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Vaccination, Specific Immunity & Testing

An Overview.

Nonspecific Defense Mechanisms

• 1.Physical barriers, such as skin and mucous membranes

• 2.Secretions, such as mucus, saliva, urine

• 3.Antimicrobial substances in body fluids and phagocytes

• 4.Inflammation

• 5.Physiological defenses, such as fever

First Line of Defense

• Structures, chemicals, processes that work to prevent pathogens entering the body

• Includes the skin and mucous membranes of the respiratory, digestive, urinary, and reproductive systems

Microbial Antagonism

• Normal microbiota help protect the body by competing with potential pathogens

• Various activities of the normal microbiota make it hard for pathogens to compete

– Secrete antimicrobial substances that limit pathogen growth

– Consumption of nutrients makes them unavailable to pathogens

– Create an environment unfavorable to other microorganisms by changing pH

Second Line of Defenses

• Operates when pathogens succeed in penetrating the skin or mucous membranes

• Composed of cells, antimicrobial chemicals, and processes but no physical barriers

– Many of these components are contained or originate in the blood

Specific Immunity

• Is acquired over time

• Antigens trigger specific immune responses

• Various cells, tissues, and organs are part of specific immunity

– Includes B and T lymphocytes

B Lymphocytes

• Arise and mature in the red bone marrow

• Found primarily in the spleen & lymph nodes.

• Small percentage of B cells circulate in the blood

• Major function is the secretion of antibodies

T Lymphocytes

• Produced in the red bone marrow and mature in the thymus

• Circulate in the lymph and blood and migrate to the lymph nodes, spleen

• Part of the cell-mediated immune response because they act directly against various antigens

Antigens

• Molecules that trigger a specific immune response

• Include components of bacterial cell walls, capsules, pili, and flagella, as well as proteins of viruses, fungi, and protozoa

• Food and dust can also contain antigenic particles

• Enter the body by various methods

– Through breaks in the skin and mucous membranes

– Direct injection, as with a bite or needle

– Through organ transplants and skin grafts

Antibodies

• Also called immunoglobulins (Ig)

• Soluble, proteinaceous molecules that bind antigen

• Secreted by plasma cells, which are B cells actively fighting exogenous antigen

• Considered part of the humoral immune response since bodily fluids such as lymph and blood were once called humors

Antibody Function

• Antigen-binding sites are complementary to antigenic determinants (epitopes)

– Due to the close match can form strong, noncovalent interactions

– Hydrogen bonds and other attractions may also be involved

Antibody Function

• Function in several ways

– Activation of complement

– Stimulation of inflammation

– Agglutination

– Neutralization

– Opsonization

Classes of Antibodies

• A single type of antibody is not sufficient for the multiple types of invaders to the body

• The class involved in the immune response depends on the type of foreign antigen, the portal of entry, and the antibody function needed

• 5 different classes of antibodies

Direct and Indirect Testing

• Direct: demonstration of the presence of an infectious agent

– Culture

– Microscopy

– Molecular methods such as PCR

• Indirect: demonstration of presence of antibodies to a particular infectious agent

– Serology

Immunization and Immune Testing

Immunizations

• 2 artificial methods to make an individual immune to a disease

– Active immunization-administration of a vaccine so that the patient actively mounts a protective immune response

– Passive immunization-individual acquires immunity through the transfer of antibodies formed by an immune individual or animal

History of Immunization

• The Chinese noticed that children who recovered from smallpox did not contract the disease a second time

• They infected young children with material from a smallpox scab to induce immunity in these children, a process known as variolation

• The use of variolation spread to England and America but was eventually stopped due to the risk of death

• Edward Jenner found that protection against smallpox could be induced by inoculation with material from an individual infected with cowpox, a similar but much milder disease

History of Immunization

• Since cowpox was also called vaccinia this process was called vaccination, and the inoculum was termed a vaccine

• Louis Pasteur developed a vaccine against Pasteurella multocida

• Practice of transferring protective antibodies was developed when it was discovered that vaccines protected through the action of antibodies

Vaccination Problems

• Socioeconomic and political problems prevent many developing nations from receiving vaccines

• Inability to develop effective vaccines for some pathogens

• Vaccine-associated risks discourage investment in developing new vaccines

Vaccine Types

• 3 general types of vaccines

– Attenuated (live)

– Killed (inactivated)

• Whole cell

• Sub-unit (bioengineered)

– Toxoid

Attenuated Vaccines

• Also called modified live vaccines

• Uses pathogens that are living but have reduced virulence so they don’t cause disease

• Attenuation is the process of reducing virulence

– Viruses often attenuated by raising them in tissue culture cells for which they aren’t adapted until they lose the ability to produce disease

– Bacteria can be made avirulent by culturing under unusual conditions or through genetic manipulation

Attenuated Vaccines

• Can result in mild infections but no disease

• Contain replicating microbes that can stimulate a strong immune response due to the large number of antigen molecules

• Vaccinated individuals can infect those around them, providing herd immunity

Problems with Attenuated Vaccines

• Attenuated microbes may retain enough virulence to cause disease, especially in immunosuppressed individuals

• Pregnant women should not receive live vaccines due to the risk of the modified pathogen crossing the placenta

• Modified viruses may occasionally revert to wild type or mutate to a virulent form

Inactivated Vaccines

• Can be either whole agent vaccines produced with deactivated but whole microbes, or subunit vaccines produced with antigenic fragments of microbes

• Both types are safer than live vaccines since they cannot replicate or mutate to a virulent form

• When microbes are killed must not alter the antigens responsible for stimulating protective immunity

• Formaldehyde is commonly used to inactivate microbes by cross-linking their proteins and nucleic acids

Problems with Inactivated Vaccines

• Do not stimulate herd immunity

• Whole agent vaccines may stimulate a inflammatory response due to nonantigenic portions of the microbe

• Antigenically weak since the microbes don’t reproduce and don’t provide many antigenic molecules to stimulate the immune response

Problems with Inactivated Vaccines

• Administration in high or multiple doses, or the incorporation of an adjuvant, can make the vaccine more effective

– Adjuvants are substances that increase the antigenicity of the vaccine

– Adjuvants may also stimulate local inflammation

– High and multiple vaccine doses may produce allergic reactions

Toxoid Vaccines

• Chemically or thermally modified toxins used to stimulate active immunity

• Useful for some bacterial diseases

• Stimulate antibody-mediated immunity

• Require multiple doses because they possess few antigenic determinants

Modern Vaccine Technology

• Research attempts to make vaccines that are more effective, cheaper, and safer

• A variety of recombinant DNA techniques can be used to make improved vaccines

Vaccine Safety

• Problems associated with vaccination

– Mild toxicity is the most common problem

• Especially seen with whole agent vaccines that contain adjuvants

• May cause pain at the injection site and in rare cases can cause general malaise or fever high enough to induce seizures

– Anaphylactic shock

• Is an allergic reaction that may develop to a component of the vaccine

Vaccine Safety

– Residual virulence

• Attenuated viruses occasionally cause disease in healthy children or adults

– Allegations that certain vaccines against childhood diseases cause or trigger autism, diabetes, and asthma

• Research has not substantiated these allegations

Passive Immunity

• Administration of preformed antibodies to a patient

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

• Immunologists remove the serum from human or animal donors that have been infected with the disease or immunized against it

• Serum used for passive immunizations is called antiserum

Limitations of Antisera

• Contain antibodies against many different antigens not just the ones of interest

• Repeated injections of antisera collected from a different species can trigger allergic reactions

• Antisera may be contaminated with viral pathogens

• Antibodies of antisera are degraded relatively quickly

Serologic Terms

• Antigen (Ag): anything that induces a specific immune response

• Antibody (Ab): a large protein molecule produced in response to an antigen that interacts specifically with that antigen, found in serum

• Antiserum: Serum containing specific antibodies

Serologic Testing

• Used to detect Ab to infectious agents for diagnosis

• Gold standard is isolation of infectious agent:

– Can have low sensitivity

– Comparatively expensive

– Can be slow (days vs. hours)

– Can be less safe than serology

Serologic Testing (2)

• Antibody can also be used to detect Antigen

– Salmonella serotypes

– Neisseria meningitidis serogroups

– Streptococcus groups

Serologic Reactions

• We need to demonstrate when Ag+Ab reaction has occurred

– Agglutination, or clumping

– “Sandwich” techniques

• IFA (Indirect Fluorescent Antibody)

• ELISA (Enzyme-Linked Immunosorbent Assay)

Serologic Testing

• Usually quantitative

• Serial dilutions indicate how much Ab can still be demonstrated

• Titer is reciprocal of dilution factor

– Dilution of 1:1280 = titer of 1280

Immune Testing

• Numerous types of serologic test

– Precipitation tests

– Agglutination tests

– Neutralization tests

– Complement fixation test

– Various tagged antibody tests

Virus Neutralization

• Mix dilutions of antiserum with known virus

• Incubate 1 hour

• Add cells of any cell line that is sensitive to this virus

• Incubate

• Observe for CPE (Cytopathic Effect)

Complement Fixation Test

• Based on the generation of membrane attack complexes during complement activation that disrupt cytoplasmic membranes

• Used to detect the presence of specific antibodies in an individual’s serum

• Can detect antibody amounts too small to be detected by agglutination

Labeled Antibody Test

• Use antibody molecules that are linked to some molecular “label” that enables them to be easily detected

• Used to detect either antigens or antibodies

• 3 examples

– Fluorescent antibody tests

– ELISA

– Western blot test

Indirect Fluorescent Antibody Test (IFA)

• Known antigen (can be viral, bacterial, or eukaryotic) is placed on microscope slides to dry

• Serial dilutions of patient serum added to individual antigen spots on slides

• Washed, antihuman serum tagged with fluorescent dye (FITC) applied, incubated, washed

• Read slides with UV microscope, look for fluorescence.

Uses fluorescent dyes as labels

• Fluorescein is the most important dye used in these test

– Chemically linked to an antibody without affecting antibody’s ability to bind antigen

– Glows bright green when exposed to fluorescent light

• Fluorescein-labeled antibodies used in 2 types of tests

– Direct fluorescent antibody test

– Indirect fluorescent antibody tests

Direct Fluorescent Antibody Tests

• Identifies the presence of antigen in tissue

– Tissue sample flooded with labeled antibody

– Antibody and antigen are allowed to bind for a short period

– Unbound antibody washed from the preparation

– Results observed under a fluorescent microscope

• Used to identify small numbers of bacteria in patient tissues

• Not a quantitative test- the amount of fluorescence observed is not directly related to the amount of antigen present

ELISA

• Stands for enzyme-linked immunosorbent assay

• Uses an enzyme as the label

– Reaction of the enzyme with its substrate produces a colored product indicative of a positive test

• Most common form of ELISA is used to detect the presence of antibodies in serum

Advantages of The ELISA

• Can detect either antibody or antigen

• Can quantify amounts of antigen or antibody

• Easy to perform, inexpensive, and can test many samples quickly

• Plates coated with antigen and gelatin can be stored for later testing

• Technique for detecting antibodies against multiple antigens in a complex mixture

• Can detect more types of antibodies and are less subject to misinterpretation than other tests

Recent Developments in Immune Testing

• Development of simple immunoassays that give results in minutes

• Generally not quantitative but are useful in determining a preliminary diagnosis

• Most common are the immunofiltration and immunochromotography assays

– Immunofiltration

• Rapid ELISA that uses antibodies bound to membrane filters rather than polystyrene plates

• Membrane filters have a large surface area making the assay quicker to complete

Recent Developments in Immune Testing

– Immunochromatography

• Very rapid and easy to read ELISAs

• Antigen solution flows through a porous strip where it encounters antibody labeled with either pink colloidal gold or blue colloidal selenium

• Antigen-Antibody immune complexes flow through a region and encounter antibody against them, resulting in a visible pink or blue line

• Used in pregnancy testing to detect human chorionic growth hormone

Immunological Tests and Some of Their Uses

Recap-
Direct and Indirect Testing

• Direct: demonstration of the presence of an infectious agent

– Culture

– Microscopy

– Molecular methods such as PCR

• Indirect: demonstration of presence of antibodies to a particular infectious agent

– Serology

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