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MGY378 > Viral vectors (Dr. Kozak) > Flashcards

Viral vectors (Dr. Kozak) Flashcards

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1
Q

Why do we use virus as vectors?

A

able to mount a robust immune response –> long lasting

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2
Q

What needs to be balanced when making a viral vector?

A

i) amount of immune repsonse it produces
ii) attenuated virulence (cannot be too strong or else have symtpoms on ppl)

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3
Q

What happened in 1950s in viral vectors?

A

first attenuated viruses for polio and influenza for vaccine use

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4
Q

What happened in 1985 in viral vectors?

A

introduce foerign gene HbsAg to vaccinia virus

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5
Q

What happened in 1990s in viral vectors?

A

Develop DNA plasmid as vector

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6
Q

What happened in 2000s in viral vectors?

A

heterologous prime boosting, replicons, virus like particles (safer)

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7
Q

Things to consider when desgining a viral vector

A

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

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8
Q

Difference between human adenovirus and poxvirus

A

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

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9
Q

Similarities between human adenovirus and poxvirus as viral vector

A

both have broad tropism

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10
Q

Why we need to attenuate the virus before using as vector?

A

i) prevent disease (especially in immunocompromised grp)
ii) deleting genes can increase carrying capacity

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11
Q

What genes are usually removed in virus for attenuation?

A

genes that help evade or counteract host immune response

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12
Q

Traditional methods of attenuation

A

do lots of passages of virus in cell lines or animals
–> try to lower immune response through lots of passages

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13
Q

con of using traditional method of attenuation

A

time consuming, inefficient

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14
Q

example of using traditional method of attenuation

A

vaccinia virus unable to replicate in mammalian cells after more than 500 passages

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15
Q

Better method of attenuation

A

selectively target key genes through deletion, recombination or generating replicon-like system

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16
Q

Things to consider when doing attenuation

A

need to look into possible side effects of deleting a gene
–> may reduce immunogenicity

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17
Q

Example of possible side effects of deleting a gene

A

E3L in vaccinia –> inhibits interferon response
–> when removed, induce interferon response

BUT, low B cell response, less antibodies

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18
Q

Ways to deliver viral vectors

A

IV, oral, intramuscular, nasal, aerolisized…

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19
Q

Why veterinay vaccines preferred over human vaccine?

A

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…)

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20
Q

Common route of admin for veterinary vaccines

A

i) admin in food or drinking water
ii) aerosol spray
iii) in ovo (injected in eggs)

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21
Q

What happened to HIV vaccine trial for Adv-5?

A

increased risk of getting HIV after vaccination

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22
Q

Why did this happen in HIV vaccine trial for Adv-5?

A

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)

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23
Q

What are solutions to pre-existing immunity?

A

i) use novel vectors
ii) use rare serotypes
iii) use animal viruses as vector
iv) modify route of immunity

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24
Q

E.g. of using animal virus as vectors to help with pre-existing immunity

A

Chimp adenovirus in AZ covid 19 vaccine

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25
E.g. of using novel vector to help with pre-existing immunity
VSV
26
E.g. of rare serotypes to help with pre-existing immunity
HAdV, AAV
27
E.g. of virus more susceptible to pre-existing immunity
human adenovirus, herpesviruses
28
What does heterologous prime boosting mean?
combining two vaccines where on their own is not effective
29
Example of heterologous prime boosting
Thai HIV Vaccine trial --> efficacy 33% after 3 years
30
Examples of adenovirus vectors used as vaccines
i) Adv-5 --> Covid ii) Adv-26 --> Covid iii) ChAdv --> Covid, Ebola
31
What did researchers do to lower the risk of pre-existing immunity in Adv-5?
use alternate route of delivery
32
What is ChAdV used in vaccine?
Covid, Ebola
33
What is Adv-5 used in vaccine?
Covid
34
What is Adv-26 used in vaccine?
Covid
35
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
36
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
37
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
38
Why did Jesse Gelsinger pass away after receiving gene therapy?
massive inflammatory response to vector --> organ failure
39
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
40
Why does our immune system also attack viral vector?
unable to distinguish between viral infection and vector for treatment --> still trigger immune response
41
Where does adeno-associated viruses integrate into our genome?
locus AAVS1
42
Features of genome of adeno-associated viruses
i) flanked by 2 T-shaped inverted terminal repeats ii) single stranded
43
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
44
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
45
When does AAV integrate into host genome?
once enter nucleus, it uncoats and genetic material can integrate after turning into double stranded
46
When is AAV discovered?
1965-66
47
When is first FDA approval of using AAV for gene therapy?
2015
48
When is AAV vector first made for gene delivery?
1984
49
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
50
Strategies to overcome humoral immunity from viral capsid of AAV
i) do serological screening of patients ii) remove neutralizing antibody epitopes
51
Strategies to overcome innate immune response from AAV infection
i) modify genome by reducing GC content ii) include TLR silencing sequence
52
What vectors are used to treat cancer?
lentiviral vectors
53
Target of lentiviral vectors for cancer treatment
hematopoietic stem cells
54
What immune response is elicited thru lentiviral vectors?
tumor specific B and T cell response
55
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
56
An improvement made to adenoviral vectors
removing E1 --> inhibits viral replication --> improve transgene carry capacity
57
Challenges for lentiviral vector to help with cancer
i) induce a robust durable response ii) overcome tolerance to self-antigens
58
When are viruses for polio adn influenza attenuated and used as vaccine?
1950s
59
When are technologies like heterologous prime boosting, replicons and virus like particles developed?
2000s
60
When is the first time scientist introduced foreign genes (HbsAg) to vaccinia virus?
1985
61
When are DNA plasmids developed as vectors?
1990s
62
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
63
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
64
Ways that oncolytic virus help destroy tumor cells
i) direct lysing of tumor cells ii) activate immune system against the tumor
65
How is oncolytic virus better than immunotherapies?
it can target tumor independent of tumor antigen expression patterns
66
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
67
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
68
features of innate immune system for antiviral immunity
increase complement production
69
features of innate immune system for antitumor immunity
increase in tumor antigen presentation
70
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
71
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
72
Methods to reduce the strength of immune response
attenuate the oncolytic virus
73
What is the first FDA-approved oncolytic virus?
T-vec
74
What is T-vec used for?
treating nodal lesions in patients with recurrent melanoma --> those that cannot be removed with surgery
75
What is the oncolytic virus for T-vec?
Herpes simplex virus (HSV)
76
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)
77
Why is y34.5 deleted from HSV?
it activates host phosphatase to dephosphorylate eIF-2a --> continue translation when the host tries to stop it
78
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
79
Why is US12 deleted HSV?
it blocks peptides from antigen processing pathway
80
What happens after US12 gene is deleted?
tumor-associated peptides is presented by MHC-1
81
What happens after HSV is engineered to express GM-CSF?
chemokine promotes anti-tumor immune response
82
How to administer T-vec to patient?
directly into the tumor --> location of tumor important
83
Effects on HSV seropositive ppl vs HSV seronegative
response rate similar --> BUT seropositive ones have more severe response due to pre-existing immunity
84
What can we do to increase the efficacy of T-vec?
combine it with targeted immunotherapies
85
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
86
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
87
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
88
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
89
Example of modification on OV
oncolytic adenovirus removed E1B55kd gene --> unable to degrade p53
90
What happens after E1B55kd is deleted from oncolytic adenovirus
can better replicate in p53 deficient cancer cells
91
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
92
Examples of gene modified for cytokine delivery
GM-CSF, IFN, IL-12, IL-2
93
Examples of gene modified for presenting tumor related antigens to immune system
CEA, PSA
94
Examples of gene modified for immune checkpoint inhibitors
PD-1, CTLA4 (T cells)
95
Examples of gene modified for increasing tumor suppressor genes
p53
96
Examples of gene modified for anti-angiogenesis
anti VEGF antibody
97
Common ways to deliver oncolytic virus
IV, inside tumor
98
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)
99
What is tested for oncolytic potential for hepatocellular carcinoma?
ARV-PB1
100
Features of ARV-PB1
i) non-enveloped ii) ds iii) segmented genome
101
Phylogenetic analysis for ARB-PB1
distinct from human reovirus --> less likely to have pre-existing immunity
102
Features for ARV-PB1 infection
syncytia formation (for other non-human reoviruses as well)
103
How did ppl test for oncolytic activity of ARV-PB1?
measure cell viability using different cancer cell lines against the virus
104
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
105
How did ppl test for safety of ARV-PB1?
See if primary hepatocytes are able to clear out the OV
106
Results from safety testing of ARV-PB1
primary hepatocytes unable to support growth of virus -> innate immune response cleared the virus
107
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
108
Why OV have poor efficacy against solid tumor?
physical barrier of endothelial cell layer
109
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
110
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

MGY378 (13 decks)

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