Chapter 18: Practical Applications of immunology

vaccine

• contains proteins from the organism to be targeted

• nucleic acids encoding those proteins (DNA or mRNA vaccines)

• disabled forms of the organism itself = body’s immune defenses against pathogen

types of vaccines

1. attenuated vaccines

2. inactivated vaccines

3. subunit vaccines

4. recombinant

5. toxoids

6. VLP

7. polysaccharide vaccines

8. conjugated vaccine

9. DNA

10. mRNA

essential elements of diagnostic tests

1. sensitivity

2. specificity

sensitivity

probability that test is reactive if the specimen is a true positive (catch disease when its there)

specificity

probability that test will NOT be reactive if a specimen is a true negative (rule out disease when its not there)

precipitation reactions

involve the reaction of soluble antigens with IgG or IgM antibodies

occurs in 2 stages / steps…

1. antigens and antibodies rapidly form small antigen-antibody complexes (tiny clumps)

2. antigen-antibody complexes form larger interlocking molecular aggregates (molecules stick together) called lattices that precipitate from the solution

3. forms the zone of equivalence (precipitin ring test)

4. involves soluble antigens

zone of equivalence (precipitin ring test)

cloudy line of precipitation will appear in the area in which the optimal ratio has been reached

agglutination reactions

involves either particulate antigens (particles such as cells that carry antigenic molecules) or soluble antigens adhering to particles

• agglutination antibodies bind to antigens on particles and cross link them into visible clumps

• (particulate antigens + antibodies = visible clumps)

• IgM = most efficient antibody, IgG = less efficient

• direct and indirect tests

particulate antigens

antigens that are already on cells

direct agglutination test

detect antibodies against naturally occurring cell bound antigens, detect antibodies against relatively large cellular antigens on RBC, bacteria, fungi

direct agglutination process

1. add antigen (bacteria) to wells

2. add diluted patient serum (contains antibodies)

3. look for clumping

titer - highest dilution of serum that still causes agglutination

indirect (passive) test

used when antigens are soluble (not naturally on cells); the antibody reacts with the attached antigen or in reverse by using particles coated with antibodies to detect the antigens against which they are specific for

(use coated particles to detect soluble antigens or antibodies)

two formats

1. detect antibodies (use antigens)

2. detect antigens (use antibodies)

indirect agglutination process

1. attach antigen or antibody to particles like latex beads

2. add patient sample

3. if target is present = beads clump

neutralization reactions

is an antigen-antibody reaction in which antibodies block the harmful effects of a bacterial exotoxin or block viruses from infecting cells

• uses antitoxin

antitoxin

antibodies to toxin, binds to the exotoxin and blocks its toxic effect before damaging cell

hemagglutination

specific type of agglutination reaction involving RBCs (clumping of RBCs caused by antibodies binding to antigens on RBCs)

ELISA

“Enzyme linked immunosorbent assay”

• is a doble antigen test

• theres 2 basic methods (direct and indirect)

variolation

small prevention procedure involving inoculation of material from dried small pox scabs into the respiratory tract or skin (1400s in china-1700s)

herd immunity

immunity in most of the population

• outbreaks are sporadic due to the lack of susceptible individuals

attenuated vaccines

• Weakened live pathogen, reduced virulence

• Closely mimic an actual infection

• Vaccine organisms replicate in the body, magnifying the effect

• Confers lifelong immunity (both humoral and cellular)

• Not given to immunocompromised patients

• Risk of mutating back to virulent form

  • Problem with the oral polio vaccine

inactivated vaccines

• Whole microbes are killed or inactivated

• Safer than attenuated vaccines

• Require repeated booster doses

• Induce mostly humoral immunity

subunit vaccines

use antigenic fragments to best stimulate an immune response (avoids dangers associated with use of live or killed pathogenic organisms)

recombinant vaccines

subunit vaccines produced by genetic modification of yeast or insects (non pathogentic microbes) to produce desired antigenic fraction

• is a product of recombinant DNAs

• ex. hepatitis B vaccine, the capsid grown in recombinant yeast

toxoids

inactivated toxins produced by a pathogen and elicit an antibody response against that particular toxin

• diphtheria, tetanus

virus like particle (VLP) vaccines

resemble intact viruses but do not contain viral genetic material

ex. HPV

polysaccharide vaccines

made from molecules in pathogen’s capsule; not very immunogenic

• ex. pneumococcal vaccine

conjugated vaccines

polysaccharide antigen is attached to a protein (made to deal with children’s poor immune response to vaccines based on capsular polysaccharides)

• Children < 2 years old, do not respond to T- independent antigens like capsular polysaccharides

• Attaching polysaccharide to a protein carrier (conjugate): makes the vaccine immunogenic in babies as young as 2 months

ex. diphtheria or tetanus toxoid

DNA vaccines

• Injected naked or encapsulated DNA into muscle (that encodes specific protein antigens)

• DNA directs the synthesis of mRNA (transcription, translation) and produces the protein antigen encoded in the DNA

• Stimulates humoral and cellular immunity

mRNA vaccines

• mRNA enclosed in a lipid nanoparticle is injected into muscle where it directs the synthesis of the encoded antigen

• COVID-19 vaccines direct synthesis of spike protein antigen

vaccine production

produced by growing pathogen in animals, embryonated eggs or cell cultures

adjuvants

• Additives to a vaccine that improve its effectiveness

  • Alum (aluminum salts)

  • Monophosphoryl lipid A (derivative of LPS)

• Improve the innate immune response, activation through Toll-like receptors

vaccine administration

• Oral vaccines: favored due to ease of administration and effectiveness against pathogens that enter through the GI tract

  • Vaccines for rotavirus, adenovirus, cholera, typhoid

• Nasal vaccine: attenuated influenza vaccine

• Skin patch vaccines: (NanopatchTM) - administers a dry formulation of a vaccine

• Multiple-combination vaccines

Immunologic-based diagnostic tests

• interactions of humoral antibodies with antigens

• Known antibody can identify an unknown pathogen

• Known pathogen can determine the presence of an unknown antibody

hybridoma

“immortal” cancerous B cell (myeloma) combined with an antibody-producing normal B cell that when grown in culture produces the type of antibody characteristic of the ancestral b cell indefinitely

• produces monoclonal antibodies (MABS)

  • Nearly unlimited quantities of identical antibody, same specificity as the ancestral normal B cell

  • No contamination by other antibodies

  • Highly specific

monoclonal antibodies

Mabs are uniform, highly specific, and produced in large quantities

• Used in diagnostic tools

• Used in human therapy

  • Treatments for multiple sclerosis, Crohn’s disease, psoriasis, cancer asthma, arthritis, COVID-19

• Often derived from mouse B cells, leading to side effects

viral hemagglutination

occurs when viruses agglutinate RBCs without an antigen-antibody reaction

Viral hemagglutination inhibition test

is used for subtyping viruses (to detect antibodies to a virus)

– Viruses and RBCs are mixed with the patient's serum, If the serum contains antibodies to a virus, they neutralize the virus and inhibit hemagglutination

(red blood cells + antiviral antibodies serum + virus = virs neutralized and hemagglutination inhibited)

Complement fixation

complement serum protein binds to and is fixed to the antigen–antibody complex (The test checks whether a patient’s serum contains antibodies to a particular pathogen)

• Detects small amount of antibody

• Works for antibodies that do not work with precipitation or agglutination reactions

• Use to diagnose certain viral, fungal, and rickettsial diseases

direct ELISA

detects antigens

1. have well filled with antibodies for antigens

2. sample containing target antigens is mixed with antibody

3. enzyme linked antibodies is mixed in and reacts with antigen bounded to original antibody

4. substrate attaches to the enzyme detecting antibody and a color is produced (positive test)

indirect ELISA

detects antibodies

1. have well filled with antigens

2. patient serum is added, complementary antibody binds to antigen

3. enzyme linked anti-HISG is added and binds to antibody bound to original antigen

4. substrate is added to enzyme linked anti HISG and reaction produces a color change (positive test)

rapid antigen test

(similar to ELISA, uses dye molecles instead of enzymes); is a double antigen test and type of lateral flow immmuno assay

• Nasal swab sample which may contain SARS-CoV-2 antigens is applied to a paper strip

• If SARS-CoV-2 antigens are present, as they flow through the strip they will be captured by labeled antibody—forming a colored line on the strip

control line

test validation

test line

forms a colored line on the strip

lateral flow assay

allow for detection of viral antigens by their binding to labeled antibodies, then attached to a paper strip