Antiviral Agents / Virotherapy

Question 1

While antiviral ______ are the classical approaches, antiviral _________ is an upcoming field of research and development for the treatment of infectious diseases.

Answer 1

Drugs; Immunotherapy

Question 2

For which viral infections do we rely on antiviral drugs?

Answer 2

For those that cannot be effectively controlled via public health measures and vaccines.

Question 3

(T/F) To date, most antiviral drugs are targeted against HIV and herpesvirus due to their clinical significance.

Answer 3

True!

Millions of lives have been saved by the use of antiviral drugs!

Question 4

What are the three major limitations in antiviral drug development?

Answer 4

1) Requirement for a high degree of SAFETY (must ensure compound does not have deleterious effects on host cell)

2) Requirement for POTENCY (modest viral growth in the presence of inhibitor is NOT good enough; allows for mutants)

3) Some viruses difficult to PROPAGATE IN VITRO in the lab or there may be a LACK of animal models to test safety + potency

Question 5

What does R and D mean for R&D of antiviral drugs?

Answer 5

R = Research and compound identification (only the beginning of the process of producing a drug)

D = Development, compromises all steps necessary to take an antiviral lead compound through safety testing, scale-up of synthesis, formulation, pharmacokinetic studies, and clinical trials

Takes 5-15 years!

Question 6

Briefly describe the descending staircase of antiviral drug discovery.

Answer 6

First you have thousands of compounds synthesized or purified from the library.

Then you have preclinical testing where you test potency and toxicity in cells and then animals.

Finally, you have clinical testing where you test toxicity and potency in humans.

In each step, the numbers of promising candidates are decreasing, resulting in a descending staircase.

It takes several years before a final product that is safe and effective is produced!

Question 7

Clinical trials occur in a series of phases. Match the phases to their definitions.

1) Phase I
2) Phase II
3) Phase III
4) Phase IV

A) focus on EFFECTIVENESS, slightly large groups (<100), still continue to monitor safety

B) once the drug is approved; further evaluation of EFFECTIVENESS and LONG TERM SAFETY, groups of hundreds to thousands

C) SAFETY assessment in small groups (20-80), early evidence of efficacy

D) focus on EFFECTIVENESS, SIDE EFFECTS compared to standard therapy, larger groups (100-thousands) who are randomly assigned to “trail arms”

Answer 7

Phase I: SAFETY assessment in small groups (20-80), early evidence of efficacy

Phase II: focus on EFFECTIVENESS, slightly large groups (<100), still continue to monitor safety

Phase III: focus on EFFECTIVENESS, SIDE EFFECTS compared to standard therapy, larger groups (100-thousands) who are randomly assigned to “trial arms”

Phase IV: once the drug is approved; further evaluation of EFFECTIVENESS and LONG TERM SAFETY, groups of hundreds to thousands

Question 8

True or False

1) It takes around 10-15 years to discover a drug. We can fast track certain drugs using phase I-II (safety + efficacy in the same phase).

2) Pharmaceutical/biotech companies pay for clinical trials in collaboration with clinical investigators.

3) Phase I and Phase II take around 1 year altogether.

Answer 8

1) True

2) True

3) False; 2-4 years

Question 9

How are antiviral compounds discovered?

Answer 9

Knowledge of viral life cycle IDENTIFIES TARGET for antiviral drug discovery:

The life cycle of many viruses are known, allowing for identification of several targets for intervention.

Viral genes essential for reproduction can be cloned and expressed in genetically tractable organisms and their products can be purified and analyzed in molecular and anatomical detail - INHIBITORS OF CRITICAL PROCESSES CAN BE IDENITFIED

Question 10

What are some of the challenges/things to consider after a drug target is identified?

Answer 10

Must determine if compound:

1) will get to the right place at the right concentration (bioavailability)

2) will persist in the body long enough to be effective (pharmacokinetics)

3) will be tolerated or toxic

Question 11

Differentiate mechanism-based screens with cell-based screens.

Answer 11

Mechanism-based screens: seeks to identify compounds that affect the function of a known viral target such as enzymes, transcriptional activators, cell surface receptors, ion channels. often screening is conducted with purified protein in formats that facilitate automated assays of many samples.

Cell-based screens: elements of the mechanism to be inhibited are engineered into an appropriate cell system. provides information not only about target inhibition, but also cytotoxicity & specificity. the use of multiple reporter molecules may also allow for detection for more than one event at a time.

Question 12

Briefly describe a mechanism based screen for inhibitors of a viral protease.

Answer 12

A FLUOROGENIC MOLECULE is covalently linked to the N-terminus of the substrate (peptide) of the protease and the C-terminus is linked to a bead.

When the protease cleaves the peptide, the fluorogenic N-terminus is released into the soluble fraction which is separated from the insoluble beads containing C-terminus fragment.

Protease activity is assayed by the appearance of soluble fluorescent peptide.

Fluorescence activity if enzyme is NOT inhibited, while there will no fluorescence activity when enzyme is inhibited.

Question 13

Briefly describe a cell based screen for inhibitors of a viral protease.

Answer 13

Tetracycline-resistant bacteria have tetracycline efflux protein on the membrane with a HIV protease site added.

When there is expression of a (HIV) protease, there is inactivation of the tetracycline efflux protein.

Inactivation causes the bacteria to be tetracycline sensitive! It leads to no bacterial growth on tetracycline-containing media.

A functional inhibitor should result in antibiotic resistant bacteria as the protease site would not be cleaved!

Question 14

(T/F) Cell based screens can be done in eukaryotic cells. However, bacterial cells are faster and cheaper!

Answer 14

True!

Question 15

Advances in protein separation techniques, _____ _______ and bioinformatics allow to determine the total protein repertoire aka ________ of virus samples.

We are now able to determine protein interactions of _____ proteins with _____ proteins, identifying critical nodes or _________ ____.

Answer 15

Mass spectrometry; proteome

viral; host; INTERACTION HUBS

*an example of this is the systematic survey of the interaction between cellular and viral proteins of HIV I. these interactions provide clues of potential targets for drugs!

Question 16

(T/F) After administration, the drug must reach the correct site in the body and must remain at an effective concentration to allow for the inhibition of viral reproduction.

Answer 16

True!

Question 17

How can we modify compounds to improve absorption? Give an example.

Answer 17

Add side chains!

ACYCLOVIR is not effectively taken up after oral ingestion. But the derivative VALACYCLOVIR has 5x oral bioavailability through the addition of a side group, which allows increased passage of drug from the intestines into the bloodstream..

Question 18

Match the following drugs to their definitions:

1) Acyclovir (Zovirax)

2) AZT (Retrovir)

3) Amantadine (Symmetrel)

4) Tamiflu

A) Targets INFLUENZA A virus M2 protein which forms a tetramer that creates a transmembrane ion channel to transport protons. By binding to the outside of the tetramer, it blocks the entry of protons.

B) Specific non-toxic drug - effective against HERPES virus. Nucleoside analog related to guanosine. Mediates chain termination of DNA synthesis.

C) Blocks the function of Neuraminidase, an INFLUENZA enzyme that cleaves sialic acid group from glycoproteins to release virion from host cell.

D) First drug licensed for the treatment of AIDS. Analog of thymidine. Mediates chain termination of DNA synthesis.

Answer 18

Acyclovir: Specific non-toxic drug - effective against HERPES virus. Nucleoside analog related to guanosine. Mediates chain termination of DNA synthesis.

AZT: First drug licensed for the treatment of AIDS. Analog of thymidine. Mediates chain termination of DNA synthesis.

Amantadine: Targets INFLUENZA A virus M2 protein which forms a tetramer that creates a transmembrane ion channel to transport protons. By binding to the outside of the tetramer, it blocks the entry of protons.

Tamiflu: Blocks the function of Neuraminidase, an INFLUENZA enzyme that cleaves sialic acid group from glycoproteins to release virion from host cell.

Question 19

Two major success stories of antiviral drug development: ______ and _________.

Answer 19

HIV; Hepatitis C

*Steps in the reproduction of HIV and HCV are targeted by antiviral drugs.

Question 20

What is immunotherapy?

Answer 20

Therapeutic strategies to stimulate or restore the ability of the patient’s immune system to fight disease (mimick immune system). These therapies normally employ biological products, known as BIOLOGICAL RESPONSE MODIFIERS.

Question 21

What are the different kinds of immunotherapies?

Answer 21

Cytokines (interferon-α) and antibodies (super-antibodies) which are PASSIVE immunotherapy.

Lymphocytes (T cells) which are ACTIVE immunotherapy.

Question 22

(T/F) When cells get infected with pathogens, they will secrete signalling molecules called CYTOKINES to neighbouring cells to notify them of infection, and also to train and boost the immune system.

Answer 22

True!

Question 23

The cytokine, interferon-α, was the first approved therapy for _________ virus and _______ virus.

The effects on _______ infection are potent, while on ________ are modest.

Answer 23

Hepatitis B; Hepatitis C

Question 24

Immunomodulatory agents, such as ___________, which can stimulate the T cell responses or ________ which stimulates Natural Killer (NK) cells, are being investigated for their ability to reduce _____ ____ and/or moderate _______ of persistent infections (HPV/HIV-1)

Answer 24

Interferon-α; Interleukin-12; viral load; complications

Question 25

What are the limitations to cytokine therapy?

Answer 25

1) Biological activity is generally short (not very stable)
2) High side effects (flu-like symptoms, bone marrow suppression, autoimmune disease)
3) Expensive

Question 26

1) What are monoclonal antibodies?

2) How long have they been in use? Are they common?

3) What are their limitations?

Answer 26

1) Monoclonal antibodies are laboratory produced molecules engineered to serve as “substitute” antibodies. They are generated by B-cell clones. They RECOGNIZE and NEUTRALIZE antigens.

2) They have been used for more than 1000 years for the treatment + prevention of infectious diseases but a single anti-viral antibody, the Respiratory syncytial virus (RSV) specific antibody is in widespread clinical use.

3) High cost; Difficulties in administration; Belief that antibodies are effective only in prophylactic (preventive) settings - which are already achieved by the cheaper vaccines.

Question 27

What are super-antibodies?

Answer 27

Super-antibodies are a new generation of highly potent and/or broadly cross-reactive human monoclonal antibodies.

They are typically generated infrequently and/or in a limited number of individuals during natural infections.

Isolation of these has been achieved by large-scale screening for suitable donors and new single B cell approaches to human monoclonal antibody generation.

Question 28

What are the potential advantages of super antibodies?

Answer 28

1) Higher potency (decreased cost, low treatment dosages, different administration routes, extended half-life)

2) Higher cross-reactivity (could target multiple viruses of a given family with a single antibody)

Question 29

What are the current approaches for Viral-specific T cells antiviral immunotherapy?

Answer 29

1) Banking T cells from donors who have already experienced certain viral infections

2) Training or genetically engineering virus-naive T cells taken from unexposed tissues. These approaches could make antiviral cellular immunotherapy more robust and more widely used.

Question 30

(T/F) The clinical data looks promising to use donor T cells as antiviral therapies for bone marrow transplant patients who are vulnerable to infections from common viruses.

Answer 30

True!

Question 31

What are the limitations of T-cell immunotherapy?

Answer 31

1) Very expensive
2) Not possible for a massive outbreak

Question 32

What is virotherapy? What are the two arms of virotherapy?

Answer 32

Virotherapy is a treatment using biotechnology to convert viruses into therapeutic agents by selecting or reprogramming viruses to treat diseases.

The two arms of virotherapy:
1) Viral vectors for gene therapy
2) Cancer-killing (oncolytic) viruses

Question 33

What is gene therapy?

Answer 33

Gene therapy is the process of removing, modifying, or replacing defective genes with healthy ones.

Though it has been around 45 years, it is still being developed as treatments are rarely making past the clinical trial stages.

Question 34

Recombinant, ___________ _____ vectors were the first molecular tool enabling efficient, nontoxic gene transfer into human somatic cells.

__________ and _________ virus have shown the most clinical promise and are the carriers (vectors) of several recently approved gene therapies.

Answer 34

Replication-defective viral vectors

Retroviruses; Adeno-associated virus (AAV)

*we can’t use just any virus for gene therapy! we have to select certain viruses.

Question 35

Gene therapy using an adenovirus vector can be used to treat or cure certain genetic diseases in which a patient has a defective gene.

What are the mechanisms of gene therapy?

Answer 35

1) Gene insertion: a new version of gene is introduced into the treated cell

2) Gene modification: an exciting gene is modified

3) Gene surgery/removal: a particular gene is excised and may also be replaced by its healthy counterpart

*modify viral vector to express healthy gene of interest
*virions infect cells + deliver the gene

Question 36

Adeno-associated viral vectors are engineered from a ________, _______ parvovirus that is naturally replication-defective. This means wildtype AAV requires another virus such as _______ or a ________ to replicate.

All viral coding sequences of AAVs are ______ with a gene expression cassette of interest.

Answer 36

Non-pathogenic; non-enveloped; adenovirus; herpesvirus

Replaced

Question 37

What are some limitations of AAV vectors?

Answer 37

1) They can not package more than ~5 kb of DNA.

2) They are predominantly NON-INTEGRATING. This lessens risks related to integration but also LIMITS long-term expression from AAV vectors to long-lived post-mitotic cells.

Question 38

Early AAV trials established safety but were limited by insufficient dosing, and anti-AAV immune responses, most likely because many people carry neutralizing antibodies and memory T cells directed against the AAV capsid.

How was this overcome?

Answer 38

1) Increase dosage

2) Use recombinant vectors that allow them to be not sensitive to the neutralizing vectors

Question 39

What are γ-retroviral vectors?

Answer 39

These vectors were used in 1st generation clinical trials to DELIVER A NORMAL COPY of a specific defective gene into the genome of T cells or HSCs from patients with immunodeficiency or blood cancers.

Question 40

What were the second generation of retroviral vectors? How are they different from the first generation?

Answer 40

LENTIVIRUSES and SPUMAVIRUSES were the second generations of retroviral vectors.

In contrast to γ-retroviral vectors, lentiviral vectors enabled gene transfer into NON-DIVIDING cells but still left quiescent G0 cells out of reach.

Lentiviral vectors also can carry larger and more complex gene cassettes than γ-retroviral vectors and thus development provided a critical advance for HEMOGLOBINOPATHIES.

Question 41

Briefly answer the following questions regarding retroviral vectors.

1) Which vectors are currently the tools of choice for most HSC applications?

2) What is another approach that decreases the risk of genotoxicity?

Answer 41

1) Lentiviral vectors

2) Removal of endogenous strong enhancer from lentiviral and γ-retroviral vectors is another approach that decreases the risk of genotoxicity.

Question 42

What must happen to a virus before being used to carry therapeutic genes into human cells?

Answer 42

It is modified to remove its ability to cause infectious diseases (disease-causing gene is excised).

Question 43

Briefly answer the following questions regarding AAVs:

1) What technique is used to produce recombinant adeno-associated virus (rAAV) vectors?

2) What is one of the central challenges of rAAV production?

Answer 43

1) rAAV vectors are produced using the triple transfection technique.

2) One of the central challenges of rAAV production is the need for a “HELPER VIRUS” to facilitate replication within a host mammalian cell.

The combined activity of the adenovirus-derived genes on the helper plasmid with the rAAV genes on the second plasmid proved sufficient to generate functional rAAV particles containing the transgene from the third plasmid.

Question 44

Gene therapy can be used to modify cells inside or outside the body.

Differentiate between ex vivo (cell-based delivery) and in vivo (direct delivery).

Answer 44

Cell-based delivery: treatment gene is added to a harmless lentivirus or retrovirus vector which is introduced to the isolated stem cells of patient. The stem cells containing the treatment gene are returned to the patient.

Direct delivery: Doctor injects the vector carrying the gene directly into the part of the body that has defective cells.

Question 45

(T/F) Tumours are composed only of cancer cells.

Answer 45

False!

Tumour microenvironment is very heterogeneous.

Tumours are more than just cancer cells. They include tumour stroma which is made of non-cancer cells such as endothelial cells, immune cells, stem cells, etc that allow the cancer cells to grow.

Question 46

What are the three types of anti-cancer therapies? Briefly describe them.

Answer 46

1) Chemotherapy (drugs that affect cells THAT ARE DOUBLING, not specific, and highly cytotoxic)

2) Cancer targeted therapies (agents that inhibit or attack a more specific target in cancer cells. attempt to take advantage of genetic changes in malignant cells. ex: small molecules, antibodies, anti-angiogenic drugs)

3) Oncolytic viruses (exploit tumour cell-specific genetic defects)

Question 47

What is oncolytic virus therapy based on?

Answer 47

Oncolytic virus therapy is based on selective replication of viruses in cancer cells and their subsequent spread within a tumour without causing damage to normal tissue.

They represent a unique class of cancer therapeutics with distinct mechanisms of action. The activity of oncolytic viruses is very much a reflection of the underlying biology of the viruses from which they are derived.

Question 48

(T/F) The same biological processes that control cell growth and death also control the ability of individual cells to fight virus infections!

Answer 48

True!

Question 49

Interferon production:

Answer 49

1) locally prevents further infection

2) prevents unrestricted cell growth

3) induces apoptosis

4) prevents angiogenesis

5) activates adaptive immunity

*not compatible with replication of cancer cells

Question 50

________ cells have an intact interferon response, therefore virus replication is prevented.

________ cells have impaired interferon production (no production), therefore virus replication is able to proceed.

Answer 50

Healthy; Cancer

*healthy cells fight virus infections by producing interferons (cytokines)

*interferons expressed at low levels without viral infections

Question 51

(T/F) Oncolytic viruses infect cancer cells only not healthy because they impair interferon production, which is desired in cancer cells.

Answer 51

True!

Question 52

(T/F) The biological processes underlying anti-viral defense are incompatible with efficient tumor evolution. Cancer driver mutations are oncolytic virus driver mutations.

Answer 52

True!

Question 53

What are the two classes of oncolytic viruses?

Answer 53

1) Viruses that naturally replicate preferentially in cancer cells and are non-pathogenic in humans often due to elevated sensitivity to innate antiviral signalling (can’t defend against interferons) or dependence on oncogenic signalling pathways.

2) Viruses that are genetically manipulated for use as vaccine vectors and/or those genetically engineered with mutations/deletions in genes required for replication in normal, but not cancer cells.

Question 54

What are the features of oncolytic viruses (OVs) that make them advantageous and distinct from current therapeutic modalities (resulting in a high therapeutic index)?

Answer 54

1) There is a low probability for the generation of resistance, as OVs often target multiple oncogenic pathways and employ multiple means for cytotoxicity

2) They replicate in a tumour-selective fashion and are non-pathogenic, only minimal systemic toxicity has been detected

3) Virus dose in the tumour increases with time due to in situ virus amplification, as opposed to classical drug pharmacokinetics that decreases with time

4) Safety features can be built in, such as drug and immune sensitivity

Question 55

Oncolytic viruses have been engineered or selected to initiate a MULTI-MODAL cancer attack that uses complementary mechanisms to destroy the tumour.

What are the three mechanisms?

Answer 55

1) Direct virus-mediated cell lysis (infect cancer cells only which die eventually)

2) Anti-tumour immunity (cancer + viral antigens released when cell dies; “cancer vaccines”)

3) Disruption of tumour vasculature (viruses infect endothelial cells in blood vessels that are present in the tumour; they shut down blood vessels; no nutrient + oxygen 4 tumour)

Question 56

_______ virus can be genetically modified to deliver therapeutic payloads against tumours. It has an excellent safety record in humans.

Answer 56

Vaccinia

Question 57

How does a systemically delivered oncolytic virus enter the tumour bed?

Answer 57

Tumors have leaky blood vessels!

Question 58

(T/F) Anti-tumor immunity is just as important of a mechanism as cell lysis of oncolytic viruses.

Answer 58

True!

Question 59

1) What happens if a mouse whose colon tumour was treated with an oncolytic virus was challenged with colon cancer cells?

2) What are athymic mice?

Answer 59

1) (Immune competent) mice will reject the colon tumours! T cell-based immunity leads to long-term immune surveillance and anti-tumour immune responses.

2) Athymic mice are strains that have genetic mutations that cause a deteriorated or absent thymus, resulting in a compromised immune system due to a greatly reduced number of cells.