Lecture 12: Viral Protection and Therapeutics

Question 1

Why are most antibiotics ineffective at targeting viruses?

Answer 1

Most antibiotics inhibit the ribosome, which viruses do not have.

Question 2

How does AZT work as an anti-viral therapeutic?

Answer 2

AZT is a nucleoside analog that has an azide group (N3) in place of the hydroxyl group (OH). This inhibits DNA chain extension during transcription. AZT = DNA CHAIN TERMINATOR

Question 3

What is the effect of a missing dose of antiviral drug?

Answer 3

Partially-resistant virus will have time to develop a second mutation to become fully drug resistant.

Question 4

How can multi-drug antiviral therapy help prevent viral resistance?

Answer 4

Multiple drugs can target different viral proteins at once so it can never become a fully resistant virus (ie. HAART therapy).

Question 5

How can monoclonal antibodies be used as an antiviral therapeutic via humanizing a mouse?

Answer 5

Mice can be humanized. Immune system destroyed via irradiation and its bone marrow re-populated with human hematopoietic stem cells. Recover B cells from this mouse.

Question 6

How are monoclonal antibodies made in the lab for therapeutics?

Answer 6

Synthesize a mouse antibody capable of targeting the chosen antigen in the lab. Chimerize the variable (Fab) region of a mouse antibody with the constant (Fc) region of a human antibody to get a human antibody that can recognize said target antigen. Express the chimeric antibody in a bacteria cell to multiply, harvest, and purify. Inject the final product directly into patient bloodstream to generate immediate immunity/therapeutic effects.

Question 7

What are nanobodies? How can nanobodies be used in antiviral therapeutics?

Answer 7

Nanobodies are the first domain of only one chain of the variable (Fab) region of an antibody. This singular domain can be used to hit valleys/nooks in the virus surface. Then, nanobodies can be detected by another antibody, making easier to target the virus.

Question 8

What is a soluble decoy receptor? How do immunotoxins and immunoadhesins act as soluble decoy receptors?

Answer 8

Soluble decoy receptors are soluble versions of the same receptor that the virus would usually bind to. That way, virus binds to these and not actual human cells. Link it to a toxin, and the toxin can kill infected cells. Link it to an adhesin or Fc portion of an antibody, and virus opsonization will induce macrophage phagocytosis.

Question 9

What is the difference between prevention and treatment?

Answer 9

Prevention occurs before acquiring the pathogen (ie. vaccination). Exception: pre-exposure drugs. Treatment occurs after contracting the pathogen (ie. drugs and therapeutic antibodies). Exception: Some treatments can consist of injecting a vaccine if the virus has a long incubation period.

Question 10

What is the host response to a vaccine?

Answer 10

Antibody production, leading to cell-mediated immunity

Question 11

When did smallpox (Variola) first get treated? What was the change in case fatality?

Answer 11

First treated in 900 Ad via nasal insufflation (grind healing scabs and snort up nose). 30-40% fatality dropped to 1%

Question 12

How did Jenner invent the smallpox vaccine in 1796?

Answer 12

In 1796, he inoculated boy’s arm with pus from healing cow pock.

Question 13

What was significant about milkmaids?

Answer 13

Milkmaids infected with cowpox never got smallpox.

Question 14

What are the 8 strategies for vaccine development? Which method is mostly a therapeutic and not so much a prevention method?

Answer 14

1. Use harmless ANIMAL VIRUS in a different host
2. Use ATTENUATED viruses, can be to the point it its unable to replicate
3. Genetically engineer an ATTENUATED virus
4. Use INACTIVATED/killed viruses
5. Use SUBUNIT viruses, consists of just protein, peptide, or empty capsid
6. DNA or RNA-BASED vaccine
7. Engineer a live virus by INSERTING ANTIGEN GENE from a different virus.
8. Adoptive transfer with mAbs, or Abs. [Therapeutic mostly]

Question 15

Describe how the live-attentuated vaccine strategy was created.

Answer 15

Pasteur took serially passaged saliva from rabid dog into the brains of live rabbits. After doing this multiple times, he dried, ground up, and inocculated the rabbit’s spinal cord into a dog and human, which gave both immune protection.

Question 16

How do passages help with creating a live-attentuated vaccine? Why is hang drying effective?

Answer 16

Serial passages allow for mutations to develop in the virus to select for its current host. Over time, the virus becomes more lethal to the rabbit/animal than the human.
Hang drying results in partial virus inactivation.

Question 17

How can a virus be inactivated?

Answer 17

Chemically (ie. formaldehyde) kill with toxic chemicals to prevent further viral replication but retain its antigenicity to stimulate the IS.

Question 18

LIve-attenuated viruses vs. Inactivated viruses

Answer 18

Live-attenuated generate longer lasting immunity, and only one round of immunization is needed. However, it needs refrigeration and possible reversion to being virulent. Inactivated does not need refrigeration, may co-inactivate other contaminating pathogens, and will not lead to reversion. However, it last for a shorter period and needs multiple immunizations.

Question 19

How are subunit vaccines created? What’s a major disadvantage?

Answer 19

An individual protein is chosen and extracted, or can be replicated by plasmid transfer into bacteria. MAJOR DISADVANTAGE: No cell-mediated immunity b/c protein isn’t synthesized inside cells

Question 20

How do genome-based vaccines work? What is a disadvantage?

Answer 20

Transfect live patient with plasmid via saline injection, gene gun w/ DNA-coated gold powder, or topical application of liposomes into mucosal organs. APCs take it up and activate many B/T cells. Leads to cell-mediated immunity. DISADVANTAGE: TRANSFECTION = LOW EFFICIENCY. Since mRNA doesn’t need to go into nucleus to be transcribed, it can be modified to prevent degradation by the immune system.

Question 21

How does engineering an attenuated virus work?

Answer 21

Manually delete remaining virulence genes, aka genes used in combating host defenses, by putting it onto a plasmid first. Then, transfect, recombine.

Question 22

How does engineering a recombinant live virus work?

Answer 22

Insert foreign gene from a disease-causing virus into genome of a benign virus “vector”. The vector virus will express the foreign gene on its surface to stimulate cell-mediated immune responses, and as it is only one gene from the pathogenic virus, it’s relatively safe.

Question 23

How does the booster shot work?

Answer 23

A second stimulation with the vaccine will call upon memory B and T cells to replicate, thus an even faster immune reaction against the virus results in a shorter amount of time. Some strategies prime with DNA first, then boost with recombinant virus. Mixing vaccine strategies prevents the vector virus from being attacked the second time. OR prime/boost with different vector viruses.

Question 24

What would be the perfect vaccine?

Answer 24

100% efficacy after single dose, no refrigeration, orally administered, life-long immunity, inexpensive to produce

Question 25

Why do vaccines fail?

Answer 25

1. No cell-mediated immune response
2. Viral strain-to-strain variation
3. Escape mutations: mutations to surface epitopes that Ab bind to
4. important epitopes are hidden from Ab. Conformational hiding (protein changes after Ab binding) or steric hiding (narrow valleys and peaks).
5. Weak Ab can bind and promote immune cells to uptake virus. Virus can replicate inside and kill the immune cell after.

Question 26

What do anti-viral therapeutics need to have?

Answer 26

Targets molecules on virus and not host. There are few virus-specific targets (ie. Reverse transcriptase, RNA replicase, integrase, viral protease, influenza neuraminidase)