Hybridoma & Vaccines

Question 1

what is the problem that Antibodies that are generated after immunization (or via natural
immune response) are polyclonal?

Answer 1

• different specificities
• different affinities
• different properties (e.g. isotype/subclass etc)

Question 2

what is the major issue with polyclonal antibodies?

Answer 2

The heterogeneity is as major issue !
* No antisera are the same.
* Limited amounts can be produced of each antisera.
* The very same reagent cannot be used in a long series of
experiments
* Complex mixtures with potentially cross-reactive antibodies.

Question 3

what is the solution for making the same type of antibodies?

Answer 3

Create an indefinite source of your Ab with homogeneous
structure and known specificity and affinity.
-> Monoclonal antibody!

Question 4

polyclonal (antisera)

Answer 4

polyclonal antibodies represents a collection of antibodies from different B cells that recognize multiple epitopes on the same antigen. Each of these individual antibodies recognizes a unique epitope that is located on that antigen.

Question 5

monoclonal antibodies

Answer 5

represents antibody from a single antibody producing B cell and therefore only binds with one unique epitope.

Question 6

STRATEGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES

Answer 6

1) Preparation of immunogen
- Purification / control of purif.
- Coupling to carrier (if needed).
2) Immunization
- Choice of animal
- Schema (e.g. Booster)
- Adjuvant
- Amount (Dose)
3) Early testing
- Specificity
- Titer
- Binding properties
- Absorption
- Boostering
4) Fusion, cloning and antibody production
5) Purification
- ammoniumsulphate precipitation
- ion-exchange chromatography
- protein A / protein G
- affinity chromatography
- etc
6) Quality control
- potential application
- isotype
- stability
- specificity
- affinity
- etc

Question 7

Scheme – production of monoclonal antibodies (MAbs)

Answer 7

Immunization
Immortalisation
HAT selection
Detection of prod. Ab
Cloning
–>Mab!!

HAT - hypoxanthine-aminopterin-thymidine

Question 8

1. Immunization.

Answer 8

 Immunize a mouse with the targeted antigen.
 Isolate Ab producing cells from the spleen.

Question 9

2. Immortalisation.

Answer 9

Spleen cells producing antibody from mouse immunized with antigen A combined with Myeloma cells (immortal) lacking antibody secretion and the enzyme HGPRT

–> mix and fuse cells with PEG
–>Gives a hybrid cell line denoted HYBRIDOMA

Question 10

What is the fusion partner in the immortalisation step?

Answer 10

Myeloma cell - immortal
- lacks its own Ab production
- sensitive for HAT (lacks HGPRT)

Potential cell lines
- mouse myeloma cell lines (Sp2/0, NS0, NS1)
(after immunization of mice)
- rat myeloma cell lines (Y3)
(after immunization of rats)

Question 11

what types of Somatic cell fusion are there in immortalisation?

Answer 11

PEG-fusion
electrofusion

Question 12

what do Hybridomas consist of?

Answer 12

B cells and myeloma cell line

Question 13

B cells

Answer 13

-produce antibodies
-no survival potential in culture

Question 14

myeloma cell line

Answer 14

• does not produce antibodies
• survive and grow in culture

Question 15

What do the cells need to be in order to grow on HAT media?

Answer 15

For cells to grow on HAT media, they need to be positive for the HGPRT enzyme.

HGPRT – hypoxanthineguanine
phosphoribosyl transferase

Question 16

3. HAT selection mechanism

Answer 16

 Aminopterin
Blocks dihydrofolatreductase and thereby purinsynthesis.
Blocks synthesis of TMP.
 Hypoxanthin
Phosphorylates HGPRT to IMP that in turn is
converted to AMP and GMP.
 Thymidine
Is phosphorylated by thymidinkinase (TK) to TMP

Question 17

name 2 ways cell division (requires synthesis of nucleotides)

Answer 17

Salvage pathway= Parts of degraded nucleotides are used

De novo pathway=
New nucleotides using small
metabolites that are present in
HAT media

Question 18

How does cell division happen in hat media?

Answer 18

In HAT media: cells cannot operate de novo pathway due to Aminopterin
So cells use the salvage pathway! BUT, they have to be HGPRT+ and they use
Hypoxanthin and Thymidin as precursors.
Remember!
B cells: HGPRT+
Myeloma cells: HGPRT-

Question 19

which cells are HGPRT+ and which are HGPRT-?

Answer 19

Normal cells are HGPRT+
– They tolerate HAT but cannot survive in culture –
Myeloma cells are HGPRT-
– They can live in culture but do not tolerate HAT –
The fused hybridoma cells are HGPRT+
– They survive in culture, even in the presence of HAT

Question 20

which cells survive in HAT?

Answer 20

Immortal hybridomas proliferate

mortal spleen cells and unfused HGPRT- myeloma cells die.

Question 21

4. Cloning

Answer 21

After HAT selection:
 select the hybridoma that produces Ab with the desired specificity.
 Clone the selected cells.
”limiting dilution” < 1 cell/well
To reassure monoclonality.

Question 22

Production of Mab.

Answer 22

 in conventional cell cultures
 in fermentors

Question 23

What is the difference between an antigen and an immunogen?

Answer 23

Antigen
An antigen is any substance that can be recognized by the immune system, specifically by antibodies, B cells, or T cells. Antigens can be proteins, peptides, polysaccharides, lipids, nucleic acids, or small molecules. The key points about antigens are:

Recognition: An antigen is defined by its ability to bind to specific receptors on immune cells (such as B cell receptors or T cell receptors) or to antibodies.
Epitope: The specific part of the antigen that is recognized and bound by an antibody or a receptor on a B cell or T cell is called an epitope or antigenic determinant.
Response: Not all antigens can elicit an immune response on their own; they may require additional signals or contexts to do so.
Immunogen
An immunogen is a type of antigen that is capable of eliciting an immune response on its own. This means that an immunogen not only binds to immune receptors but also triggers the activation and proliferation of immune cells, leading to an adaptive immune response. Key characteristics of immunogens include:

Immune Response: An immunogen induces a specific immune response, leading to the production of antibodies, the activation of T cells, or both.
Size and Complexity: Typically, immunogens are larger and more complex molecules. Proteins and larger polysaccharides are common immunogens.
Foreignness: The immune system is more likely to recognize molecules as immunogens if they are foreign (not naturally occurring within the host).
Key Differences
Capability to Elicit Immune Response:

Antigen: Can be recognized by the immune system but does not necessarily elicit an immune response by itself.
Immunogen: Always elicits an immune response upon recognition by the immune system.
Size and Complexity:

Antigen: Can be small molecules or simple structures, including small peptides, nucleic acids, or even small haptens (when bound to a larger carrier molecule).
Immunogen: Generally larger and more complex structures like proteins or polysaccharides that can stimulate an immune response.
Functionality:

Antigen: Any molecule that binds specifically to an antibody or a receptor on a B cell or T cell.
Immunogen: A type of antigen that can induce an adaptive immune response by activating lymphocytes.

Practical Example
Hapten: A small molecule that, by itself, is an antigen but not an immunogen. When conjugated to a larger carrier protein, the hapten-carrier complex becomes immunogenic and can elicit an immune response. The hapten is recognized by antibodies (antigen), but the immune response is generated against the hapten-carrier conjugate (immunogen)

Question 24

What is the difference between antisera technology and hybridoma technology?

Answer 24

Antisera refers to blood serum containing polyclonal antibodies, which are produced by immunizing an animal with an antigen. The resulting serum contains a mixture of antibodies that recognize multiple epitopes on the antigen.

Key Characteristics:
Production:

Immunization: An animal (commonly rabbits, goats, or horses) is immunized with an antigen.
Immune Response: The animal’s immune system produces antibodies against the antigen.
Serum Collection: Blood is collected from the animal, and the serum (containing the antibodies) is separated.
Antibody Type:

Polyclonal Antibodies: The antibodies in antisera are polyclonal, meaning they are a heterogeneous mix of antibodies that recognize multiple epitopes on the antigen.
Advantages:

Broad Recognition: Polyclonal antibodies can recognize multiple epitopes, making them useful for detecting antigens with high variability.
Ease of Production: Relatively simple and cost-effective to produce in large quantities.
Disadvantages:

Variability: Batch-to-batch variability due to differences in immune responses between individual animals.
Cross-Reactivity: Higher risk of cross-reactivity with other antigens, leading to potential non-specific binding.
Hybridoma Technology
Hybridoma technology involves the production of monoclonal antibodies by fusing an antibody-producing B cell with a myeloma (cancer) cell, resulting in a hybrid cell line (hybridoma) that can produce large quantities of a single type of antibody indefinitely.

Key Characteristics:
Production:

Immunization: An animal (commonly a mouse) is immunized with an antigen.
Cell Fusion: B cells from the immunized animal’s spleen are fused with myeloma cells to create hybridomas.
Screening and Cloning: Hybridomas are screened for the production of the desired antibody, and positive clones are isolated and expanded.
Antibody Type:

Monoclonal Antibodies: Hybridomas produce monoclonal antibodies, which are homogeneous and recognize a single epitope on the antigen.
Advantages:

Specificity: High specificity and uniformity, as all antibodies produced by a hybridoma are identical and recognize the same epitope.
Consistency: Consistent production of antibodies with minimal batch-to-batch variation.
Unlimited Supply: Hybridomas can be cultured indefinitely, providing a continuous supply of monoclonal antibodies.
Disadvantages:

Complexity and Cost: More complex and expensive to produce compared to polyclonal antisera.
Limited Epitope Recognition: Monoclonal antibodies recognize only a single epitope, which may not be effective against antigens with high variability or multiple epitopes.

Antisera Technology:

Used in research for detecting a wide range of antigens.
Common in immunohistochemistry and Western blotting where broader detection is needed.
Hybridoma Technology:

Used for the production of highly specific monoclonal antibodies for research, diagnostics, and therapeutic applications.
Essential for creating consistent and reliable diagnostic tests and therapeutic agents (e.g., monoclonal antibody drugs).

Question 25

What are the six main strategy points to be followed for the production of a
monoclonal antibody (mAb)? Mention and elaborate on them.

Answer 25

Immunization
Immortalisation
HAT selection
Detection of prod. Ab
Cloning
production of Mab

1. Immunization
   Goal: To elicit a strong and specific immune response in the host animal.

Antigen Selection: Choose an antigen relevant to the intended application. The antigen should be well-characterized and capable of eliciting a robust immune response.
Immunization Protocol: Administer the antigen to the host animal (commonly mice) along with an adjuvant to enhance the immune response. Multiple injections (boosts) are often given over several weeks to ensure a high titer of antibodies.
Monitoring Response: Collect blood samples periodically to measure antibody titers and ensure that the immune response is developing as expected.
2. Immortalization
Goal: To create hybrid cells (hybridomas) that can produce antibodies indefinitely.

Harvesting B Cells: After a strong immune response is confirmed, spleen cells (rich in antibody-producing B cells) are harvested from the immunized animal.
Cell Fusion: Fuse the harvested B cells with myeloma cells using a fusion agent like polyethylene glycol (PEG). This process combines the antibody-producing capability of B cells with the immortality of myeloma cells.
Hybridoma Formation: The fused cells (hybridomas) can grow indefinitely and produce the specific antibody of interest.
3. HAT Selection
Goal: To select hybridoma cells that have successfully fused and are capable of producing antibodies.

Selective Medium: Hybridomas are cultured in HAT (Hypoxanthine-Aminopterin-Thymidine) medium. This medium selects for successfully fused hybridomas by exploiting the fact that unfused myeloma cells cannot survive in HAT medium, while normal spleen cells have a limited lifespan.
Screening for Viability: Only hybridomas (fused cells) can survive and proliferate in HAT medium, allowing for the selection of successful fusions.
4. Detection of Productive Antibody (Screening)
Goal: To identify hybridoma clones that produce the desired antibody.

Primary Screening: Use techniques like ELISA, Western blotting, or immunofluorescence to screen the culture supernatants from hybridoma wells for the presence of antibodies specific to the target antigen.
Secondary Screening: Further characterize positive clones for specificity, affinity, and functionality to ensure they produce high-quality antibodies.
5. Cloning
Goal: To ensure monoclonality of the antibody-producing cells.

Limiting Dilution: Perform limiting dilution or other cloning methods to isolate single hybridoma cells. This step ensures that each colony originates from a single cell, resulting in monoclonal antibodies.
Verification: Confirm the monoclonality of the clones by additional rounds of screening and testing to ensure that the antibodies produced are homogeneous and specific.
6. Production of Monoclonal Antibodies (mAb)
Goal: To produce and purify monoclonal antibodies at a larger scale.

Expansion: Once stable hybridoma clones are identified, they are expanded in culture to produce larger quantities of the antibody.
Purification: Purify the antibodies from the culture supernatant using affinity chromatography (e.g., protein A/G columns), ensuring high purity and concentration.
Quality Control: Conduct rigorous quality control tests to verify the purity, concentration, and functionality of the monoclonal antibodies. This includes testing for contaminants, aggregation, and consistent performance in relevant assays

Question 26

Why can’t we use B cells alone to create a mAb?

Answer 26

1. Limited Lifespan of B Cells
   Finite Lifespan: Primary B cells have a limited lifespan in culture and cannot proliferate indefinitely. This makes it impractical to use them for long-term production of antibodies.
   Senescence: Even if B cells could be cultured for a short period, they would eventually undergo senescence and die, ceasing antibody production.
2. Insufficient Yield
   Low Antibody Production: Primary B cells produce antibodies, but the yield is typically low and not sufficient for large-scale production required for therapeutic, diagnostic, or research applications.
3. Lack of Immortality
   No Continuous Cell Division: B cells do not have the ability to divide indefinitely. To produce monoclonal antibodies continuously, a cell line with the capability for unlimited division (immortalization) is required.
4. Variability and Instability
   Genetic Instability: B cells can be genetically unstable and may lose their antibody-producing capability over time.
   Variability in Antibody Production: Different B cells produce different antibodies, and without a way to isolate and continuously propagate a single antibody-producing cell, it is impossible to maintain consistency

Question 27

What is the importance of using myeloma cells that lack the enzyme HGPRT?

Answer 27

1. Selection in HAT Medium
   HAT Medium Composition: HAT medium contains hypoxanthine, aminopterin, and thymidine.

Hypoxanthine: A precursor for the salvage pathway of nucleotide synthesis.
Aminopterin: A folic acid antagonist that blocks the de novo synthesis of purines and pyrimidines.
Thymidine: A precursor for DNA synthesis.
Role of HGPRT: HGPRT is an enzyme involved in the salvage pathway of purine synthesis. It allows cells to utilize hypoxanthine to produce nucleotides when the de novo pathway is blocked by aminopterin.

2. Selective Growth of Hybridomas
   Myeloma Cells Without HGPRT: Myeloma cells are chosen to lack HGPRT to ensure they cannot use the salvage pathway to synthesize nucleotides in the presence of aminopterin. Without this pathway, they cannot survive in HAT medium.
   B Cells With HGPRT: Normal B cells, which have functional HGPRT, can use the salvage pathway to survive in HAT medium, but they have a limited lifespan and will eventually die.
   Hybridoma Survival:

Fusion of B Cells and Myeloma Cells: When B cells (which have HGPRT) are fused with myeloma cells (lacking HGPRT), the resulting hybridoma cells inherit the HGPRT enzyme from the B cells.
Selective Growth: Only successfully fused hybridomas, which have both immortality from myeloma cells and the HGPRT enzyme from B cells, can survive in HAT medium. Unfused myeloma cells die because they cannot use the salvage pathway, and unfused B cells die due to their limited lifespan.
3. Ensuring Monoclonality and Specificity
Elimination of Unfused Cells: The use of HGPRT-deficient myeloma cells ensures that only hybridomas survive in HAT medium, eliminating unfused myeloma cells and any other non-hybrid cells.
Hybridoma Stability: Hybridomas are stable and capable of indefinite proliferation, continuously producing the specific monoclonal antibody of interes

Question 28

What is a vaccine?

Answer 28

A vaccine is a biological product that can be used to safely induce an immune
response that confers protection against infection and/or disease on subsequent
exposure to a pathogen.

Question 29

What must a vaccine contain?

Answer 29

The vaccine must contain antigens that are either derived from the pathogen or
produced synthetically to represent components of the pathogen

Question 30

Why do we need vaccines?

Answer 30

To protect us, through engagement of adaptive immunity, against disease or conditions
that possess a threat to our health.

Question 31

What do vaccines stimulate?

Answer 31

Vaccines stimulate a protective immune response. After a mild infection there will be protective immunity so that immunological memory remembers the pathogen if we get re-infected.

Question 32

What is the most effective way of controlling infectious diseases?

Answer 32

Vaccination is the most effective mean of controlling infectious diseases

Question 33

Active immunization

Answer 33

Initiation of an immune response against a certain pathogen by injection of a vaccine, a dead or
attenuated (non-pathogenic) form of the pathogen or something else that may induce immunity
against it.

Question 34

Passive immunization

Answer 34

Transfer of an active component (e.g. antiserum or purified antibodies) that protects against disease
in the short term, as long as the transferred antibodies remain active in the body.

Question 35

What do we aim to accomplish through vaccination?

Answer 35

Short term–> Antibody response (B cells)
T helper cells
Cytotoxic T cells

Herd immunity
Prevention of infection vs disease possibly vaccines prevent infection and the development of disease after infection with a pathogen (difficult to distinguish)

Non-specific effects: immunization with some vaccines perturbs the immune system in such a way that there
are general changes in immune responsiveness that can increase protection against unrelated pathogens.

Immune memory
Long-term

Question 36

Generation of immune response from vaccine: step by step

Answer 36

1) Vaccine injected into muscle
2) Protein Ag taken up by DCs
(activated through pattern recognition receptors by danger
signals in the adjuvant)
3) Trafficked to draining lymph nodes
4) Peptide presentation- MHC -> T cell activation
+ signalling (by soluble Ag) through B cell receptor
➔ T cells drive the B cell development in the LN
➔ Maturation of the Ab response increases Ab
affinity and induces different Ab isotypes
➔ Production of short-lived plasma cells, secrete
vaccine protein- specific Abs, increase the
serum Ab levels.
➔ Memory B cells are produced
➔ Long-lived plasma cells that can produce Abs
for decades, travel to bone marrow niches
➔ CD8+ memory T cells: proliferate rapidly when
encountering a pathogen
➔ CD8+ effector T cells eliminate infected cells

Question 37

name Applications of vaccines

Answer 37

o Infectious agents (viruses, bacteria, parasites and their toxins) e.g. polio, tetanus, measles, Hepatitis B etc
o Allergy
o Cancer

Question 38

explain how allergy immunotherapy works

Answer 38

o Injection of allergen in small doses
o Gradually increased levels of allergen
o Works well e.g. for allergy against grass pollen.
o Results in production of blocking antibodies, commonly of IgG4 type. These bind to the
allergen and block the otherwise subsequent allergic reaction.
o Shifts the T cell response away from TH2 and towards TH1
➢ Grazax® indicated for disease modifying treatment of grass pollen (Phleum pratense or
allergens cross reacting with P. pratense) induced allergic rhinitis with or without
conjunctivitis in adults, adolescents and children above the age of 5 years.

Question 39

Applications of vaccines: cancer

Answer 39

Prophylactic cancer vaccines: Gardasil®, Gardasil-9®, Cervarix®, HEPLISAV-B® (HBV-related liver
cancer)
➢ Can prevent viral infection which can cause cancer
Therapeutic cancer vaccines (TCV): Bacillus Calmette-Guérin (early-stage bladder cancer),
Provenge® (prostate cancer)
o Help the immune system recognise, target and eliminate cancer cells
o Do not target the underlying cause of cancer
o Target commonly expressed antigens
o Personalised TCVs: ie. autologous therapy: using antigens expressed by a patient’s individual
tumor
o capable of providing co-stimulatory signals so that the effector cells can attack the tumor
cells that lacks such signals.

Question 40

Cancer vaccines mode of action

Answer 40

1. Vaccination with tumor antigen
2. Transport of antigen to lymph nodes
3. Activation of B cells and T-cells.
   4.Activated immune cells mount an immune response against the tumor cells.
4. Tumor cell death

Question 41

What do effective vaccines have to be?

Answer 41

Effective vaccines must induce long-lasting protection while being safe and inexpensive

A benefit of an effective vaccination program is the ‘herd immunity’ that it confers on the general population.

Question 42

Herd immunity

Answer 42

Herd immunity gives population scale immunity when critical level of vaccination has been achieved

• Protection conferred to unvaccinated individuals in a
  population produced by vaccination of others and
  reduction in the natural reservoir for infection.
• Virus spread stops when the probability of infection
  drops below a critical level.
• This critical level is virus- and population- specific
  o population % that needs to be vaccinated to achieve
  herd immunity is 80-85% for polio and 90-95% for
  measles (highly contagious)
• None of the vaccines is or will be 100% effective

Question 43

Effective vaccines about certain diseases are still missing, give an example

Answer 43

-HIV/AIDS
-Measels

• Effective vaccine for measles is available, but it
  is heat sensitive, which limits its use in tropical
  countries
  *Measles vaccination averted 56 million deaths being between 2000
  and 2021.
  *Even though a safe and cost-effective vaccine is available, in 2021, there were an estimated 128 000 measles deaths globally, mostly among unvaccinated or under vaccinated children under the age of 5 year