Microbio Topic 15: Immunization & Serology

Immunization (artificial active/passive, herd)

Artificial active: vaccine
Artificial passive: anti sera
Herd immunity: one person not getting vaccinated and relying on everyone else to get vaccinated

History of Immunization

Variolation: first used in 10th century china for small pox (used crusted puss and shot it up peoples noses)

Edward Jenner: father of immunology developed smallpox vaccine

Passive was developed in the 20th century once the nature of antibodies became known

Vaccination Problems

Economic problems still prevent people in developing countries from having adequate access to vaccines

Inability to develop effective vaccines for some pathogens

Financial considerations

Live Attenuated Vaccines

Living avirulent (not pathogenic) used to trigger immunity

Live Attenuated Vaccines Advantages

very effective at triggering strong active immune responses leading to long term immunity (memory cells),
contact immunity (vaccinate some, share immunity)

Live Attenuated Vaccines Disadvantages

short shelf life, expensive,
risk to pregnant and immunecompromised recipients, small chance of reversion

Inactivated Vaccines

Intact killed/inactivated pathogens

Inactivated Vaccines Advantages

Longer shelf life, easy to store
Safer than live: no potential for replication/reproduction or reversion

Inactivated Vaccines Disadvantages

Weak: must be high dosage or series of boosters or combination with adjuvants
Adjuvants can trigger allergic responses
No possibility for contact immunity

Subunit Vaccines

Antigenic component of a pathogen

Subunit Vaccines Advantages

Safer than live: no potential for replication/reproduction or reversion

Subunit Vaccines Disadvantages

Reduced shelf life vs whole agent vaccines
Reduced antigenic diversity
Weak: must be high dosage or series of boosters or combination with adjuvants
Adjuvants can trigger allergic responses
No possibility for contact immunity

Conjugate Vaccines

Advanced form of subunit vaccine

Antigenic protein conjugated with an antigenic capsule polysaccharide
-protein triggers T-dependent polysaccharide triggers T-independent

Conjugate Vaccines Advantages

Safer than live
More effective than subunit vaccines involving polysaccharide antigens for triggering immunity in children under 2

Conjugate Vaccines Disadvantages

Similar to subunit and more expensive

Toxoid Vaccines

Inactivated bacterial toxins

Toxoid Vaccines Advantages

Safer than live
Stimulates antibody mediated immunity to the toxin

Toxoid Vaccines Disadvantages

Doesn't prevent infection
Requires multiple doses often in combination w/ adjuvants (aluminum salts to help stimulate body's response)
Shorter duration of immunity
No possibility for "contact" immunity

mRNA Vaccines

mRNA encoding for antigen introduced and expressed

mRNA Vaccines Advantages

Safer than live
Faster to develop and easily scalable

mRNA Vaccines Disadvantages

Can require multiple doses
Shorter immunity
No possibility for contact immunity
Cryogenic storage and transportation required

Recombinant DNA Technologies for Immunization: Expression of Antigens by Non-Pathogenic Microbes

Harvesting of purified antigen expressed by the recombinant microbe

Use of the recombinant microbe itself as the vaccine

Recombinant DNA Technologies for Immunization

Removal of virulence genes to create attenuated strains with no potential reversion

Insertion of antigenic genes into crops

Vaccine Safety

Mild toxicity/ allergy at injection site
Anaphylactic shock (severe allergic response)
Reversion
Andrew Wakefield claims the MMR causes autism (false)

Passive Immunization: administration of antibodies to patient

Used when protection against a recent infection or an ongoing disease is needed quickly

Passive Immunization: Antisera

Contains antibodies collected from donors (polyclonal) or produced in cell cultures (monoclonal)

-antitoxins
-antivenins
-antivirals

Limitations of Antisera

Polyclonal antibodies bind many different antigens, increasing chances of cross reactivity

Antiserum could be contaminated with viral pathogens

Antibodies provided by Antisera are degraded relatively quickly

Patient can have an immune response to animal antibodies

Serological Testings

Using know antibodies/antigens to detect specific antibodies/antigens

Precipitin Reactions Principle

SOLUBLE antigens and matching antibodies mixed in the proper proportion will agglutinate, forming large complexes (precipitins) that precipitate out of solution

Precipitin Reactions Interpretation

If preciptin is observed the solution contains a matching antibody/antigen pair

Precipitin Reactions problem

Preciptin only observed when antigen:antibody ratio is within the equivalence zone

Precipitin Reactions Ring Test

Used to determine titer (relative amount of an antibody in serum)

Titer is the reciprocal of the highest dilution giving a positive result

Double Immunodiffusion (Ouchterlony) Assay

Antibody and antigen solutions are loaded into wells on an agar plate

Diffusion yields concentration gradients radiating outward from each well

Precipitin arcs form between matched pairs

Radial Immunodiffusion (RID) Assay

Agar contains a known antibody (ex rabbit anti human antibodies) concentration

Antigen solution of increasing concentration (ex serum containing human antibodies) loaded into standard wells and allowed to diffuse

Zones of precipitation will form around the standard wells

Additional sample well loaded with serum. Zone diameter of sample well can be measured and compared to standard wells -> quantifying antigen concentration

Immunoelectrophoresis

Antigen mixture loaded onto a central well in a gel

Electrophoresis separates antigens into discreet regions within the gel (by charge and mass)

Antibodies loaded into a trough parallel to the row of separated antigens

Precipitin arcs form where pairings occur

Flocculation Assays Principle

INSOLUBLE antigens in suspension (lipids) and antibodies mixed in the proper proportion will form lattice that results in flocculation (macro- or microscopic foaming)

Flocculation Assays Interpretation

If flocculation (clumping) is observed-> the solution contains a matching antibody/antigen pair = positive result

Agglutination Assays

Similar to Precipitin assays, but antigens are associated with solid cells or particles

Agglutination causes clumps to appear in solution (visible to naked eye)

Agglutination Assays Direct

Involved antibodies reacting directly with an antigen

• clumping of bacterial cells due to bound antibodies

Agglutination Assays Indirect

Linking antibodies to latex beads that connect those antibodies to antigens (vise versa)
Greater sensitivity

Hemagglutination Assay

Clumping of RBC (blood typing, cross matching)

Hemagglutination Assay Direct Coombs Test

Direct Antiglobulin Test (DAT)

Blood sample taken from patient hemolytic anemia with antibodies attached to RBC -> add coombs reagent (anti human antibodies) -> agglutination reaction (clumping) is visible after cross-linkage of antibodies

Antibodies added to blood

Hemagglutination Assay Indirect Coombs Test

Indirect Antiglobulin Test

Patient serum containing antibodies -> donor blood added -> patient's antibodies bind to donors RBC -> coombs reagent mixed in with sample -> agglutination (clumping) is observed

Viral Hemagglutination

Clumping of RBC as they become cross-linked by virions

Viral Hemagglutination Direct Hemagglutination Assay (HA)

Detects viruses

• patients serum mixed in with RBC. Of virus is present, Hemagglutination may be observed (flu, mumps, rubella)

Viral Hemagglutination Hemagglutination Inhibition Assay (HIA)

Detects antibodies against a virus
-patients serum mixed with virus, the RBC added. If antibodies against virus are present, the virus will be neutralized and Hemagglutination will NOT occur

Neutralization Assays

Toxins (and some viruses) can alter cellular appearance
-cytopathic effects: visible changes in the morphology of virally-infected cells

Neutralization Assays Testings

Toxin/virus is mixed with patients serum, then inoculated to cell culture plate

If antibodies to the toxin/virus were present in the serum, morphological change will not be observed

Complement Fixation Test Use (complement mediated immunoassay)

Detection of very low concentrations of antibodies in serum

• too low to cause precipitation, agglutination, or neutralization

Complement Fixation Test Basis (complement mediated immunoassay)

Even a few bound antibodies are enough to trigger complement: patients serum is heated to destroy pre-existing complement proteins -> antigen added -> complement proteins are added -> sheep's RBC and anti sheep antibodies are added -> examine for lysis of sheep's RBC (negative result, non reactive)

Labeled Antibody Tests: FEIAs and fluorescent immunoassays

Immunohistochemistry: Stain tissues

Immunocytochemistry: stain subcellular structures

Direct Immunofluorescence: used to stain specific cells (TB)

Labeled Antibody Tests: Indirect Immunofluorescence

Used to detect specific antibodies in serum

Antigen -> patient serum added, if antibodies are present they'll bind to antigen -> second antibody (marker) binds to patient's antibody

Labeled Antibody Tests: ELISA

enzyme-linked immunosorbent assay

Enzyme is used as the label
-enzyme catalyzes a color changing reaction indicates positive test

Used to detect the presence of specific antibodies in serum

Labeled Antibody Tests: "antibody sandwich" ELISA

Modification of the ELISA technique, allowing for detection of antigens rather than antibodies

Antigen being tested for is "sandwiched" between two antibody molecules (the first being bound to the well)

Labeled Antibody Tests: western blot

Electrophoresis-based method used for detecting specific antibodies or antigens in a complex mixture

Western blot testing

Battery of antigens in solution are separated on a gel using electrophoresis -> antigen bands transferred from gel to nitrocellulose paper -> paper is washed with antibodies, antibodies will bind to matching antigen -> labeled anti-antibodies applied. Bind to any antibodies which are already bound to antigen bands -> observation identifies which specific antibodies or antigen were present in serum

Immunochromatographic Assay (lateral flow, strip test)

Fluid (potentially containing antigens) is applied to absorbent pad and flows by capillary action

fluid flows past region embedded with beads (gold or latex) with attached antibodies. If antigens present, complexes form

beads mobilized and flow over two more stripes
-TEST stripes with immobilized anti-antigen antibodies: captures bead with attached complexes
-CONTROL stripe captures any beads not bound by the test stripe (those lacking complexes)

Color changes at test and control stripe to give result

Flow cytometry

Counts fluorescently- labeled cells and unlabeled cells

Fluorescence activated cells sorters are FCs that can physically sort cells based on fluorescence differences