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05-01-12 Applications of the Immune Response

Principals of immunization

Vaccines

Immunizations

Monoclonal antibodies

Immunological tests

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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)

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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 donorsvariety of Absgiven to travelers and immunosuppressed individuals

Hyperimmune globulin – sera from donors with high levels of specific Abs

eg anti tetanus, rabies, hepatitis A and hepatitis Bgiven during disease incubation period to prevent

disease development

Herd immunity – inability of a pathogen to spread; no hosts

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Vaccination History:

The first known vaccination procedures were performed by the Chinese during the Sung dynasty (A.D. 960-1280) 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.

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Figure 12-6

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

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New York Times on Measles

http://www.nytimes.com/packages/khtml/2006/04/28/health/20060430_BRINK_AUDIOSS.html

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Vaccines

• Toxoids – inactivated toxins• Protein subunit vaccines (and recombinant vaccines)

contain key protein antigensreduced unwanted side effects

• Polysaccharide vaccines – T-independent antigensconjugate vaccine – polysaccharide plus protein =

T-dependent vaccine – protection for childrenagainst H. influenza type b (meningitis) and S. pneumoniae (variety of infections)

• Adjuvant – enhances immune response to antigens, provide “danger signals”

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Vaccines (cont.)

• Attenuated - weakened form of the disease-causing agentAgent replicates, may cause mild diseaseMimics wild type strain, controls infectionLonger antigen exposure than inactivated vaccinesCan cause disease in immunocompromised peopleeg Sabin polio vaccine

• Inactivated - unable to replicate; retains immunogenicitycannot cause infections or revert to dangerous formno amplification of dose in vivo; boosters requiredInactivated whole agent vaccines – killed microorganismseg Salk vaccine is a mixture of inactivated forms

of the three types of poliovirus

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Viral vaccines are made from whole viruses orviral components

“Varioloation” – dried pustules from people with milder forms of small pox administered intranasally orintradermally – 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 againstviruses 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)

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Figure 12-1

Vaccination with cowpox protects against smallpox(P14.1)

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Figure 12-2 part 1 of 2

Attenuated viruses are selected by growinghuman viruses in non-human cells (P14.2)

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Figure 12-2 part 2 of 2

Attenuated viruses are selected by growinghuman viruses in non-human cells (P14.2)

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Bacterial vaccines are made from whole bacteria,capsular polysaccharides or secreted toxinsToxoid – purified toxin inactivated with formalin, effective

against diphtheria and tetanusDTP combination vaccine active against diphtheria, tetanus

and pertussis (whooping cough)Pertussis bacteria act as adjuvantDTP replaced by DTaP (acellular pertussis)

Polysaccharide vaccines for encapsulated bacteria designed to elicit complement fixing Abs that bind strain-specificand 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 antigenby coupling to carrier protein that binds T cells“conjugate vaccines”

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Adjuvants nonspecifically enhance the immuneresponses

Vaccination must create a state of inflammation created bybacterial products that activate macrophages

Purified proteins do not elicit a strong immune response

The response can be enhanced by adding substances thatinduce 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

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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 3people per million vaccinated

One of the three polio virus strains in Sabin vaccine differsfrom natural polio virus by only 10 nucleotidesubstitutions; can mutate to the natural strainand cause disease

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Table 18.1 - Vaccines

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Table 18.1 - Vaccines

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Table 18.2 Characteristics of Attenuatedand Inactivated Vaccines

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Table 18.3 - Effectiveness of immunizations

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Table 17.5 - Future immunizations

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Figure 12-8

(P14.8)

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Immunizations

• Paralytic poliomyelitis

• Effectiveness of immunizations

• Recommended immunizations

• Future immunizations

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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

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New York Times on polio

http://video.on.nytimes.com/index.jsp?fr_story=77549ceb1b1ae1777e43d4eedfaf7bcff47b0a31

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Immunological testing

• Monoclonal antibodies

• Serology

• Quantifying antigen – antibody reactions

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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 trials18 MAbs approved for use in the US$5-6 billion in revenues in 2003may triple in next five yearspossibly 32% of biotech market by 2008

Genentech produces three anticancer MAbs:HerceptinAvastinRituxan$2.7 billion in revenues in 2004

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Perspective 18.1Monoclonal Antibodies

Movie on Monoclonals

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Problems and solutions encountered during MAb drug development:

1. Removal of inappropriate Abs from antiserasolution: MAbs (Milstein and Kohler, 1984)produce single mouse Ab

2. Mouse MAbs were immunogenic in humanssolution: “humanization” of mouse MAbsMouse 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

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4. 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 trialseg panitumumab – targets EGF receptor to combat metastatic colorectal cancer

150 HuMab-Mouse MAbs in developmenteg MDX-010 – anti-CTLA-4 formetastatic melanoma in clinical trials

binding of CTLA-4 blocksinhibition of T-cell proliferationeffector T cells eliminate tumors

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4. 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 than100 billion different phage antibodies

screen to select for strong binding to specific Ag

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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

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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)

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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

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

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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

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• 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

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Movie on ELISA

(18.11)

17-41Direct ELISA test for pregnancy (18.12)

Detects human chorionic gonadotropinPresent only in pregnant women

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Western blot for HIV (18.13)

Ags separated by electorphoresis

Transferred to membrane

Probed with specific Abs

Abs detected indirectlyusing anti-HGG

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gp 160

gp 120

p66

p55gp41

p39

p31

p24

p17

Serumcontrol

(a) Strong reactive control(b) Weak reactive control(c) Non-reactive control

Courtesy of Genelabs Diagnostics

ca b