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Flashcards in TD: Application of biotechnology Deck (39)
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1
Q

What cell produces ABs?

What are monoclonal ABs?

A

Plasma B-lymphocytes produce ABs - these only detect a small part of the molecule called the epitope.

A monoclonal antibody is the product of a single clone of plasma cells and will therefore react with a single specific epitope on an antigen.

2
Q

In 1975 Kohler and Milsten developed a method for the production of …

A

In 1975 Kohler and Milsten developed a method for the production of unlimited amount of ABs of a pre-determined specificty from a single clone of cells (basically all cells produce that 1 monoclonal AB)

3
Q

What are the properties of these cells

What is good about their production?

A
  • The cell lines are immortal and can be grown in any lab to purify the AB.
  • These AB are very much like a chemical reagent. They behave in a predictable way and are reproducibe from one lab and one time to the next
4
Q

Although these AB are specific for only one epitope on one antigen, what problem can occur?

A

Cross-linking can occur i.e recognises a very similar sequence of AA therefore validation must be done

5
Q

Describe the use of AB in diagnositic tests - what is used and why?

A

Monoclonal AB used in diagnostic tests often requires a cocktail of more than one AB, to avoid loss of detection due to mutation affecting one epitope

6
Q

Describe the production on monoclonal AB

A

The animal of choice for monoclonal antibody production is the mouse, due mainly to the large number of mouse myeloma (plasma cell tumour) cell lines available for hybridoma formation. The initial stages of producing an immune response are identical to those required for polyclonal antibody production an antigen mixed with an adjuvant such as Freund’s, is introduced by subcutaneous or intraperitoneal injection into the mouse, followed by a secondary boost several weeks later. The mouse will produce an immune response but instead of bleeding the mouse to harvest the circulating antibody content, it is sacrificed and the antibody source, the B cells (from the lymph nodes and/or the spleen) are removed for processing.

The memory B lymphocytes contain the information allowing each individual cell to produce antibody of single specificity against a particular epitope (one epitope). Antibody producing B cells will produce the specific antibody required but cannot be maintained in vitro long enough to produce usable quantities of antibody.

In contrast, a mouse myeloma cell line will grow indefinitely in vitro. Myeloma cell lines from a syngeneic (genetically identical or closely related) mouse should ideally be selected as they do not secrete endogenous immunoglobulins. Hybrid cells can be formed between a memory B lymphocyte and a syngeneic mouse myeloma cell which will give rise to a hybridoma.

This hybridoma will possess the immortality of the myeloma cell and grow and divide in culture, with the ability to produce and secrete antibody, of a predetermined single specificity, from the B lymphocyte.

One of the problems in the production of monoclonal antibodies was the development of a system which would separate these hybrid cells from single myeloma cell and single B lymphocytes. In 1975 Kohler and Milstein developed a system which overcame these problems and forms the basis of most current fusion techniques.

Sendai virus was originally used as the fusion agent to bring the myeloma cell and the B lymphocyte into close proximity allowing the two membranes to fuse forming a hybridoma. Polyethylene glycol is now the fusion agent of choice as it is much easier to handle. The separation of hybridomas from myeloma cells is performed by selecting myeloma cell lines for the fusion process that are deficient in the enzyme hypoxanthine phosphoribosyl transferase (HPRT).

HPRT deficient cells will die in culture medium which contains hypoxanthine, aminopterin and thymidine, referred to collectively as HAT. In HAT medium, aminopterin acts to block the main pathway of DNA synthesis and the salvage pathway which utilises exogenous hypoxanthine and thymidine depends on the presence of the enzyme HPRT. Since unfused single B lymphocytes which do contain HPRT will die in culture anyway and single myeloma cells, which do not contain HPRT, die in HAT medium, the only cells which survive are hybrid cells in which the myeloma provides immortality in culture while the B lymphocyte provides the enzyme HPRT to allow the cell to utilise the DNA salvage pathway. This provides a system of cell fusion together with a method for positively selecting the hybridomas that are produced.

7
Q

What needs to be done to these mice AB and what is this process called?

A

Genetic engineering of AB is required as otherwise the human body would evoke an immune response to the mice AB and produce AB against these

8
Q

Describe the process of genetic engineering

A
  • Genetic engineering can be used to modify monoclonal antibody genes to give them attributes more appropriate for particular applications.
  • Human hybridomas are difficult to produce, so one can begin with a mouse antibody, produce probes for the CDR and insert the DNA for the CDR into genes for a human Ab.
  • This allows human antibodies of a particular specificity to be produced in quantity.
  • They can be used for therapy in humans without inducing an immune response as mouse antibodies would.
9
Q

Fragments of mice AB can also be used as opposed to whole ABs - why might this be better?

A

•Other engineered versions include Fv (fragment variable). This includes no constant regions. Its pharmacological properties (more rapid and widespread distribution and longer half-life) are better than whole antibodies or even Fab fragments.

10
Q

Describe the production of AB-phage display libaries

A
  • By taking gene segment of antigens or antibodies and fusing them to the protein coat of phages, these phages will now express the anti-body in a fusion protein
  • Phage Display Libraries of antigens can be created to create anti-body phage display libraries
11
Q

Describe the steps of phage supply

What does this produce?

What is this used for?

A
  • Different sets of genes are inserted into the genomes of multiple phages
  • Proteins and peptides are fused to the Capsid (surface) of the phage
  • The combination of the phage and peptide is known as a Fusion Protein

Used for cloning foreign genes anoung other applications

Once these Phages are isolated and recovered they can be used to infect bacteria such as Escherichia coli which will create a particle similar to a monoclonal antibody

12
Q

Summary of phage disaply

A

Summary: Phage display

Use bacteriophages that propagate in E. coli to express various ligands/antibodies/peptides

Target gene is cloned into phage genome upstream of the genes encoding phage coat protein

Bacteriophage-expressed ligands are “displayed” on the surface of the phage particle produced in and released from E. coli.

Can be used for selection against a given target.

Cons:

Small- to mid-size proteins only

Lacks eukaryotic post translational modification may not be functional

13
Q

What can mAB be used for?

A

•Diagnostic Applications
Biosensors & Microarrays

•Therapeutic Applications

•Future Applications
Fight against Bioterrorism

•Clinical Applications
Imaging the target

14
Q

Possible problems with immunotherpay?

A
  • Can you get enough antibody to the site required?
  • Long term treatment causing alterations in the immune response
  • Cost
15
Q

What are the stages of vaccine development for tranditional vaccines

A
  • Several stages in vaccine development prior to licencing
  • Initial stage: identify and isolate natural or synthetic antigens.
  • Antigen products are then in a cell culture or animal models
  • Further assay development to determine the purity of raw materials, stability of the vaccine and potency of the vaccine product
  • New vaccine must then seek appropriate approval from governing bodies such as Medicines and Healthcare products Regulatory Agency (MHRA) in the UK, Food and drug administration (FDA) in the US and European Medicines Agency (EMA).
  • Once approval has been granted the process moves to clinical development where the focus is on the safety of the new medicine and whether the vaccine has any effects on the body or the body on the medicine
16
Q

Limitations of tranditional vaccines?

A
  • Limitations To Traditional Vaccines:
  • can’t grow all organisms in culture
  • safety to lab personnel
  • Expense
  • insufficient attenuation (living agent altered to become harmless or less virulent)
  • reversion to infectious state
  • need refrigeration
  • do not work for all infectious agents
17
Q

Describe new generation vaccines

A
  • Recombinant DNA technology is being used to produce a new generation of vaccines.
  • Virulence genes are deleted and organism is still able to stimulate an immune response.
  • Live nonpathogenic strains can carry antigenic determinants from pathogenic strains.
  • If the agent cannot be maintained in culture, genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e.g. E. coli.

18
Q

Describe passive vaccines

A
  • Unlike active vaccines that deliver antigens for the body to create an immune response that results in the production of antibodies,
  • Passive vaccines contain IgG antibodies that offer immediate immunity.
  • Since these antibodies come from an external source rather than being generated via an immune response, the immunity given, though instantaneous, is short-lived (Weeks to months) as the individuals immune system has not learned to make them.
  • The acquired antibodies are obtained by collecting the serum from a donor whose previously had the virulent virus, the IgG is then isolated and concentrated prior to vaccination
19
Q

What are whole inactived vaccines?

A

•Infectious virus denatured stopping the virus from being infectious, but the virus still contains antigenic properties enabling the host to develop an immune response against the virus.

20
Q

What denaturring agents/ techniques used?

A
  • Chemical inactivation agents: Hydrogen peroxide, Ascorbic acid, Psoralens, Formaldehyde, Ethylenimine derivatives, β-Propiolactone (BPL).
  • Physical inactivation methods: UV, Heat, Gamma radiation, pH
21
Q

Disavantage of WIV?

A

•Major disadvantage: large volume of antigens needed to elicit an antibody response. Therefore, further inoculations, known as “booster doses”required throughout to maintain the immunity.

22
Q

Example of WIV

Where are they grown?

Adv of WIV?

A
  • Example of such a vaccine seasonal influenza vaccine. Yearly analysis performed by WHO and CDC to determine which strains of H1N1 and H3N2 influenzas are going to be the most virulent of a given year.
  • Grown in an egg embryo for several days and are chemical.
  • Safer than attenuated viruses as the virus is killed and no longer poses any infection.
23
Q

Describe live vaccine

Adv

Disadv

Example

A
  • Attenuated vaccines: pathogenic strains in which the virulent genes are deleted or modified.
  • Live vaccines are more effective than a killed or subunit (protein) vaccines.
  • Examples of vaccines produced using method are yellow fever vaccine, measles, mumps and rubella.
  • Cost-effective, easy to mass produce, provides long-term immunity widely used
  • No Booster shot required as the virus is still alive.
  • But vaccine could potentially revert back to its more virulent form. i.e Sabin oral polio vaccine (OPV).
  • Can revert back and lead to Vaccine-Associated Paralytic Poliomyelitis cases (VAPP) and the emergence of Vaccine Derived Polioviruses strains (VDPV).
  • Rare 1 case per 6.9 million doses and mainly in children.
24
Q

Describe the use of subunit/peptide vaccines

A
  • The capsid or envelope proteins are enough to cause an immune response:
    • Herpes simplex virus envelop glycoprotein O.
    • Foot and mouth disease virus capsid protein (VP1)
    • Extracellular proteins produced by Mycobacterium tuberculosis.
  • Antibodies usually bind to surface proteins of the pathogen or proteins generated after the disruption of the pathogen.
  • Binding of antibodies to these proteins will stimulate an immune response.
  • Therefore proteins can be use to stimulate an immune response.
25
Q

Give an example of a peptide/subunit vaccine

A
  • Tuberculosis is caused by Mycobacterium tuberculosis.
  • The bacterium forms lesions in tissues and organs -> causing cell death. e.g. lung.
  • Approximately 2 billion people are infected ~3 million deaths annually.
  • Currently tuberculosis is controlled by a vaccine called BCG (Bacillus Calmette-Guerin) which is a strain of Mycobacterium bovis.
  • M. bovis often responds to diagnostic test for M. tuberculosis.
  • Six extracellular proteins of M. tuberculosis were purified.
  • Separately and in combinations these proteins were used to immunized guinea pigs.
  • These animals were then challenged with M. tuberculosis.
  • After 9-10 weeks examination showed that some combinations of the purified proteins provided the same level of protection as the BCG vaccine.
26
Q

What peptides are used for subunit vaccines?

What also might be required and why?

A

•Use discrete portion (domain) of a surface protein as Vaccine

•These domains are ‘epitopes’ (antigenic determinants) -> are recognized by antibodies

  • CARRIER PROTEINS
  • Problem Small Peptides are often Digested no strong immune response
  • Carrier Proteins Make more Stable + stronger immune response
  • Make fusion protein of carrier + vaccine peptide inert carrier or highly immunogenic carrier (hepatitis B core protein)
27
Q

What are challenges of vaccine development?

A
  • Predicting: specific antigen
    • e.g., Influenza (haemagglutinin & neuraminidase variants)
  • Not knowing the virulence determinants
    • e.g., Tuberculosis
  • Antigenic variation
  • Promoting T-cell stimulation
28
Q

What are the properties of good candidates?

A
  • •Organism – causes significant illness
    • e.g. not cold
  • •Organism – no oncogenic potential
    • e.g. not cause cancer
  • •Antibodies – block infection / systemic spread
  • •Vaccine – heat stable
29
Q

What are boosters and why are they used?

A

•Booster Shots: same vaccine given at a later date (e.g. DT given every 10 years)

–to refresh the memory cell population

30
Q

What are adjuvants in vaccines?

Why are the used and in what type of vaccines are they used in?

A

•Adjuvant: chemicals in the vaccine solution that enhance the immune response

–Vaccines such as attenuated and WIV have naturally occurring adjuvants with them, however subunit vaccines, with only components of viruses, are unable to elicit an immune response so need assistance.

–help the body by mimicking inflammatory effects of a virulent virus

– slow the diffusion of the vaccine from the injection site allowing “antigen assimilation” over a longer period of time.

31
Q

Positive/negatives of oral vaccines?

A

•Non-evasive, safe no training required. Few approved due to first line of defence i.e. stomach acid and proteolytic enzymes

32
Q

positive/negatives of intranasal vaccines

A
  • Non-invasive strong local microbial-specific immune response within the mucosal surface - nasal-associated lymphoid tissue (NALT) (Waldeyer’s ring) located in the nasopharynx at the back of the throat. IgA produced combating infection and creating immunity.
  • But lack of human compliant mucosal adjuvant, large dose of vaccine required and rapid clearance of the nasal passage which may not give the vaccine enough time to create a sufficient immune response.
33
Q

Positive/negative of IM vaccines

A
  • Direct injection into the muscle – rapidly absorbed into the vasculature decreasing the time for an immune response. Training required to elicit an immune response, recommended sites are the deltoid in the arm, Vastus lateralis muscle of the thigh, Ventrogluteal muscle of the hip and Dorsolateral muscles of the buttocks (NHS 2017)
  • The needles required for an IM injection are much larger than those required for other injectable routes
34
Q

Positives/negatives of intradermal vaccines?

A
  • Injected directly into the upper most layer of skin (dermis) more effective than intramuscular or subcutaneous injections at eliciting an immune response. Due to the greater numbers of dendritic cells (DC’s) in the dermis compared to the other layers. Dendritic cells phagocytose the antigens and present them to T-cells activating them.
  • Increased skill level required as it is a more delicate process due to the thinness of the dermis.
  • ID syringes which are thinner and smaller than average syringes.
  • ID injections also appear to have an increased amount of adverse effects compared to subcutaneous and intramuscular injections
35
Q

Describe delivery via SC route

A

•Vaccine injected into the subcutaneous layer. Training and specialised equipment is needed to specifically reach this layer. Once administered are transported from the injection site (Interstitial space) by diffusion to the lymphatic capillaries resulting in an immune response.

36
Q

Describe vaccine delivery by IV

A

•Tends not to be used to deliver vaccines due to a low immune response and potential adverse effects.

37
Q

Describe Anti-body titer

A
  • A test to measures the presence and amount of antibodies in blood against a particular type of tissue, cell, or substance
  • Titer determines if you have adequate protection against a disease
  • May need to give booster if titer too low
  • E.g., happens with HepB vaccine
38
Q

Dewscribe herd immunity

A
  • Every virus has a herd immunity threshold depending on how contagious they are
  • Herd immunity is generally achieved when 70-90% of the population is immune
  • How contagious a virus is, is determined by the basic reproductive number R0
  • Indirect protection from infection among susceptible members of a population, and the protection of the population as a whole, due to the presence of the immune individuals
  • Therefore, leads to reduction of transmission in a population (sometimes can lead to the disappearance of the disease)
39
Q
A