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Animal biotechnology > Vaccines & monoclonal antibodies > Flashcards

Vaccines & monoclonal antibodies Flashcards

1
Q

What are the five types of antibodies and which is the most abundant?

A

IgG (most abundant), IgM, IgA, IgD, IgE

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2
Q

Which is the largest + function?

A

IgM - Produced first upon antigen invasion and indicates an acute immune response

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3
Q

IgG is classified into four subclasses (IgG1 - 4) , what is its role?

A

Highest opsonization and neutralization activities involved in the immune response and long-term immunity. Found in the blood for a long time and indicates chronic disease.

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4
Q

Opsonization = an immune process which uses opsonins to tag foreign pathogens for elimination by phagocytes. Without an opsonin, such as an antibody, the negatively-charged cell walls of the pathogen and phagocyte repel each other.

A
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5
Q

Where is IgA expressed?

A

Mucosal tissues where it forms dimers after secretion (gastrointestinal tract, respiratory tract, and genitourinary tract). Can be found in tears, saliva, and breast milk.

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6
Q

IgE?

A

Involved in allergy

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7
Q

Which antibody then is the only immunoglobulin not specifically found in the bloodstream?

A

IgA (found in the first line of defence)

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8
Q

IgD can be found on the surface of B cells and can activate them.

A
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9
Q

These antibodies can then determine the locations of specific diseases within an organism.

A
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10
Q

What is the source of polyclonal therapeutic antibodies (first used to treat toxin mediated infectious diseases (Tetanus, Diphtheria) and snake bites?

A

Immune serum

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11
Q

Why are immune serums important?

A

An immune system may not respond fast enough after infection (not produce enough/quickly antibodies) so a boost of antibodies is needed for efficacy.

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12
Q

What type of immunity is this?

A

Passive immunity

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13
Q

What are three types of sources used for obtaining polyclonal antibodies?

A
  1. Pooled norma plasma (individuals plasma containing numerous antibodies)
  2. Pathogen specific plasma
  3. Antisera from immunised animals
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14
Q

What is antisera?

A

Blood serum obtained from animals which have been intentionally immunised against a specific pathogen

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15
Q

Three advantages of using polyclonal antibodies

A
  1. Cost effective
  2. The clonal and biophysical diversity allows for great sensitivity (specificity)
  3. Able to recognise many epitopes on an antigen
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16
Q

Four disadvantages of using polyclonal antibodies

A
  1. Finite supply
  2. Mediate transmission of Hep. B, HIV, and other pathogens such as prions if patient screening is poor
  3. Low batch consistency and reproducibility (difficult on large scale, may not always be specific)
  4. Can induce allergic reactions and anaphylactic shock
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17
Q

For (mouse hybdridoma) monoclonal antibody production, what is the first step?

A
  1. The mouse is immunised with the antigen of choice to stimulate an immune response and antibody production
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18
Q

2nd

A
  1. Antibody producing cells are isolated from the immunised spleen
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19
Q

3rd & 4th

A
  1. Non-antibody producing, HAT sensitive myeloma cells, are grown in tissue culture.
  2. Antibody cells (B cells) fused with the myeloma cells to form hybridomas
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20
Q

Why is a tumour cell-line needed?

A

Because the B cells don’t proliferate on their own naturally and would therefore need a large amount of B cells to be obtained from the mouse, but the myeloma cells do.

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21
Q

Why is the HAT medium important?

A

Allows for selection of hybridomas over unfused parental B cells and tumour cells. B cells on their own do not proliferate often and have a short life span, so when fused, they have the enzyme (HGPRT) needed for survival and myeloma cells enable proliferation.

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22
Q

Last step

A

Hybdridoma cells are screened for Antibody production and these cells are cloned. Then isolate the monoclonal antibodies.

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23
Q

What are the three structures of fragments derived from mAbs?

A
  1. Fabs (antigen binding fragment)
  2. scFvs (single-chain variable fragment)
  3. 3Gs (third generation or minituarized ATBs)
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24
Q

What is a Bispecific antibody?

A

When two antigen binding domains like Fabs or scFVs are linked together to simultaneously bind to different antigens.

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25
Q

Common uses of mAbs:
- Cancer
- Autoimmune diseases
- Anti-infective
- Cardiovascular system
- Eye disorder treatment

A
26
Q

Chimeric antibodies are also being produced, why is this effective?

A

Because 90% of the antibodies being produced within the animal (mice mainly, now goats and cows), is human sequence, making it unlikely to be rejected or cause an allergic reaction for the patient. The mouse component has greater binding affinity than human antibodies.

27
Q

How are chimeric antibodies produced?

A

Splicing of genes responsible for producing the antigen-binding region of the antibody (variable regions) from a mouse antibody and combine with human constant regions. Hybrid genetic material inserted vector, then into bacteria/mammalian cells to act as cellular factories to produce chimeric antibodies.

28
Q

What are three benefits of engineering chimeric antibodies using mouse genetic material?

A
  1. Reduced immunogenicity
  2. Increased affinity - mouse antibodies have a higher affinity than human antibodies for their target antigens
  3. Increased specificity - can recognise and bind to unique targets like cancer cells more effectively than human antibodies
29
Q

Why are most therapeutic antibodies humanised?

A

Engineered to have a structure that closely resembles a human antibody to reduce the potential for immune reactions when the antibody is used as a therapeutic agent in humans

30
Q

What are the first two steps of protein production via phage display libraries?

A
  1. Collection of DNA fragments from the same pathogen and are placed into a plasmid vector which encode for part of the antigen.
  2. Each plasmid will represent a different part of the protein, transfect plasmid per phage.
31
Q

What does the phage do with the DNA?

A

It will express the protein on its surface (coat) = phage library can be produced

32
Q

3rd step

A
  1. Pool of antibodies will determine specific binding affinities on the different phages, determining which antibody is suited for the different proteins expressed on the surface to different epitopes
33
Q

This is useful for if someone has a fever and you need to know what the disease is. Use serum across the known phages which represent different diseases, this will allow you to see the binding affinity of the serum to the phages = disease is determined. Can also determine the efficacy of the mAbs produced in the lab.

A
34
Q

1st & 2nd step for production of human monoclonal ATBs using phage display

A
  1. Human is exposed to pathogen to cause the B lymphocytes to produce specific antibodies. Isolate the B cells.
  2. Extract DNA fragments from the B cells and PCR to amplify the immunoglobulin genes.
35
Q

3rd & 4th

A
  1. Clone DNA fragments (each represent different part of antibody) into phages. Phages present antibody on surface.
  2. This produces a library. Screen known antigens from pathogens against the phages to determine binding efficacy.
36
Q

Three benefits of this method

A
  1. Rapid
  2. Cheap
  3. No use of animals
37
Q

Can then combine the two methods presented to see which has the best binding. Screen and then produce more of the specific antibody which is most effective = mAb production

A
38
Q

Naked antibodies are used for cancer treatment. What are naked antibodies and how do they work? (category 1 therapeutic antibodies)

A

Naked antibodies are traditional mAbs that are NOT conjugated to drugs or therapeutic agents. They bind specifically to specfic proteins on cancer cell surfaces and mark them for their destruction by the immune system. They also interfere with signalling pathways required for cancer cell survival.

39
Q

CAR-T therapeutic antibodies are category 2. How do they work?

A

They can have drugs conjugated to them.

40
Q

A radionuclide destroy cancer cells but not T or B cells, why is that?

A

Because cancer cells are actively dividing unlike the immune cells. Therefore, they uptake the radionuclide and are destroyed.

41
Q

What is immunisation?

A

Making a person or animal immune to infection by inoculation.

42
Q

What is vaccination?

A

Introduction of a vaccine into the body to produce immunity to a specfic disease.

43
Q

What type of passive immunity is ingested 24-36 hours after birth and why?

A

Colostrum. Animals do not receive antibodies through the placenta. They have an open epithelium for the absorption of antibodies and it seals after the time frame.

44
Q

What happens if you administer too much of an antibody rich serum?

A

Develop “serum sickness”. Immune response against donor Igs. Can also cause disease transfer if screening was not properly carried out of the donor.

45
Q

What are the three types of vaccines in use?

A
  1. Live, attenuated vaccines
  2. Inactivated vaccines
  3. Subunit vaccines
46
Q

How are attenuated vaccines produced?

A

Growing the pathogen under suboptimal conditions.

47
Q

What are subunit vaccines?

A

Antigenic macromolecules (part of the pathogen) from the surface of the pathogen.

48
Q

What are two advantages of using mRNA vaccines?

A
  1. No genetic alteration in the host. Lack of mutagenic effects.
  2. Modified RNA molecules encode for antigenic components where the cellular machinery rapidly increases the amount of mRNA for protein to be produced so it elicits a strong and long-lasting immune response.
49
Q

Two disadvantages

A
  1. Requires significant engineering to ensure powerful immune modulation, without degrading mRNA
  2. Minor side-effects like autoantibodies
50
Q

What are live vaccines?

A

Attenuated forms of the pathogen.

51
Q

They can be deletion mutants. Good Ab response, but potential reversion to virulence.

A
52
Q

What is a disadvantage of inactivated vaccines?

A

They elicit a good immune response where antibodies are produced rapidly and in large amounts but there is not a good ‘memory’ and only last 6-12 weeks.

53
Q

Five examples of inactivated vaccines

A
  1. Whole pathogen
  2. Inactivated toxins
  3. Subcellular fragments
  4. Subunit vaccines
  5. Naked DNA
54
Q

Why are inactivated vaccines more stable than attenuated live vaccines?

A

Because attenuated require stable storage conditions to prevent the living organism being easily inactivated.

55
Q

Which have better antigen production between live attenuated and inactivated?

A

Live attenuated as it has all the antigens present as its a whole pathogen. Inactivation with a chemical can change the composition of the antigen, lowering its efficiency.

56
Q

Attenuated have endogenous antibody presentation whereas inactivated vaccines have exogenous presentation.

A
57
Q

What are adjuvants and which type of vaccine requires it?

A

Additions to the vaccines to boost the immune response, attracts immune response to ares (inflammation). Inactivated vaccine.

58
Q

Disadvantage of adjuvants

A

Excessive inflammation, non-specific.

59
Q

Modern adjuvants: Toll-like Receptors target - what is this?

A

Receptors in dendritic cells provide early detection of the pathogen as they can detect different components of the pathogen like LPS and single stranded RNA. The adjuvent can have these toll-like receptors present to determine more components of the pathogen. Smaller volumes of the vaccine are required.

60
Q

Antigenic variation can be caused by drift and shift. What is drift?

A

Point mutations in the DNA sequences leading to different amino acid sequences and different structures for the surface proteins on the pathogen.

61
Q

What is shift?

A

Reassortment of segments in the genome between different strains of the same pathogen, leading to dramatic changes in the expressed protein. The protein is no longer recognised by the immune response

Animal biotechnology (8 decks)

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