Touro PA Microbiology Lecture 13 Immunology And Serology Fall 2023.ppt

Immunology and Serology © McGraw Hill, LLC 1 A Glimpse of History Variolation dates from Sung dynasty (960 to 1280) • Powdered scabs from smallpox lesions inhaled or placed in skin; produced mild disease, gave immunity • Occasionally fatal; person also contagious Because of dangers, many Europea...

Immunology and Serology © McGraw Hill, LLC 1 A Glimpse of History Variolation dates from Sung dynasty (960 to 1280) • Powdered scabs from smallpox lesions inhaled or placed in skin; produced mild disease, gave immunity • Occasionally fatal; person also contagious Because of dangers, many Europeans remained unprotected until 1796 when Jenner exposed boy to cowpox; 6 weeks later exposed him to smallpox • Boy did not develop smallpox; he was immune • Jenner and others worked to spread variolation using less dangerous cowpox material Pasteur used the word vaccination for protective inoculation In 1967, WHO started smallpox vaccination program; last wild type case occurred in Somalia, Africa, in 1977 • Virus still present in laboratories; U.S. has vaccine stockpile © McGraw Hill, LLC 2 Immunization Immunization is a process of inducing immunity • Has probably had greatest impact on human health of any other medical procedure • Immunotherapies are methods designed and used to enhance or suppress immune responses as a means to treat certain diseases. • Useful applications of immunological reactions in diagnostic tests Access the text alternative for slide images. © McGraw Hill, LLC 3 Principles of Immunization 1 Naturally acquired immunity is gained through normal events, such as illness Artificially acquired immunity is gained through immunization Active immunity results from exposure to antigen • Lymphocytes are activated; memory cells provide longlasting protection • Natural by infection; artificial through vaccination Passive immunity results from addition of other’s antibodies • Temporary protection; no memory cells formed © McGraw Hill, LLC 4 Principles of Immunization 2 Natural passive immunity • During pregnancy, mother’s IgG antibodies cross placenta • Breast milk contains secretory IgA Artificial passive immunity • Injection of antiserum containing antibodies • Can prevent disease before or after likely exposure • Limit duration of certain diseases • Block action of microbial toxins • Antitoxin is antiserum that protects against a toxin • Hyperimmune globulin (antibodies to specific disease) • Immune globulin (IgG fraction from many donors; variety of antibodies) © McGraw Hill, LLC 5 Principles of Immunization (top left): ©SPL/Science Source; (top right): ©Jose Luis Pelaez Inc/Blend Images LLC/Getty Images; (bottom left): Jill Braaten/McGraw-Hill Education; (bottom right): ©SPL/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 6 Vaccines and Immunization Procedures Vaccine is preparation of a pathogen, or its products used to induce active immunity • Protect individual; prevent spread in population • Herd immunity develops when critical portion of population is immune to disease; infectious agent unable to spread due to insufficient susceptible hosts • Responsible for dramatic declines in childhood diseases • Diseases sometimes reappear and spread as result of failure to vaccinate children • Effective vaccines should be safe, have few side effects • Give long lasting protection • Ideally low in cost, stable, easy to administer © McGraw Hill, LLC 7 Table 17.1 A Comparison of Characteristics of Attenuated and Inactivated Vaccines Two general categories of vaccines • Attenuated vaccine (agent can replicate): stronger immune response, but may cause disease • Inactivated vaccine (agent cannot replicate): weaker immune response, but cannot cause infections © McGraw Hill, LLC 8 Attenuated Vaccine Weakened form of pathogen • Grown under conditions that foster mutations, or genetically manipulated to replace genes – both lower pathogenicity • Advantages: • Single dose usually induces long-lasting immunity due to microbe multiplying in body • Can inadvertently immunize others by spreading • Disadvantages: • Can sometimes cause disease in immunosuppressed individuals • Can occasionally revert or mutate, become pathogenic • Generally, not recommended for pregnant women • Usually require refrigeration • Examples: measles, mumps, rubella, chickenpox, yellow fever © McGraw Hill, LLC 9 Inactivated Vaccine Unable to replicate, but still immunogenic • Advantage: cannot cause infections or revert to pathogenic forms • Disadvantage: no replication, so no amplification; several booster doses usually needed Often contains adjuvant that enhances immune response to antigens • Thought to provide danger signals to dendritic cells • Some hold antigen and release it slowly over time • Some trigger inflammatory response © McGraw Hill, LLC 10 Types of Inactivated Vaccines 1 Inactivated whole agent vaccines: contain killed microorganisms or inactivated viruses • Treated with formalin or other chemical that does not significantly change surface epitopes • Includes influenza, rabies, hepatitis A Toxoid vaccines: toxins treated to destroy toxic part, retain antigenic epitopes • Includes diphtheria, tetanus Subunit vaccines: consist of key protein antigens or antigenic fragments from pathogen • Avoids cell parts that may cause side effects • For example, acellular pertussis (aP) vaccine © McGraw Hill, LLC 11 Types of Inactivated Vaccines 2 Recombinant subunit vaccines: subunit vaccines produced using genetically engineered microorganisms • For example, hepatitis B virus; yeast cells produce part of viral protein coat VLP (virus-like particle) vaccines: empty capsids produced by genetically engineered organisms • For example, human papillomavirus (HPV) Polysaccharide vaccines: contain polysaccharides from capsules • Not effective in young children; polysaccharides are Tindependent antigens, which elicit poor response • For example, pneumococcal vaccine for adults © McGraw Hill, LLC 12 Types of Inactivated Vaccines 3 Conjugate vaccines: polysaccharides linked to proteins • Converts polysaccharides into T-dependent antigens • Haemophilus influenzae type b (Hib) has nearly eliminated Hib meningitis in children Nucleic acid-based vaccines: • Segments of naked DNA or RNA from infectious agent • Some cells express genes for a short period after injection • Induce immune response Many inactivated vaccines contain an adjuvant, that enhances the immune response to antigens © McGraw Hill, LLC 13 The Importance of Vaccines 1 Benefits greatly outweigh very slight risk • Child with measles has 1:2,000 chance of serious brain inflammation versus 1:1,000,000 chance from vaccine Prior to vaccinations, numerous deaths, disabilities Many still become ill or die from preventable diseases • Some parents refuse to vaccinate children, fear harm • No evidence of link between vaccines and autism • Vaccines have become victims of their own success • People no longer see how serious these diseases are • Reports of adverse effects of vaccine lead some to falsely believe that the risk of vaccination is greater than the risk of diseases © McGraw Hill, LLC 14 The Importance of Vaccines • Vaccination dramatically reduces incidence of diseases • Drops in immunization rates lead to disease outbreaks Disease Cases per Year Before Immunization Decrease After Immunization Diphtheria 175,885 (1920 to 1922) Nearly 100% Haemophilus influenzae type b invasive disease 20,000 (estimated) 98.8% Measles 503,282 (1958 to 1962) Nearly 100% Mumps 152,209 (1968) 99.2% Pertussis (whopping cough) 147,271 (1922 to 1925) 77.6%* Poliomyelitis 16,316 (1951 to 1954) 100% Rubella (German measles) 50,230 (1966 to 1969) Nearly 100% Smallpox 48,164 (1900 to 1904) 100% Tetanus 1,314 (1922 to 1926) 98% © McGraw Hill, LLC 15 The Importance of Vaccines 2 Some routine vaccines are administered in combination, thereby minimizing the number of injections required • MMRV is a vaccine against measles, mumps, rubella, and varicella • DTaP-IPV-Hib is used against diphtheria, tetanus, pertussis, polio, and Haemophilus influenzae type B disease • In late 2018 hepatitis B (HepB) was added to that mix U.S. Centers for Disease Control and Prevention (CDC) regularly publishes recommended immunization schedules for children, adolescents, and adults © McGraw Hill, LLC 16 The Campaign to Eliminate Poliomyelitis 1 Three serotypes of poliovirus; entry is via the mouth • Virus infects throat and intestinal tract, invades blood • From bloodstream, can invade nerve cells, cause disease Salk vaccine (mid-1950s) contains inactivated viruses of all three types: inactivated polio vaccine (IPV) • Dramatically lowered rate of disease but required series of injections for maximum protection Sabin attenuated vaccine (1961): oral polio vaccine (OPV) • Cheaper oral administration; induces better mucosal immunity (secretory IgA response); better herd immunity • Attenuated viruses can mutate; approximately 1 out of 2.4 million doses results in poliomyelitis © McGraw Hill, LLC 17 The Campaign to Eliminate Poliomyelitis 2 To avoid vaccine-related poliomyelitis, use IPV • IPV allows wild poliovirus to replicate in intestines • Disease can be transmitted to others, spread rapidly To eradicate disease completely, use OPV • Stops spread of wild poliovirus OPV campaign successful in eliminating wild poliovirus from United States by 1980 • To avoid vaccine-related disease, children are first given IPV, followed by OPV Efforts continue toward global eradication • Type 2 polio strain eradicated in 2015; new OPV contains only serotypes 1 and 3 © McGraw Hill, LLC 18 Immunotherapies • Immunotherapies modify specific immune responses as a means to treat diseases • Primarily used in cancer treatments; treat other diseases also • Recent advances have been dramatic, with dozens of treatment options currently approved by the U.S. Food and Drug Administration (FDA) • Many are still in development • Therapies often have significant side effects, in which case their use is limited to only disabling or life-threatening diseases © McGraw Hill, LLC 19 Immunotherapies for Cancer Cancers are “self” cells that multiply and spread without control Cancerous cells often express proteins referred to as tumor antigens Should be eliminated by the immune system but they have ways to evade detection • Some cancer cells interfere with immune response by exploiting the peripheral tolerance mechanisms the system uses to avoid responses against “self” cells • Down-regulate production of MHC molecules so that less antigen is presented Cancer immunotherapies attempt to overcome those evasion mechanisms © McGraw Hill, LLC 20 Immunomodulators Therapies that either boost or suppress the immune response •Cytokines direct immune cell activities • Interleukin 2 (IL-2) stimulates proliferation of T cells • Interferon alpha increases tumor cell surveillance by T cells •Adjuvants boost the immune system. • One approved for cancer treatment targets a toll-like receptor pathway; used to treat a type of skin cancer •Checkpoint inhibitors have revolutionized the treatment of certain types of cancer © McGraw Hill, LLC 21 Monoclonal Antibodies (mAb) A preparation of antibody molecules produced by clones of a single B cell • All molecules recognize a single epitope An important part of immunotherapies Obtained by taking B cells from an immunized animal and fusing them with other cells that will divide repeatedly in culture Medications composed of mAbs have names ending in “-mab” mAbs are “humanized”; animal-derived antibodies are replaced with human equivalents • Immune system is less likely to recognize them as foreign and destroy them • Humanized mAbs have names ending in “-zumab” © McGraw Hill, LLC 22 Monoclonal Antibodies 2 Naked antibodies. These are simply mAbs without attached components By binding to a cancer cell, the mAbs mark the cell for destruction via antibody-dependent cellular cytotoxicity (ADCC) • Rituximab is used to treat some B-cell cancers. • Binds to a B-cell surface protein called CD20, thereby marking B cells for destruction Some interfere with growth factors that are required for cancer cell proliferation • Trastuzumab treats certain breast cancers which express high amounts of human epidermal growth factor receptor 2 (HER2) • By binding to HER2, the mAb interferes with the cancer cells’ growth © McGraw Hill, LLC 23 Monoclonal Antibodies 3 Conjugated antibodies have been constructed to deliver a toxin or other molecule to a cancerous cell •Some are derivatives of naked antibodies • Rituximab has been conjugated to a cell-damaging molecule so that the B cell is destroyed when the conjugated antibody binds to it •Bi-specific antibodies have been constructed to have two different antigen-binding sites • Blinatumomab treats some types of acute lymphocytic leukemias • Has one antigen-binding site that binds to a protein on T cells and another that binds to a protein found on some cancerous B cells involved in the leukemia • Increases the chance that the T cell will destroy the B cell © McGraw Hill, LLC 24 Checkpoint Inhibitors 1 • Interfere with immune checkpoints that help prevent cytotoxic T cells from killing “self” cells • Checkpoints are cell surface proteins that can block inappropriate responses that could otherwise lead to autoimmunity, inflammatory damage, and allergies • Also interfere with the immune system’s ability to destroy cancer cells • Allow the immune system to attack cancer cells but increase the likelihood of attack on normal host cells © McGraw Hill, LLC 25 Checkpoint Inhibitors 2 The FDA-approved checkpoint inhibitors are monoclonal antibodies that fall into two groups: Inhibitors of the PD-1/PD-L1 pathway - block a peripheral tolerance pathway that involves a surface protein on TC cells • The PD-1/PD-L1 interaction acts as a T cell “off switch” C •Several are mAbs that bind to PD-1 on the T-cell surface or to PD-L1 on the interacting cells and inhibit the “off switch” pathway Inhibitors of the CTLA-4 pathway - blocks CTLA-4, a T-cell surface receptor, that binds co-stimulatory molecules on APCs and prevents T-cell activation •By blocking CTLA-4, the checkpoint inhibitor makes T-cell activation more likely to occur © McGraw Hill, LLC 26 Chimeric Antigen Receptor (CAR) T Cells A patient’s own T cells are genetically engineered to express a modified T-cell receptor • Can recognize and respond to a specific epitope without antigen presentation or co-stimulation CAR T cells take several weeks to create • Leukocytes are obtained from the patient’s blood and T cells then collected • Cells are then genetically altered in vitro then induced to proliferate • When CAR T cells are put back into a patient they seek out and destroy host cells that have the epitope on their surface © McGraw Hill, LLC 27 Oncolytic Viruses • A genetically modified virus has been approved by the FDA for use in cancer therapy • Destroy cancerous cells directly and encodes the production of a protein that stimulates an anti-tumor immune response • Its presence increases the antigenicity of the tumors, increasing the likelihood that the immune system will attack the tumor cells © McGraw Hill, LLC 28 Therapeutic Vaccines A therapeutic cancer vaccine is used to treat prostate cancer Leukocytes are collected from the patient’s blood and then exposed to a genetically engineered protein consisting of two parts: • GM-CSF (granulocyte-macrophage colony-stimulating factor), which promotes the development of antigenpresenting dendritic cells; • An antigen that characterizes prostate cancer. • Some of the cells develop into dendritic cells that will present the prostate cancer antigen to T cells • These are then infused back into the patient so that they can activate T cells that recognize the antigen on the cancer cells © McGraw Hill, LLC 29 Immunotherapies for Immunological Disorders 1 Immunotherapies are used to treat some immunological disorders such as autoimmune disease and hypersensitivity Autoimmune diseases result from immune reactions that attack “self” molecules or cells Immunotherapies for these diseases suppress components of the immune response The immunotherapy used depends on the components responsible for the damage of that particular disease • Some are primarily T cell driven, others involve B cells The main damage often results from the inflammatory response © McGraw Hill, LLC 30 Immunotherapies for Immunological Disorders 2 Immunomodulators mechanisms of action include: • Use CTLA-4 to interfere with the activation of T cells • Trigger apoptosis of lymphocytes • Interfere with lymphocyte migration from lymph nodes • Inhibit the secretion of pro-inflammatory cytokines • Interfering with the function of tumor necrosis factor (TNF), a pro-inflammatory cytokine Monoclonal antibodies • If the damage is the result of B cells, mAbs that bind to the Bcell surface protein CD20 may decrease the numbers of those cells • If damage is due to inflammation, a mAb that binds the proinflammatory cytokines TNF or IL-6 may be used © McGraw Hill, LLC 31 Focus Your Perspective Kohler and Milstein won a Nobel prize in 1984 for their work on monoclonal antibodies. Access the text alternative for slide images. © McGraw Hill, LLC 32 Immunotherapies for Infectious Diseases mAbs are now being used to treat infectious diseases The use of therapeutic antibodies (passive immunotherapy) does not modify immune response, it provides necessary components • Two mAbs are used to treat inhalational anthrax; both function by neutralizing a bacterial protein required for the entry of anthrax toxins into host cells • Another mAb was approved to treat HIV infections that resist treatment with other medications • It binds CD4 and prevents a conformational change required for the virus to enter cells • mAbs are also proving effective in treating Ebola and COVID-19 © McGraw Hill, LLC 33 Immunological Testing Uses the specificity of antibody-antigen interactions to diagnosis disease The tuberculin skin test (PPD test or the Mantoux test) is used to detect Mycobacterium tuberculosis infection • Infected people develops a strong cell-mediated response to the bacterium and its products • When a purified protein derivative (PPD) from the organism is injected into the skin, a swollen, red area develops at the injection site • Uninfected people show little response unless they have received the BCG vaccine against tuberculosis © McGraw Hill, LLC 34 Principles of Immunoassays Immunoassay: in vitro test that uses antibody-antigen interactions to detect or quantify given antigens or antibodies in a sample • Binding of known antibodies identifies unknown pathogen • Binding of patient’s antibodies to known pathogen demonstrates current or previous infection Access the text alternative for slide images. © McGraw Hill, LLC 35 Serology The in vitro study of antibody-antigen interactions • Serum is fluid portion of blood after blood clots • Plasma is fluid portion of blood treated to prevent clotting Serological testing - examining a patient’s blood for antibodies to diagnose a disease • Seronegative: individual not yet exposed to antigen; has no antibodies to that pathogen • Seropositive: individual has been exposed; has antibodies to pathogen Seroconversion, process of producing antibodies; rise in titer is characteristic of active infection • Low, steady antibody level indicates previous exposure © McGraw Hill, LLC 36 Quantifying Antigen-Antibody Reactions Antibody concentration determined by serial dilution • Sequential 2- or 10-fold dilutions • Antigen added to each dilution • Titer (concentration) is reciprocal of last dilution that gives a detectable antigen-antibody reaction • For example, positive observed in 1:256 but not 1:512, antibody titer is 256 • Usually done in plastic microtiter plates Lisa Burgess/McGraw-Hill Education © McGraw Hill, LLC 37 Obtaining Known Antibodies Antibodies can be monoclonal or polyclonal Monoclonal recognize one epitope on antigen Polyclonal antibodies bind to multiple epitopes • Animals immunized with whole or partial agent; resulting antibodies are collected from animal’s serum • Multiple B cells respond, produce mix of antibodies to variety of epitopes • More complex antigens will yield more antibodies • Some may bind closely related organisms (for example, Shigella sp. and E. coli outer membrane proteins) • Anti-human IgG antibodies produced by animals immunized with IgG from human serum • These and other anti-human antibodies available commercially © McGraw Hill, LLC 38 Common Types of Immunoassays Immunoassays that use labeled antibodies • Markers are enzymes, fluorescent dye or radioactive isotopes • Sensitive Immunoassays that involve visible antigen-antibody aggregates • Simple, cheaper • Largely replaced by labeled antibody tests © McGraw Hill, LLC 39 Immunoassays That Use Labeled Antibodies Labeled antibodies can detect a given antigen, or can detect antibodies that bind to a given antigen • Direct immunoassays: identify unknown antigen • Antigen attached to solid surface • Labeled antibodies of known specificity added • Washing removes unbound; binding identifies antigen • Indirect immunoassays: detect specific antibodies • Known antigen attached to surface; serum added • Washing removes unbound primary antibodies (from serum) • Secondary antibodies (labeled anti-human IgG) added • Washing removes unbound; binding identifies primary antibody bound to antigen © McGraw Hill, LLC 40 Common Types of Immunoassays • Direct immunoassays identify unknown antigen • Indirect immunoassays detect specific antibody in patient’s serum Access the text alternative for slide images. © McGraw Hill, LLC 41 Fluorescent Antibody (FA) Test Use fluorescence microscopy to locate labeled antibodies bound to antigens fixed to microscope slide • Size/shape of antigens seen, but procedure is time-consuming • Using different colored labels can detect more than one antigen • Detect rabies virus in brain tissue, antibodies to agent that causes syphilis • Can be direct or indirect Source: CDC Access the text alternative for slide images. © McGraw Hill, LLC 42 Enzyme-Linked Immunosorbent Assay Direct ELISA, “sandwich method” • Antigen “captured” by antibodies attached to surface • Enzyme-labeled antibody added to detect antigens • Commercial modifications available, including rapid group A strep tests, home pregnancy kits Indirect ELISA • Detects antibodies in human blood and serum • Blood routinely screened for antibodies to HIV • May yield false positives; more reliable test (Western blotting) used to confirm © McGraw Hill, LLC 43 Enzyme-linked immunosorbent assay – Figure 17.10 Access the text alternative for slide images. © McGraw Hill, LLC 44 Enzyme-Linked Immunosorbent Assay (ELISA) Antibodies labeled with enzyme (such as peroxidase) • Enzyme converts colorless substrate to colored product; detected using colorimetric assay • Relatively easy; basis of many rapid tests (e.g. pregnancy test), blood screening • Often in microtiter plate to test many samples at once ©Raimund Koch/The Image Bank/Getty Images © McGraw Hill, LLC 45 Western Blotting First separates proteins of antigen by size using SDS-PAGE • Proteins then transferred to nylon membrane (blotted) • Serum added; unbound antibodies washed off • Enzyme- or radioactively labeled secondary antibodies added; unbound washed off • Label is detected • Indicates which proteins on the blot were recognized by antibodies in the serum © McGraw Hill, LLC 46 Western blotting b: ©Hank Morgan/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 47 Common Types of Immunoassays Fluorescence-activated cell sorter (FACS) • Counts, sorts cells labeled with fluorescent antibodies • For example, can track progression of HIV by determining serum levels of CD4 T cells Access the text alternative for slide images. © McGraw Hill, LLC 48 Immunoassays That Involve Visible Antigen-Antibody Aggregates Antibodies that cross-link antigens create larger “mouthfuls” for phagocytic cells Clumped antigen-antibody complexes form the basis of: • Agglutination reactions • Precipitation reactions © McGraw Hill, LLC 49 Agglutination reactions Agglutination reactions cross-link relatively large particles such as cells • Direct agglutination test (DAT): antibody mixed with antigen (cells, microorganisms); binding yields clumping • Hemagglutination used for blood typing b. Lisa Burgess/McGraw-Hill Education Access the text alternative for slide images. © McGraw Hill, LLC 50 Agglutination Reactions Passive agglutination used when antigens are small Antibodies or antigens attached to particles (latex beads) to form larger aggregates • Beads mixed with drop of body fluid or microbial culture b. Lisa Burgess/McGraw-Hill Education Access the text alternative for slide images. © McGraw Hill, LLC 51 Precipitation Reactions Antibodies bound to soluble antigens can cross-link, form precipitate • Certain relative concentrations of antibody and antigen • Proper concentrations determined by placing separate suspensions near each other in gel, allow molecules to diffuse • Precipitate will form in “zone of optimal proportions” Access the text alternative for slide images. © McGraw Hill, LLC 52 Precipitation Reactions Although largely replaced, Ouchterlony technique illustrates immunodiffusion test Source: Schadler, D. L. 2003. Antigen-antibody testing: A visual simulation or virtual reality. The Plant Health Instructor. DOI: 10.1094/PHI-K-2003-0224-01 Access the text alternative for slide images. © McGraw Hill, LLC 53