus more selective than RNA ones
Difference between choosing enveloped and non-enveloped viruses
i) ability to kill tumor: higher in non-enveloped
ii) cleared by immune system: enveloped has a higher chance compared to non-enveloped
Difference between choosing small vs large viruses
i) enter the tumor: smaller ones can better diffuse into tumor
ii) carrying capacity: larger ones have more carrying capacity
Example of modification on OV
oncolytic adenovirus removed E1B55kd gene
–> unable to degrade p53
What happens after E1B55kd is deleted from oncolytic adenovirus
can better replicate in p53 deficient cancer cells
What are the strategies behind modifying oncolytic viruses?
i) increase cytokine delivery
ii) increase presentation of tumor associated antigens to immune system
iii) remove immune checkpoint inhibitors
iv) increase tumor suppressor genes
v) increase expression of anti-angiogenesis genes
Examples of gene modified for cytokine delivery
GM-CSF, IFN, IL-12, IL-2
Examples of gene modified for presenting tumor related antigens to immune system
CEA, PSA
Examples of gene modified for immune checkpoint inhibitors
PD-1, CTLA4 (T cells)
Examples of gene modified for increasing tumor suppressor genes
p53
Examples of gene modified for anti-angiogenesis
anti VEGF antibody
Common ways to deliver oncolytic virus
IV, inside tumor
Why are animal viruses considered for OV therapy?
i) rarely have pre-existing immunity
ii) great inducers of innate immunity (interferon)
iii) safe (no human infections)
What is tested for oncolytic potential for hepatocellular carcinoma?
ARV-PB1
Features of ARV-PB1
i) non-enveloped
ii) ds
iii) segmented genome
Phylogenetic analysis for ARB-PB1
distinct from human reovirus
–> less likely to have pre-existing immunity
Features for ARV-PB1 infection
syncytia formation (for other non-human reoviruses as well)
How did ppl test for oncolytic activity of ARV-PB1?
measure cell viability using different cancer cell lines against the virus
Results to oncolytic activity of ARV-PB1
virus able to grow well in these cancer cell lines
–> able to spread to other cancer cells in vivo
How did ppl test for safety of ARV-PB1?
See if primary hepatocytes are able to clear out the OV
Results from safety testing of ARV-PB1
primary hepatocytes unable to support growth of virus
-> innate immune response cleared the virus
Limitations to OV
i) poor efficacy to solid tumors
ii) poor vascular/lymphatic access to tumor
–> limit viral delivery + access
iii) Pre-existing immunity to OV
iv) solid tumor secrete immunosupressive cytokines
–> reduce immune cell recruitment
Why OV have poor efficacy against solid tumor?
physical barrier of endothelial cell layer
Challenges for OV therapy
i) need to be made in high titers to hv an effect
–> hard for lots of viruses
ii) hard to measure efficacy due to expansion of immune effectors aginst tumor
–> need long time
iii) lack understanding between virus dose, therapeutic response, toxicity
iv) lack understanding in viral biodistribution
v) Viral proliferation after admin may affect therapeutic effect
Regulatory challenges
i) lack validated clinical end pt or biomarkers
ii) hard to choose a control (Lack protocol for intra-tumoral agents)
iii) lack pre-clinical data showing major diff between viruses used as OV
–> hard to set up guidelines
