Applications of the Immune Response Principals of immunization Vaccines Immunizations Monoclonal antibodies Immunological tests

17-2 Principals of immunization Active immunity – immune response in an individual upon exposure to antigen; naturally from active infection or artificially via vaccination Passive immunity – occurs during pregnancy (natural) and when Abs are supplied from another individual or animal (artificial)

17-3 Active and passive immunity (18.2)

17-4 Antitoxin – antibody preparation against a specific toxin Antiserum – a preparation of serum containing protective antibodies Immune serum globulin – passive immune preparation containing IgG (gamma globulin) pooled blood serum from many donors variety of Abs given to travelers and immunosuppressed individuals Hyperimmune globulin – sera from donors with high levels of specific Abs eg anti tetanus, rabies, hepatitis A and hepatitis B given during disease incubation period to prevent disease development Herd immunity – inability of a pathogen to spread; no hosts

17-5 Vaccination History: The first known vaccination procedures were performed by the Chinese during the Sung dynasty (A.D ) against smallpox. “Variolation” used small amounts of powdered crusts from smallpox pustules which were inhaled or placed in small cuts in the skin. A mild disease was usually produced, followed by immunity to smallpox. Variolation was practiced in Europe, but was expensive and sometimes disease resulted (1 in 100 died), so many people were not treated. Edward Jenner, in 1796, deliberately introduced material from a cowpox lesion on a milkmaid to a scratch on the arm of a young boy (our word dairy comes from “dey-ery” the room in which the “dey”, or woman servant, made milk into butter). Jenner subsequently exposed the boy to the pus of a smallpox victim; the boy did not develop smallpox. Pasteur coined the word “vaccination” to describe any type of protective inoculation.

17-6 Figure 12-6 Increase in measles in the UK compared with uptake of MMR vaccine (P14.6) (MMR = Measles, Mumps, Rubella)

17-7 New York Times on Measles

17-8 Vaccines Toxoids – inactivated toxins Protein subunit vaccines (and recombinant vaccines) contain key protein antigens reduced unwanted side effects Polysaccharide vaccines – T-independent antigens conjugate vaccine – polysaccharide plus protein = T-dependent vaccine – protection for children against H. influenza type b (meningitis) and S. pneumoniae (variety of infections) Adjuvant – enhances immune response to antigens, provide “danger signals”

17-9 Vaccines (cont.) Attenuated - weakened form of the disease-causing agent Agent replicates, may cause mild disease Mimics wild type strain, controls infection Longer antigen exposure than inactivated vaccines Can cause disease in immunocompromised people eg Sabin polio vaccine Inactivated - unable to replicate; retains immunogenicity cannot cause infections or revert to dangerous form no amplification of dose in vivo; boosters required Inactivated whole agent vaccines – killed microorganisms eg Salk vaccine is a mixture of inactivated forms of the three types of poliovirus

17-10 Viral vaccines are made from whole viruses or viral components “Varioloation” – dried pustules from people with milder forms of small pox administered intranasally or intradermally – sometimes caused disease and death Cowpox virus (“vaccinia”) was used by Jenner to produce immunity to smallpox; eradicated smallpox Killed or inactivated vaccines are used for immunity against viruses for which no natural safe counterpart exists Subunit vaccines contain only antigenic viral surface proteins Viral vaccine development is hampered by evasion of the host immune system (HIV), multiple genetic strains (flu) and weak immune responses (common cold)

17-11 Figure 12-1 Vaccination with cowpox protects against smallpox (P14.1)

17-12 Figure 12-2 part 1 of 2 Attenuated viruses are selected by growing human viruses in non-human cells (P14.2)

17-13 Figure 12-2 part 2 of 2 Attenuated viruses are selected by growing human viruses in non-human cells (P14.2)

17-14 Bacterial vaccines are made from whole bacteria, capsular polysaccharides or secreted toxins Toxoid – purified toxin inactivated with formalin, effective against diphtheria and tetanus DTP combination vaccine active against diphtheria, tetanus and pertussis (whooping cough) Pertussis bacteria act as adjuvant DTP replaced by DTaP (acellular pertussis) Polysaccharide vaccines for encapsulated bacteria designed to elicit complement fixing Abs that bind strain-specific and pathogenicity-causing surface polysaccharides Effective against pneumococcus, salmonellae, meningococci, H. influenzae, E. coli, Klebsiella pneumoniae, B. fragilis T-independent antigen is converted to T-dependent antigen by coupling to carrier protein that binds T cells “conjugate vaccines”

17-15 Adjuvants nonspecifically enhance the immune responses Vaccination must create a state of inflammation created by bacterial products that activate macrophages Purified proteins do not elicit a strong immune response The response can be enhanced by adding substances that induce inflammation called adjuvants (“helpers”) Freund’s complete adjuvant is an emulsion of killed mycobacteria and mineral oil Adjuvants cause soluble protein antigens to aggregate and precipitate to facilitate phagocytosis by APCs

17-16 Vaccination can inadvertently cause disease Live attenuated viruses simulate actual infections and elicit the best immune stimulation Live attenuated viruses can revert to a pathogenic form Sabin polio vaccine induces polio and paralysis in 3 people per million vaccinated One of the three polio virus strains in Sabin vaccine differs from natural polio virus by only 10 nucleotide substitutions; can mutate to the natural strain and cause disease

17-17 Table Vaccines

17-18 Table Vaccines

17-19 Table 18.2 Characteristics of Attenuated and Inactivated Vaccines

17-20 Table Effectiveness of immunizations

17-21 Table Future immunizations

17-22 Figure 12-8 (P14.8)

17-23 Immunizations Paralytic poliomyelitis Effectiveness of immunizations Recommended immunizations Future immunizations

17-24 Paralytic poliomyelitis 1950 – Salk vaccine (inactivated virus) no herd immunity – immunized people can be carriers requires multiple injections over time 1960 – Sabin vaccine (attenuated virus) Salk vaccine is safe, but wild type virus can replicate and spread Sabin vaccine provides herd immunity given orally, induces mucosal immunity stops spread, gives herd immunity can cause vaccine-related polio in some individuals

17-25 New York Times on polio

17-26 Immunological testing Monoclonal antibodies Serology Quantifying antigen – antibody reactions

17-27 Therapeutic MAbs: 1895 – French physicians use antiserum from dogs to treat cancer some patients improved but had immunogenicity problems nobody cured 2005 – 100 therapeutic MAbs in clinical trials 18 MAbs approved for use in the US $5-6 billion in revenues in 2003 may triple in next five years possibly 32% of biotech market by 2008 Genentech produces three anticancer MAbs: Herceptin Avastin Rituxan $2.7 billion in revenues in 2004

17-28 Perspective 18.1 Monoclonal Antibodies Movie on Monoclonals

17-29 Problems and solutions encountered during MAb drug development: 1. Removal of inappropriate Abs from antisera solution: MAbs (Milstein and Kohler, 1984) produce single mouse Ab 2. Mouse MAbs were immunogenic in humans solution: “humanization” of mouse MAbs Mouse variable regions fused to human constant regions = chimeric MAb reduced HAMA 3. Chimeric drugs Rituxin (rituximab; binds CD-20 on B cell to combat non-Hodgkin lymphoma); Remicade (infliximab) have been associated with serious allergic reactions solution: insert mouse complementary-determining regions (CDRs) into human Ab = humanized MAbs 90-95% human fewer HAMA responses

Humanized MAbs technically demanding, reduced Ag affinities solution: fully human MAbs to evade human immune response a. change the mouse: Xenomouse, HuMab-Mouse are transgenic animals with human Ab genes 11 xenomouse MAbs in clinical trials eg panitumumab – targets EGF receptor to combat metastatic colorectal cancer 150 HuMab-Mouse MAbs in development eg MDX-010 – anti-CTLA-4 for metastatic melanoma in clinical trials binding of CTLA-4 blocks inhibition of T-cell proliferation effector T cells eliminate tumors

Humanized MAbs technically demanding, reduced Ag affinities solution: fully human MAbs to evade human immune response b. skip the mouse: in vitro phage display technology used to produce human MAbs libraries of human MAbs contain more than 100 billion different phage antibodies screen to select for strong binding to specific Ag

17-32

17-33

17-34 Principles of Immunological Testing Terms –Seronegative Person not yet exposed to antigen and has no specific antibodies –Seropositive Person with exposure and actively producing antibody –Titer Concentration of antibody in serum Indicates previous exposure

17-35 Using Labeled Antibodies to Detect Interactions Detectable markers can be attached to specific antibodies –Marked antibodies used to detect presence of given antigen Tests include –Fluorescent Antibody (FA) test –Enzyme-Linked Immunosorbant Assay (ELISA) –Western blotting –Fluorescence Activates Cell Sorter (FACS)

17-36 Using Labeled Antibodies to Detect Interactions Fluorescent antibody test –Relies on fluorescent microscopy to locate labeled antibodies fixed to a microscope slide –Fluorescent polarized immunoassay uses beam of polarized light to rate spin of labeled antibodies Works under principle that bound antibodies are heavier then unbound and will spin more slowly

17-37 Fluorescent Antibody (FA) tests (18.10)

17-38 Using Labeled Antibodies to Detect Interactions Enzyme-Linked Immunosorbant Assay –Employs antibody that has been labeled with detectable enzyme Commonly horseradish peroxidase –Labels antibody bonds to antigen Binding can be direct or indirect –Antigen location is determined using colormetric assay

17-39 Enzyme-Linked Immunosorbant Assay –Direct ELISA Looks for specific antigen –Specimen placed in wells of microtiter plate »Wells treated with antibody for antigen –Indirect ELISA Looks for antibody in patient serum –Human IgG –Wells of plate treated with known antigen Using Labeled Antibodies to Detect Interactions

17-40 Movie on ELISA (18.11)

17-41 Direct ELISA test for pregnancy (18.12) Detects human chorionic gonadotropin Present only in pregnant women

17-42 Western blot for HIV (18.13) Ags separated by electorphoresis Transferred to membrane Probed with specific Abs Abs detected indirectly using anti-HGG gp 160 gp 120 p66 p55 gp41 p39 p31 p24 p17 Serum control (a) Strong reactive control (b) Weak reactive control (c) Non-reactive control Courtesy of Genelabs Diagnostics cab