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MICR 270 > module 5- vaccines and translational immunology > Flashcards

module 5- vaccines and translational immunology Flashcards

1
Q

immunological techniques

A
  • ELISA
  • FLOW cytometry
  • monoclonal antibodies
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2
Q

what is ELISA

A

enzyme-linked immunosorbent assay

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3
Q

what does ELISA do?

A

is a fundamental tool of clinical immunology based on the principle of antigen-antibody interactions
- a applicable technique that can be codified to detect and quantify substances such as peptides, proteins, antibodies, hormons +

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4
Q

what does flow cytometry do

A

is a method of detechting and quantifying different cell types in a mixed cell suspension

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5
Q

what does a monoclonal antibodies do

A
  • the production of monoclonal antibodies is a technique that was developed by george kohler and cesar milsteins in 1975
  • are antibodies that are produced by a single clone of a B-cell that are specific for a single epitope
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6
Q

epitope

A

the portion of the antigen that is recognized and bound by an antibody

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7
Q

what is ELISA highly specific

A
  • based on the principle of antigen-antibody interactions
  • antigen binding site is very specific to one antigen
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8
Q

how does ELISA work? step 1

A
  • the bottom of the wells are coated with an antigen that is specifically recognized by the antibody you wish to measure (refereed to as the primary antibody in this procedure )
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9
Q

how does ELISA work? step wash (1)

A

the wells are washed to remove any excess antigen not attached to the bottom of the well

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10
Q

how does ELISA work? step 2

A

the sample containing the antibody to be measured (serum) is added to the well. the primary antibodies if present will bind to the antigen attached to the bottom of the well

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11
Q

how does ELISA work? step wash (2)

A

the wells are washed again to remove excess primary antibody not attached to the bound antigen as well as any other sample components that might interfere with subsequent steps

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12
Q

how does ELISA work? step 3

A

an enzyme-conjugated secondary antibody is added to the well
- this secondary antibody will bind to the FC portion of the primary antibodies already present in the well
- secondary antibody used specifically recognizes antibodies from a particular animalr

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13
Q

how does ELISA work? step wash (3)

A

the wells are washed to remove any excess secondary antibody not attached to the primary antibody

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14
Q

how does the ELISA work? step 4

A
  • substrate of the enzyme attached to the secondary antibody is added to the well
  • the reaction substrate (a chromogen) and the enzyme produces a coloured product which can be measured by absorbance
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15
Q

chromogen

A

a substance that can be readily converted into a dye or other coloured compound

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16
Q

what is flow cytometry?

A

a technique designed to detect and quantigy different immune cells in a mixed cell suspension

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17
Q

what does the flow cytometry measure

A
  • physical properties of a cell
  • detect specific antigens on or inside a cell
  • the total number of cells in the suspension, the suspension and the overall composition of the suspension can be readily determined
  • used to determine complete blood counts (CBC)
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18
Q

how does flow cytometry work?

A
  1. narrow stream of cells in single file is passed through a laser light source
  2. the way the laser light is scattered is unique to each cell type; this can be detected and analyzed
  3. measuring FCS allows for the discrimination of cells by size
  4. FCS intensity is proportional to the diameter of the cell
  5. SSC provides information about the internal complexity of cell
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19
Q

what else can flow cytometry be used to determine?

A

the proportion of cells expressing a particular antigen (cells labelled with specific antibody)
- antibody is coupled wit ha flurorescent marker (can be excited by a light of a specific wavelength
- fluorescent marker emits a light with a characteristic different wavelength (only cells expressing the antibody in question will emit light of this specific wavelength

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20
Q

what is the production of monoclonal antibodies used for?

A
  • research
  • diagnosis
  • therapeutic purposes
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21
Q

what does monoclonal antibodies measure

A
  • not a specific measurement it is a tool
  • there are clinical application of monoclonal antibodes
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22
Q

what are the clinical applications of monoclonal antibodies?

A

immunotoxins and radiolabelled antibodies

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23
Q

immunotoxins

A
  • consist of a tumor-specific monoclonal antibody attached to a deadly toxin
  • technique still under investigation but a long-term objective is to use immunotoxins to target and eliminate tumor cells and treat cancer
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24
Q

radiolabeled antibodies

A

monoclonal antibodies tagged with a radioactive isotope can be used to diagnose tumors earlier than other methods
- can bind to antigens on a tumor thereby allowing the precise location of a tumor to be visualized

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25
Q

how does monoclonal antibodies work?

A
  • produced in lab by hybridomas, immortal cells that produce unlimited quantities of one identical antibody
  • hybridomas are the result of fusion between a plasma cell and canerous (myeloma) cell (share properties of both)
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26
Q

hybridoma cell

A

a perpetual source of antibodies against one antigen

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27
Q

types of vaccines

A
  • live attenuated vaccine
  • killed -inactivated vaccine
  • toxoid vaccine
  • subunit vaccine
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28
Q

what is a vaccine

A
  • is a type of biological preparation which provides active artificial immunity to a particular disease-causing agent
  • dependent on the nature of the disease and how the immune system recognizes and response to the infection
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29
Q

characteristics of live-attenuated vaccine

A
  • contains a modified strain of the disease-causing agent which has lost its pathogenic ability
  • retains it capacity to replicate within the host
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30
Q

pathogenic

A

ability of an organism to harm the host by causing a disease

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31
Q

advantages of live-attenuated vaccine

A
  • provides a prolonged exposure to the disease-causing agent
  • suitable to generate cell-mediated immunity
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32
Q

disadvantages of live-attenuated vaccine

A
  • potential to revert to a virulent form
  • requires specific storage and transport conditions
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33
Q

examples of live-attenuated vaccine

A
  • small pox vaccine
  • oral poliovirus vaccine (sabin)
  • measles vaccine
34
Q

characteristics of killed-inactivated vaccine

A
  • contains a strain of the disease-causing agent that has been inactivated by heat, chemcials or radiation
  • has the ability to generate an immune response, but unable to replicate
35
Q

advantages of killed-inactivated vaccine

A
  • safer option as it cannot mutate back to a virulent form
  • easy to store and transport
36
Q

disadvantages of killed-inactivated vaccine

A
  • generally requires multiple booster doses to maintain immunity
  • generally must be administered by injection
37
Q

examples of killed-inactivated vaccine

A
  • rabies vaccine
  • flu vaccine
38
Q

characteristic’s of toxoid vaccine

A
  • contains an inactivated toxin which is a product from the pathogen that is causing the disease
39
Q

advantages of toxoid vaccine

A
  • safe as it is not living organism that can divide, spread and/or revert
  • stable as they are less susceptible to changes in temp, humidity and light
40
Q

disadvantages of toxoid vaccine

A
  • may requie several doses and usually need an adjuvant
41
Q

adjuvant

A

a substance that enhances the bodys immune response to an antigen

42
Q

examples of toxoid vaccine

A
  • tetanus vaccine
  • diphtheria vaccine
43
Q

characteristics of subunit vaccine

A

contains only a small part of fragment of the disease-causing agent

44
Q

advantages of subunit vaccine

A
  • safest type of vaccine, can be used on everyone, including immunocompromised, pregnant, and elderly populations
45
Q

disadvantages of subunit vaccine

A
  • rarely successful at inducing long-lasting immunity, which means it will require multiple booster doses to maintain immunity and might need to be conjugated to a carrier
46
Q

carrier

A
  • a stronger antigen than the desired target antigen
  • by covalently attaching a strong antigen to a poor antigen, the overall immunological response is strengthened and hopefully, response to the poor antigen is also improved
47
Q

example of subunit vaccine

A

hepatitis B vaccine

48
Q

about mRNA vaccines

A
  • most recent vaccine type that have changes the fueld of vaccinoogy
  • known for their use against SARS-CoV-2 (covid)
  • used in several formulations to fight the virus (boosters, vaccines)
49
Q

the princple of the assay (mRNA)

A

relies on the use of mRNA to produce viral proteins and recruit immune cells to respond to the antigenic target
- proteins then displayed on surface of an APC to induce B-cells and T-cell immunity

50
Q

RNA and DNA based vaccines

A

involve making genetic material only
- they do not require the use of the whole virus

51
Q
  1. vaccine production of mRNA vaccine
A
  • mRNA is made in lab from a DNA template of the virus
  • it encodes an antigen of the virus
    (for Covid, the mRNA encodes the spike protein on the surface of the virus)
  • the mRNA is incorporated into a formulation that can be administered as a vaccine
52
Q

mRNA vaccine mechanisms

A
  1. vaccine production
  2. host cell
  3. APC
  4. immune response
53
Q
  1. host cell
A

once inside the body, mRNA enters the host cell and uses host cell machinery to produce the spike protein

54
Q
  1. APC
A
  • newly formed spike exits the cell and is recognized by an APC
  • APC internalized the spike protein and processes it into a peptide (antigen)
  • APC then displays the antigen on the surface of the cell via MHC
55
Q
  1. immune response
A
  • the antigen is recognized by a T-cell helper, which initiates an immune response
  • b cells produce antibodies that stop the virus from infecting cells
  • t-cell destroys cells infected with the virus
56
Q

antivirals medications against COVID-19

A

(only treated it once you already tested positive)
- does not prevent covid

57
Q

what are the antiviral medications for covid?

A

polyermase inhibitor
protease inhibitor

58
Q

polyermase inhibitor

A
  • the first antiviral meducation
  • is a enzyme that plays a crucial role in viral replication and transcription

molnupiravir- is a polymerase inhibitor used to treat covid. it increases the frequency of viral RNA mutations and impairs replication’s of the virus

59
Q

protease inhibitor

A

protease cuts proteins into smaller, more workable pieces
- often adminsitered in combination
- nirmatrelvir stops protease from cutting viral proteins into functional peices
- ritonvair protects nirmatrelivir from destrcution by the body and allows it to keep working
- stops it from replicating and infecting other cells

60
Q

evolution of vaccines

A

Dr. Edward Jenners use of extracts prepared from cowpox lesions as a means of protecting his patients against smallpox infection
- vaccination derived from the latin word for cow and was first coined by louis pasteur in 1881 to honor jenners accomplishment
- jenns was 100 years before discovery of virus by Dmitry Ivanovsky 1892

61
Q

vaccine success story: HPV VLP vaccines

A

cerival cancer and genital warts are caused by prior infection with certain types of human papillomavirus (HPV)
- 2002, clincial trial demonstrated that a virus like particle (VLP) vaccine could protect against infection by a type of HPV often associated with cervical cancer
- the efficacy reported in this clinical was 100% and subsequent larger scale clincial trials continued to report efficacies approaching 100%

62
Q

VLPs

A

are composed of the structural proteins of HPV, which can self assemble into particles that resemble the natural virus both structurally and immunologically
- as they do not contain viral DNA, VLPs are not infectious

63
Q

what are the three HPV vaccines currently authorized

A
  • cervrix
  • gardasil
  • gardasil 9 (9 means can protect against 9 different types of HPV)
64
Q

success story: ebola vaccines

A
  • ebola virus (EBOV) and marburg virus (MARV) are part of the filoviridae family of viruses that cause hemorrhagic fever with high mortality rate in humans and nonhuman primates
  • these virus produce transmembrane glycoproteins thought to play a role in the virulence of these viruses
65
Q

2000 ebola

A

Winnipeg inadvertently discovered that mice infected with EBOV and MARV glycoproteins became protected against infection from live virus

  • development of petenially viable vaccine using glycoproteins from EBOV and putting them into a live attenuated recombination vesicualr stomatitis virus (VSV) that expresses the transmembrane glycoproteins of EBOV and MARV
66
Q

2005 ebola vaccines

A

canadia research team tested this vaccine the 2000 vaccine on macaques (old world monkeys) and found it to be 100% effective against EBOV infection

67
Q

failure story: genital herpes vaccine

A

a subunit vaccine was developed which was composed of a prominent structural protein of HSV-2 (the type most commonly associated with genital infections)
- the vaccine showed promise in early trials with 70% efficacies in women who were seronegative for both HSV-1 and HSV-2 at the beginning of the trial
- larger trial was 20% efficacy and no protection from HSV-2

68
Q

what did the genital herpes vaccine cause

A

researchers in the field to consider developing live attenuated vaccines for genital herpes and has spurred basic research efforts aimed at better understanding and augmenting immune responses in the genital tract

69
Q

what is genital herpes

A
  • caused by infection with herperes simplex virus (HSV) types 1 or 2
  • are permanent
  • require lifelong mamgement
  • serious health threat to new borns through vertical transfer
  • emotional distress in infected individuals
  • increase susceptibility to and spread of HIV infection
70
Q

vertical transfer

A

is the passage of a disease-causing agent from mother to baby during the period immediately before and after birth

71
Q

seronegative

A

giving a negative result for a test of blood serum for the presence of a host antibody response against a foreign particle/pathogen, such as HSV-1 and HSV-2 virus
- informs you of 1. IgG vs IgM levels, indicating when the host was exposed to the pathogen 2. if the host has ever been exposed to a pathogen 3. if host has ever been vaccinated

72
Q

phases of vaccine development

A
  1. lab studies
  2. preclinical
  3. clinical phase I
  4. clinical phase II
  5. clinical phase III
  6. health Canada approval
73
Q
  1. lab studies
A
  • identify the infectious agent causing the disease and select a strain (or subtype)
  • that is relevant to the target population
  • dependent on research carried out in the laboratory utilizing assays, which may involve exhaustive screening to identify a suitable antigen
  • creation of vaccine concept
74
Q

what else does lab studies involve

A
  • developing and testing the manufacturing process of the vaccine according to GOOD MANUFACTURING PRACTICE standards
75
Q
  1. preclinical
A
  • involves research carried out in animal models to evaluate the pharmacological aspects of the product
76
Q

clinical phase I

A
  • due to rigorus testing requirements, only a very small % of vaccines progress to licensing, making the costs of vaccine research and development extremely high
  • invloves 3 phases spanning over servela years
77
Q

phase I clinical trails for vaccines

A

involve small scale trials in humans to asses vaccine safety by evaluating local and systemic reactions after administration
- provides preliminary data on the immunogenicity of the vaccine and the immune response it evokes

78
Q

clinical phase II

A
  • occur at bigger scale (50-500) to collect data on the safety, side-effects, and efficacy of the vaccine
  • evaluates the dosage requirements of the vaccine
  • mointor the effects of increasing vaccine dosage and conduct challenge tests to define the optimal dose and vaccination schedule
79
Q

clinical phase III

A
  • involve multiple geographic sites with many hundreds of subjects (300-30000) to evaluate efficacy under natural disease condition
  • researchers are required to demonstrate the efficacy in target populations and complete a safety assessment for the vaccine
  • if successful manufactur will then apply to the regulatory authorities for a license to market
80
Q

health canada approval

A
  • is the regulatory authority in Canada responsible for ensuring the quality, safety, and efficacy of all biologic drugs, including vaccines for human use
  • regulation is necessary
  • vaccine candidates must be submitted to health Canada to be considered for approval with sufficient scientific and clinical evidence to show that it is safe

MICR 270 (4 decks)

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