Immunology & Immunological Preparations
By: Bijaya Kumar Uprety 1
Immunology Branch of biological science concerned with the study of immunity, •
•Or concerned with the structure and
function of immune system.
Immunity •Latin term immunis meaning “exempt”. • Immunity means the state of
protection from infectious disease.
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Year
Name
Event
430 B.C – Earliest written reference to the phenomenon of immunity.
Thucydides (great historian of the Peloponnesian war).
In describing plague in Athens, he wrote during the war, only who had recovered from the plague could nurse the sick because they would not get the disease for the second time.
Chinese and Turks
Dried crust derived from small pox pustules were either inhaled into the nostrils or inserted into small cuts in the skin (technique known as variolation).
Lady Mary Wortley (wife of british ambassador to Constantinople)
Performed variolation on her own children after realizing the technique was effective among her native people.
Edward Jenner (physician)
Propounded an idea that introducing fluid from a cowpox pustule into people might protect them from smallpox and he tested his idea on eight year old kid which was successful.
15th century- First record to induce immunity deliberately.
1718
1798
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Louis Pasteur grew the fowl cholera causing
bacterium in culture and when this was injected it into chicken they developed cholera but later on when he once again injected them with the same old culture they got ill but recovered later on. He grew fresh culture and tried it on same chickens. They completely recovered. Hence, Hypothesized and proved that aging had weakened the virulence of the pathogen and concluded that the attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine.
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Continue….. Later extended his findings to other diseases
and demonstrated it is possible to attenuate, or weaken a pathogen and administer them to use it as a vaccine.
In 1881, Pasteur vaccinated one group of sheep with
heat-attenuated anthrax bacillus (bacillus anthracis) and left another group of unvaccinated sheep . All unvaccinated sheep died while other lived. This was the beginnings of
the discipline of immunology. In 1885, he first administered his first vaccine to a human (a young boy) against rabies. 5
Pasteur proved vaccination worked but didn’t know
how it worked. In 1890, Emil von Behring and Shibasaburo Kitasato
gave first insight into the mechanism of immunity Got nobel prize in 1901. They demonstrated that serum from animals
previously immunized to diptheria could transfer the immune state to unimmunized animals.
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During next decade, it was demonstrated by various
researchers that an active component from immune serum could neutralize toxins, precipitate toxins and agglutinate bacteria and active agent was named for its activity it exhibited: antitoxin, precipitin and agglutinin resp. Initially different serum component was thought to be
responsible for each activity but during 1930, Elvin Kabat (mainly him) found that gamma-globulin (now immunoglobulin, also a fraction of serum) was responsible for all these. 7
The active molecules in the immunoglobulin fraction
are called antibodies. Because the immunity was mediated by antibodies contained in the fluids (known at that time as humors), it was called humoral immunity. In 1883, Elie Metchnikoff demonstrated that cells also contribute to the immune state of an animal. He hypothesized that cells rather than serum components were major effector of immunity.(term phagocytes was coined and an idea of cell-mediated immunity dvpt). Controversy developed between two concepts. 8
But latter proved that both were correct. Immunity requires both humoral and cellular responses. In 1950, lymphocyte was identified as the cell responsible
for both cellular and humoral immunity and experiments on chicken pioneered by Bruce Click at Mississippi State University indicated that there are two types of lymphocytes. 1. T- lymphocytes derived from thymus mediated cellular immunity. 2. B-lymphocytes from bursa of Fabricius were involved in humoral immunity. Both these systems work hand in hand to protect our body against various foreign attack. 9
Introduction to Immune system Remarkably versatile defense system that protect
animals against various invading micro-organisms and cancer. Able to generate enormous variety of cells and
molecules capable of specifically recognizing and eliminating large variety of foreign invaders. Invaders human body immune system respond eliminate or destroys the invaders 10
Body endowed with different defense system. At first, external defense system comes into play which
includes, skin, secretion of mucus, ciliary action, lavaging action of bactericidal fluids (e.g. tears), gastric acid and microbial antagonism. If penetration occurs, bacteria are destroyed by
soluble factors such as lysozyme and by phagocytosis with intracellular digestion. 11
Functionally, immune response can be divided into
two related activities1. Recognition – Remarkable for its specificity. 2. Response.
Immune system is able to recognize subtle chemical differences that recognize one foreign pathogen from another.
Able to discriminate between foreign molecules and the body’s own cells and proteins. 12
Once a foreign organism has been recognized, it recruits a
variety of cells and molecules to mount an appropriate response, called effector response, to eliminate or neutralize the organism. The immune response enables the elimination or
neutralization of the cells/molecules (pathogens) from the body. Convert initial recognition event variety of effector responses eliminate or neutralize particular pathogen. 13
Later exposure to the same foreign organism induces a
memory response, characterized by a more rapid and heightened immune reaction that serves to eliminate the pathogen and prevent disease.
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Types of immunity Two types of immunity:
1. Innate or nonspecific immunity. 2. Acquired or specific immunity. Innate immunity: It is the basic resistance to diseases that an individual has from the time of its birth. Not specific to any one pathogen but rather constitutes a first line of defense. 15
It consists of following four types of defensive barriers:
Anatomic barriers 2. Physiologic barriers 3. Endocytosis /phagocytosis barriers 4. Inflammatory barriers 1.
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• Endocytosis-
Process of cellular ingestion of macromolecules by invagination of plasma membrane to produce an intracellular vesicle which encloses the ingested material. • 3 types Phagocytosis (for particulates), Pinocytosis (liquid), Receptor mediated endocytosis
(LDL) . • Most phagocytosis (most common) is
done by blood monocytes, neutrophils, and tissue macrophages.
Fig. Steps in phagocytosis of a bacterium. 18
Fig 2 showing the major events in the inflammatory response.[ vasoactive and chemotactic factors i.e kinin and histamine. Additionally , bradykinins which is 19 a type of kinin stimulate pain receptors and fibrin-clot]
Acquired Immunity Also known as adaptive immunity. Capable of recognizing and selectively eliminating
specific foreign microorganisms and molecules (i.e. foreign antigens). Displays four characteristic attributes: 1. Antigenic specificity 2. Diversity 3. Immunologic memory 4. Self/nonself recognition. 20
Components of Acquired Immunity Involves the following two major groups of cells 1. Lymphocytes which includes B and T lymphocytes.
2. Antigen presenting cells (APCs) Group of B-cells, dendritic cells and macrophages. They express class II MHC molecules on their membranes & They are able to deliver a co-stimulatory signal that is necessary for TH cell activation.
APC have Class II MHC (major – histocompatibility complex) molecules on their surface MHC molecules bind to antigen derived peptides present them to lymphocytes immune system activated. 21
B lymphocytes B lymphocytes mature within the bone marrow; when they leave it, each expresses a
unique antigen-binding receptor on its membrane . This antigen-binding or B-cell receptor is a membrane-bound antibody molecule. Antibodies are glycoproteins that consist of two identical heavy polypeptide chains and two identical light polypeptide chains. Each heavy chain is joined with a light chain by disulfide bonds, and additional disulfide bonds hold the two pairs together. The amino-terminal ends of the pairs of heavy and light chains form a cleft within which antigen binds. When a naive B cell (one that has not previously encountered antigen) first encounters the antigen that matches its membrane bound antibody, the binding of the antigen to the antibody causes the cell to divide rapidly; its progeny differentiate into memory B cells and effector B cells called plasma cells. Memory B cells have a longer life span than naive cells, and they express the same membrane-bound antibody as their parent B cell. Although plasma cells live for only a few days, they secrete enormous amounts of antibody during this time. It has been estimated that a single plasma cell can secrete more than 2000 molecules of antibody per second. Secreted antibodies are the major effector molecules of humoral immunity. 22
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T lymphocytes T lymphocytes also arise in the bone marrow. Unlike B cells, which mature within the bone marrow, T cells migrate to the thymus gland to mature. During its maturation within the thymus, the T cell comes to express a unique
antigen-binding molecule, called the T-cell receptor, on its membrane.
Unlike membrane-bound antibodies on B cells, which can recognize antigen alone, T-cell receptors can recognize only antigen that is bound to cellmembrane proteins called major histocompatibility complex (MHC) molecules. MHC molecules that function in this recognition event, which is termed “antigen presentation,” are polymorphic (genetically diverse) glycoproteins found on cell membranes.
There are two major types of MHC molecules: Class I MHC molecules, which are expressed by nearly all nucleated cells of vertebrate species, consist of a heavy chain linked to a small invariant protein called 2-microglobulin. Class II MHC molecules, which consist of an alpha and a beta glycoprotein chain, are expressed only by antigen-presenting cells. 25
When a naive T cell encounters antigen combined with a MHC molecule on a cell,
the T cell proliferates and differentiates into memory T cells and various effector T cells. There are two well-defined subpopulations of T cells: T helper (TH) and T
cytotoxic (TC) cells. Although a third type of T cell, called a T suppressor (TS) cell, has been postulated, recent evidence suggests that it may not be distinct from TH and TC subpopulations.
T helper and T cytotoxic cells can be distinguished from one another by the
presence of either CD4 or CD8 membrane glycoproteins on their surfaces . T cells displaying CD4 generally function as TH cells, whereas those displaying CD8 generally function as TC cells. TH cells generally recognize antigen combined with class II molecules, whereas TC cells generally recognize antigen combined with class I molecules. After a TH cell recognizes and interacts with an antigen– MHC class II molecule
complex, the cell is activated—it becomes an effector cell that secretes various growth factors known collectively as cytokines. The secreted cytokines play an important role in activating B cells, TC cells, macrophages, and various other cells that participate in the immune response.
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Differences in the pattern of cytokines produced by activated
TH cells result in different types of immune response. Under the influence of TH-derived cytokines, a TC cell that recognizes an antigen–MHC class I molecule complex proliferates and differentiates into an effector cell called a cytotoxic T lymphocyte (CTL). In contrast to the TH cell, the CTL generally does not secrete many cytokines and instead exhibits cell-killing or cytotoxic activity. The CTL has a vital function in monitoring the cells of the body and eliminating any that display antigen, such as virusinfected cells, tumor cells, and cells of a foreign tissue graft. Cells that display foreign antigen complexed with a class I MHC molecule are called altered self-cells; these are targets of CTLs. 27
ANTIGEN-PRESENTING CELLS Activation of both the humoral and cell-mediated branches of the
immune system requires cytokines produced by TH cells. It is essential that activation of TH cells themselves be carefully regulated, because an inappropriate T-cell response to selfcomponents can have fatal autoimmune consequences. To ensure carefully regulated activation of TH cells, they can recognize only antigen that is displayed together with class MHC II molecules on the surface of antigen-presenting cells (APCs). These specialized cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by two properties: (1) they express class II MHC molecules on their membranes, and (2) they are able to deliver a co-stimulatory signal that is necessary for TH-cell activation. Antigen-presenting cells first internalize antigen, either by phagocytosis or by endocytosis, and then display a part of that antigen on their membrane bound to a class II MHC molecule. The TH cell recognizes and interacts with the antigen–class II MHC molecule complex on the membrane of the antigen-presenting cell .An additional costimulatory signal is then produced by the antigenpresenting cell, leading to activation of the TH cell. 28
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Humoral Immune Responses It is based on antibodies. It can be conferred on nonimmune individuals by
administration of serum antibodies from an immune individual. Antibodies act as an effector of humoral response. They bind to the antigens and facilitate their elimination. Elimination could be in various ways.
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Fig showing the structure of Antibody. 32
1. By forming clusters through cross-linking of antigen molecules, which are readily ingested by phagocytic cells.
2. By binding of antibodies to a microorganism can activate the complement system, which lyses the mo’s. 3. Antibodies bind to toxins and viral particles, and prevent their subsequent binding to host cells.
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Cell-mediated Immune Responses Based on T cells, which are a type of lymphocyte. T cells are of the following two types:
T helper (TH) 2. T cytotoxic (TC) cells. 1.
TH cell interacts with an antigen –MHC II molecule complex present on an APC cytokines secreted cytokines activate B cells, Tc Cells, and various phagocytic cells.
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Activated phagocytic cells able to kill mo’s (bacteria
and protozoa). When Tc cell interacts with an antigen-MHC I
complex, the Tc cell proliferates under the influence of cytokines produced by activated TH cells. These
Tc cells differentiate into cytotoxic T lymphocytes (CTLs). The CTLs kill all such cells that display foreign antigens complexed with MHC I molecules. Such cells are called altered self-cells, they are usually virus-infected cells, tumor cells and foreign tissue cells. 35
Thus TH cells and CTLs are effectors of the cell-
mediated immune response.
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Fig 2. Overview of humoral and cell mediated immune responses.
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Passive Immunization It is the administration of preformed antibodies (usually
IgG) either intravenously or intramuscularly. Used to provide rapid protection in certain infections such
as diptheria or tetanus or in the event of accidental exposure to certain pathogens such as hepatitis B. Also used to provide protection in immune compromised
individuals who are unable to produce appropriate antibody response or in some instances incapable of making any antibody at all (i.e. severe combined immunodeficiency). 38
Antibodies given to immune deficient patients are
usually IgG- derived from pooled normal plasma and are administered on a continuous basis (ideally every three weeks) as they are continuously catabolized and are effective only for short duration. Preformed antibodies from animals, notably horse are
also administered for some diseases but it presents a danger of immune complex formation and serum sickness (if repetitively injected).
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Active immunization Administration of vaccines containing microbial
products with or without adjuvants in order to obtain long term immunological protection against the offending microbe. 2 types:
Systemic Immunization. 2. Mucosal Immunization. 1.
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Systemic Immunization This is the method of choice at present for most
vaccinations. Carried out by injecting vaccine subcutaneously or
intramuscularly into the deltoid muscle. Ideally all vaccines given soon after birth but some
deliberately delayed. Common eg includes vaccines for measles, mumps, and
rubella usually given at the age of 1. If given earlier maternal antibody would decrease their effectiveness. 41
But
carbohydrate vaccines for Pneumococcus, Meningococcus, and Haemophilus infections are given at about 2 years as before this age they respond poorly to polysaccharides unless they are associated with protein components that can act to recruit T cell help for development of anti- polysaccharide antibody.
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Mucosal Immunization Most of the infectious agents gain entry to the systemic
system through mucosal route and the largest source of lymphoid tissue is also present at the mucosal surfaces. Thus recent vaccination approaches have focussed on the
mucosal route as the site of choice for immunization either orally or through the nasal associated immune tissue (NALT). Moreover, it eliminate the need for painful injection and
allow for self-administration of certain vaccines such as those for immunization against influenza. 43
Adjuvant vaccines and live vector vaccines have been
used to target mucosal immune system with some success. Attenuated strains of salmonella can act as a powerful immune stimulus as well as acting as carriers of foreign antigens. This approach has been used to immunize against mucosal surfaces against herpes simplex virus and human papilloma virus. Bacterial toxins, eg those derived from cholera, E. coli etc which posses immunomodulatory properties are also being exploited in the dvpt of mucosally active adjuvants. 44
Table 1. Passive immunization
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Vaccine Vaccine is a preparation containing
a pathogen (disease producing organism) either in attenuated or inactivated state. This preparation is introduced into an individual to induce adequate antibody production against the pathogen in question so that the individual becomes protected against infection, at a later date, by that pathogen. The introduction of a vaccine in an individual is called vaccination or immunization as it leads to the development of immunity in the vaccinated individuals to the concerned pathogen. 46
The immunity is induced by the antigens of pathogen
origin present in the vaccine. Conventionally, various vaccines can be broadly classified into two groups: 1. Vaccines containing killed or inactivated pathogens, i.e. most bacteria vaccines and some virus vaccines (e.g. influenza virus inactivated by formalin, rabies virus inactivated by phenol and β- priolactone). 2. Those containing live but attenuated pathogens, e.g., most virus vaccines.
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Attenuation means a drastic reduction in the virulence
of a pathogen which is achieved as follows: Several consecutive passages through an animal, which is not the usual host of the pathogen, e.g., small pox virus in calf. Several passages through cultured cells of the host, e.g., rabies virus in human diploid cell culture, or of a different species, e.g., rabies virus, yellow fever virus in chick embryo cell culture. Selection of less virulent strains of pathogens, e.g., a mutant strain of polio virus. 48
Treatment of the pathogen with some chemicals, e.g.,
B.C.G. (Bacillus of Calmette Guerien) vaccine produced by culturing the bacteria on a medium containing bile. Culturing pathogens under unfavourable conditions like high temp, e.g., anthrax vaccine obtained by cultivation of the bacterium (Bacillus anthracis) at 4050 0C. In general, inactivation of virus is always coupled with attenuation to minimize the accidental presence of active virulent particles which could cause disease in the vaccinated individuals. 49
The different vaccines differ in their composition,
efficacy and the duration of effective protection to the vaccinated individuals. These are one of the earliest examples of biotechnological intervention in human and animal health care.
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Types of various vaccines There are various types of vaccines, 1.
Whole-Organism Vaccines (Conventional vaccines)
2.
Purified Macromolecules as Vaccines (Conventional )
3.
Subunit Vaccine
4. Recombinant- Vector Vaccines 5.
DNA Vaccines 51
Whole organism vaccines Many vaccines now available for humans, and animal
use are made using whole organisms (bacteria or virus), either in the inactivated (killed) form or attenuated (live but avirulent) form. Examples: Salmonella typhi (killed bacteria)against
Typhoid, Salmonella paratyphi (killed bacteria) against paratyphoid, Vibrio cholerae (killed cells or cell extract) against cholera, Attenuated virus against yellow fever, measles, mumps, rubella and polio.
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Preparation and storage of Typhoid-Paratyphoid A and B Vaccine [TABVaccine] Typhoid fever (enteric fever) is an acute generalized infection caused by Salmonella typhi ; whereas, paratyphoid fever is caused by Salmonella paratyphi A and Salmonella paratyphi B. Preparation
(1) The vaccine is prepared by the general process and contains the following in each millilitre : Typhoid bacilli (Salmonella typhi) : 1000 million Paratyphi A bacilli (S. paratyphi A) and Paratyphi B bacilli (S. paratyphi B) : 500 or 750 million. (2) The smooth strains of the three organisms known to produce the full complement of O somatic antigens should be used. This specific strain of S. typhi must contain the virusassociated antigens (Vi-antigen). (3) It has been duly established that when the organisms were killed with 75% ethanol and the resulting vaccine preserved with 22.5% ethanol, the potency of the alcohol treated vaccine was found to be almost double to that of the heat-treated vaccine, there by minimizing the possibility of both local and constitutional reaction with the relatively smaller dose. Besides, alcohol treated vaccines did possess definitely and predominantly longer life under the optimal storage conditions [viz., storage between 2-4° C without allowing the vaccine to freeze]. 53
Purified macromolecules as vaccine The purified antigenic portions from the bacterial cell
wall, or viral coat protein are used as vaccines, and they can elicit immune reaction. Examples of macromolecule vaccines are: 1. Capsular polysaccharides 2. Surface antigens 3. And inactivated exotoxins called toxoids.
The macromolecule vaccines are generally safe since they don’t contain live organism. Example is given in next slide. 54
Preparation of Meningococcal Polysaccharide Vaccine The Meningococcal Polysaccharide Vaccine consists
of one or more purified polysaccharides obtained from appropriate strains of Neisseria meingitidis group A, group C, group Y and group W135 that have been adequately proved to be capable of producing polysaccharides that are absolutely safe and also capable of inducing the production of satisfactory levels of specific antibody in humans. The vaccine is prepared immediately before use by reconstitution from the stabilized dried vaccine with an appropriate prescribed sterile liquid. It may either contain a single type of polysaccharide or any mixture of the types. 55
The various Preparation steps adopted are as stated under : (1) The preparation of the vacccine is based on a seed-lot system. Each seed-lot is subjected to microbiological examination by culture in an appropriate media and microscopic examination of Gram-stained smears. (2) The polysaccharide shown to be free from contaminating bacteria is precipitated by the addition of cetrimonium bromide and then purified. (3) Each polysaccharide is dissolved under aseptic conditions in a sterile solution containing lactose or another suitable stabilizing medium for freeze drying. (4) The solution is blended, if appropriate, with solution of the polysaccharides of any or all of the other groups and passed through a bacteria-retentive filter. (5) Finally, the filtrate is freeze dried to a moisture content shown to be favourable to the stability of the vaccine
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Limitation of Conventional Vaccines Not all infectious agents can be grown in culture and no vaccines
have been developed for a number of diseases, where the infectious agent is nonculturable. Production of animal and human viruses requires animal cell culture, which is expensive. Yield and rate of production of animal viruses is low. Extensive laboratory precautions are needed while dealing with highly infectious agents. In spite of the best precautions, some batches of vaccines may not be completely killed or attenuated. Attenuated strains may revert to pathogenic state, occasionally, and may cause actual disease against which protection was sought. Not all infectious disease are preventable by traditional vaccines (e.g. AIDS). Have limited Shelf-life thus requires refrigeration.
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Vaccines made through recombinant DNA technology Recombinant DNA technology can be best used in the
following ways in vaccine development. 1. Virulence genes can be deleted from the infectious agent retaining the immunogenic properties. 2. An organism (non-pathogenic) carrying antigenic determinants can be created by insertion of the genes coding for the antigenic proteins. 3. For non-culturable agents, genes for the protein (critical antigenic determinants) can be cloned and expressed in an expression vector (e.g. E. Coli or a mammalian cell line). 4. A targeted cell-specific killing system that kills only the infected cells can be designed. In this technique, gene for a ‘fusion protein’ is constructed. First, one part of the fusion protein binds to the infected cell. Then the other part kills the infected cell. 58
Subunit Vaccines For viruses, it has been shown that specific protein from
the coat or envelope is enough to elicit the immune response. Vaccines with components of a pathogenic organism rather than the whole organism are called subunit vaccines. rDNA technology is best suited to develop subunit vaccines. Purified proteins are more stable and are chemically precise and safe from side effects. However, purification of protein can be expensive and sometimes purification can alter the configuration of protein and alter its antigenicity!!! These factors have to be assessed before making a protein preparation. One of the example of subunit vaccines developed through rDNA tech will be discussed in the upcoming slides. 59
Subunit vaccine for foot and mouth disease virus (FMDV) Formalin-killed FMDV was used as vaccine earlier. The
genome of FMDV is single stranded RNA (ssRNA). The cDNA complementary to this ssRNA, 8000
nucleotide long is prepared . It is digested with restriction enzymes, and the fragments are cloned in E. coli.
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Recombinant vector vaccines Genes that encode major antigens of especially virulent
pathogens can be introduced into attenuated viruses or bacteria. The attenuated organism serves as a vector, replicating within
the host and expressing the gene product of the pathogen. A number of organisms have been used for vector vaccines,
including vaccinia virus (it is most strong candidate as it is efficient in delivery and expression of cloned genes), the canarypox virus, attenuated poliovirus, adenovirus, attenuated strains of Salmonella, the BCG strain of Mycobacterium bovis and certain strains of streptococcus that normally exist in the oral cavity. 61
Vaccinia virus has been widely employed as a vector
vaccine. This large, complex virus, with a genome of about 200 genes, can be engineered to carry several dozen foreign genes without impairing its capacity to infect host cells and replicate. The process of producing a vaccinia vector that carries a foreign gene from a pathogen is outlined in figure below. The genetically engineered vaccinia expresses high level of the inserted gene product, which can then serve as a potent immunogen in an inoculated host. 62
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Like the smallpox vaccine, genetically engineered vaccinia
vector vaccines can be administered simply by scratching the skin, causing localized infection in the host cells. Antigen genes introduced into animal cells through vaccinia virus genome inclues Rabies virus G protein, Hepatitis B surface antigen, Influenza virus NP and HA proteins, etc. If the foreign gene product expressed by the vaccinia is a viral envelope protein, it is inserted into the membrane of the infected host cell, inducing development of cellmediated immunity as well as antibody mediated immunity. Similar to vaccinia vector vaccines other vector vaccines which have been recently tried include canarypox virus. 64
DNA vaccines/ Gene vaccine (Genetic Immunization) Plasmid DNA encoding antigenic proteins is injected
directly into the muscle of the recipient. Muscle cells take up the DNA and the encoded protein antigen is expressed, leading to both a humoral and cell-mediated response. The DNA either integrate into the chromosomal DNA or to
be maintained for long periods in an episomal form. It offers advantage over many of the existing vaccines few of
which are listed below: 1 . The encoded protein is expressed in the host in its natural form- there is no denaturation or modification . Due to this the immune response is therefore directed to the antigen exactly as it is expressed by the pathogen. 65
2. It induces both humoral and cell mediated immunity. 3. DNA vaccines cause prolonged expression of the antigen, which generates significant immunological memory. 4. Refrigeration is not required for handling and storage of the plasmid DNA (thus lowers cost and complexity of delivery). 5. The same plasmid vector could be custom tailored to make variety of proteins, so the same manufacturing techniques can be used for different DNA vaccines, each encoding an antigen from a different pathogen. 66
An improved method of administering these vaccines
involves coating microscopic gold beads with the plasmid DNA and then delivering the coated particles through the skin into the underlying muscle with an air gun (called gene gun). This will allow rapid delivery of a vaccine to large populations without the requirement for huge supplies of needles and syringes. Test of DNA vaccines in animal models have shown
these vaccines to be effective against various viral diseases including influenza virus. 67
Antigen-Antibody Interaction The antigen-antibody interaction is a biomolecular
association similar to an enzyme-substrate interaction. However, it doesn’t lead to an irreversible chemical
alteration in either the antibody or the antigen. The association between an antigen and antibody
involves various noncovalent interactions between the antigenic determinant (epitope) of the antigen and the variable-region (VH/VL ) domain of the antibody molecule, particularly the hypervariable regions, or complementarity- determining regions (CDRs). 68
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The noncovalent interactions that form the basis of
antigen-antibody binding include hydrogen bonds, ionic bonds, hydrophobic interactions, and vander Waals interactions. Since these interactions are individually weak (compared with a covalent bond), a large number of such interactions are required to form a strong Ag-Ab interaction. Furthermore, each of these noncovalent interactions operates over a very short distance (1 angstrom or 1 x 10-7 mm). Hence a strong Ag-Ab interaction depends on a very close fit between the antigen and antibody. Such fit require a high degree of complementarity between antigen and antibody. 70
Cross-reactivity Although Ag-Ab reactions are highly specific, in some cases
antibody elicited by one antigen can cross- react with an unrelated antigen. Such cross-reactivity occurs if two different antigens share an identical or very similar epitope. Cross- reactivity is often observed among polysaccharide antigens that contains similar oligosaccharide residues. The ABO blood-group antigens, for example, are glycoproteins expressed on RBCs. Subtle differences in the terminal residues of the sugars attached to these surface proteins distinguish the A and B blood group antigens. RBC glycoprotein sugars attached to the terminal end of it subtle difference in the terminal residues of these sugars distinguish A and B blood group antigens. 71
An individual lacking one or both of these antigens will
have serum antibodies to the missing antigen(s). The antibodies are induced not by exposure to red blood cell antigens but by exposure to cross-reacting microbial antigens present on common intestinal bacteria. These microbial antigens induce formation of antibodies in individuals lacking the similar blood-group antigens on their RBCs. The blood-group antibodies, although elicited by microbial antigens, will cross-react with similar oligosaccharides on foreign RBCs. This provides the basis for blood typing tests and accounts for the necessity of compatible blood types during blood transfusions. Type A individual has anti- B antibodies, type B has anti- A and type O has anti- A & B . 72
Numerous viruses and bacteria have epitopes identical
or similar to normal host-cell components. In some cases, these microbial antigens have shown to elicit antibody that cross-reacts with the host-cell components, resulting in a tissue- damaging autoimmune reaction. For e.g. bacterium Streptococcus pyrogenes, express cell wall proteins called M antigens . Abs produced against these antigens have shown to cross react with several myocardial and skeletal muscle proteins causing kidney and heart damage following streptococcal infections. Some vaccines also exhibit cross-reactivity. 73
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1. Precipitation Reactions Antibody and soluble antigen interacting in aqueous solution form a
lattice that eventually develops into a variable precipitate.
Antibodies that aggregate soluble antigens are called precipitins. Although formation of the soluble Ag-Ab complex occurs within
minutes, formation of the visible precipitate occurs more slowly and often takes a day or two to reach completion.
Formation of Ag-Ab lattice depends upon the valency of both: 1. Ab should be bivalent; a precipitate will not form with monovalent
Fab fragments.
2.
Ag must be either bivalent or polyvalent i.e. it must have at least two copies of the same epitope or have different epitopes that react with different antibodies present in polyclonal antisera.
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A. Precipitation reaction in fluids Precipitation reaction in fluids yields a precipitin
Curve. A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amount of antigen to the tubes. At one time this method was used to measure the amount of antigen or antibody present in a sample of interest. Once precipitate is formed each tube centrifuged to pellet the precipitate supernatant poured off amount of precipitate is measured. 76
Plotting
the amount of precipitate against increasing antigen concentrations yields a precipitin curve. The figure below shows that the excess of either antigen or antibodies interferes with maximal precipitation, which occurs at equivalence point. Maximal precipitation occurs at equivalence point. As a large macromolecular lattice is formed at equivalence, complex increases in size and precipitate out. Follow figure and draw it!!!!it is important to draw figure. 77
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B. Precipitation Reaction in Gels Precipitation rxn in gels yields visible precipitin Lines. Immune precipitates can form not only in solution but also
in agar matrix. When antigen and antibody diffuses towards one another in agar, or when Ab is incorporated into the agar and antigen diffuses into the antibody containing matrix, a visible line of precipitation will form. As in precipitation reaction in fluid, visible precipitation occurs in the region of antibody or antigen excess. Two types of immunodiffusion reactions can be used to determine relative concentration of antibodies or antigen , to compare antigens, or to determine the relative purity of an antigen preparation. 79
Two types of immunodiffusion reactions both of which
are carried out semisolid medium such as agar. 1 Radial immunodiffusion(Mancini method): In this method, an Ag sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of antiserum. As antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation, a precipitin ring, forms around the well. The area of precipitin ring is proportional to the concentration of antigen. By comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known concentrations of the antigen), the concentration of the antigen sample can be determined. 80
2. Double immunodiffusion (the Ouchterlony method): In this method both the antigen and antibody diffuse, radially from the wells towards each other , thereby establishing a concentration gradient. As equivalence is reached, a visible line of precipitation, a precipitin line is formed.
Please refer figure in next slide!!!!!!!!!!!!!!
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2. Agglutination Reaction The interaction between antibody and antigen results
in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reaction are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens. Just as an excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition is called prozone effect.
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Its application
a. Hemagglutination is used in blood typing: Agglutination reactions are routinely performed to type RBCs. In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood-group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide. Determination of which antigens are present on donor and recipient RBCs is the basis for matching blood types for transfusions. b. Bacterial agglutination is used to diagnose infection. c. Passive agglutination is useful with soluble antigens. 84
Immuno-assay Techniques Radioimmunoassay
ELISA Western Blotting Immunofluorescence
Immunoelectron Microscopy.
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Immunoassays The
exquisite specificity of antigen-antibody interactions has led to the development of a variety of immunologic assays, which can be used to detect the presence of either antibody or antigen.
Immunoassays have played vital roles in diagnosing
diseases, monitoring the level of the humoral immune response, and identifying molecules of biological or medical interest. These assays differ in their speed and sensitivity, some are strictly qualitative, other are quantitative.
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Radioimmunoassay One of the most sensitive techniques for detecting antigen or
antibody is radioimmunoassay (RIA). Principle: The principle of RIA involves competitive binding of
radiolabeled antigen(usually labeled with gamma emitting isotope such as 125I but beta emitting isotopes such as tritium 3H are also routinely used as labels) to a high- affinity antibody. The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. Then
test samples of unlabeled antigen of unknown concentration are added in progressively larger amounts. The antibody doesn’t distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. 87
As the concentration of unlabeled antigen increases, more
labeled antigen will be displaced from the binding sites. The decrease in the amount of radiolabeled antigen bound to
specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample.[Note: To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to separate it from free antigen, and the radioactivity in the precipitate is measured. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of test sample), and from this plot the amount of antigen in the test mixture may be precisely determined.] A microtiter RIA has been widely used to screen for the presence
of the hepatitis B virus. 88
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Enzyme-Linked Immunosorbent Assay Commonly known as ELISA (or EIA). Its principle is similar to RIA but depends on an enzyme
rather than a radioactive label. An enzyme conjugated with an antibody reacts with a
colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline phosphatase, horseradish peroxidase, and β- galactosidase. These assays approach the sensitivity of RIAs and have the advantage of being safe and less costly. 90
Types: 3 types. Indirect ELISA: used in determination of serum Abs
against HIV. Sandwich ELISA Competitive ELISA In
one of the version of ELISA using chemiluminescence, a luxogenic (light-generating) substrate is used in place of chromogenic substrate (used in conventional ELISA). Its advantage is increased sensitivity. 91
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Indirect ELISA Antibody can be detected or quantitatively determined with an indirect ELISA (Figure 6-10a). Serum or some other sample containing primary antibody (Ab1) is added to an antigen-coated microtiter well and allowed to react with the antigen attached to the well. After any free Ab1 is washed away, the presence of antibody
bound to the antigen is detected by adding an enzymeconjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody.
Any free Ab2 then is washed away, and a substrate for the
enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds. 93
Indirect ELISA is the method of choice to detect the presence of serum
antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS. In this assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to
epitopes on these viral proteins. Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection.
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SANDWICH ELISA Antigen can be detected or measured by a sandwich ELISA
(Figure 6-10b). In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well. A sample containing antigen is added and allowed to react
with the immobilized antibody. After the well is washed, a second enzyme- linked antibody
specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.
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COMPETITIVE ELISA Another variation for measuring amounts of antigen is competitive ELISA (Figure 6-10c). In this technique, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen coated
microtiter well. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific
for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well as in an indirect ELISA. In the competitive assay, however, the higher the concentration of
antigen in the original sample, the lower the absorbance.
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Western blotting Indentification of a specific protein in a complex mixture of proteins can be
accomplished by a technique known as Western blotting. In western blotting, a protein mixture is electrophoretically separated on an SDSpolyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate (SDS), a dissociating agent. The protein bands are transferred to a nylon membrane by electrophoresis and the individual protein bands are identified by flooding the nitrocellulose membrane with radiolabeled or enzyme linked polyclonal or monoclonal antibody specific for the protein of interest. The Ag-Ab complexes that is formed on the band ,containing the protein recognized by the antibody, can be visualized in a variety of ways. If the protein of interest was bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of x-ray film, a procedure called autoradiography. However, the most generally used detection procedures employ enzyme-linked antibodies against the protein. After binding of the enzyme antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen. The site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along with suitable enhancing agents is used to produce light at the antigen site.
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Western blotting can also identify a specific antibody in a mixture. In
this case, known antigens of well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. The separated bands of known antigens are then probed with the sample suspected of containing antibody specific for one or more of these antigens. Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary antibody that is specific for the species of the antibodies in the test sample. The most widely used application of this procedure is in confirmatory testing for HIV, where Western blotting is used to determine whether the patient has antibodies that react with one or more viral proteins.
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Immunoprecipitation
The immunoprecipitation technique has the advantage of
allowing the isolation of the antigen of interest for further analysis. It also provides a sensitive assay for the presence of a particular antigen in a given cell or tissue type. An extract produced by disruption of cells or tissues is mixed with an antibody against the antigen of interest in order to form an antigen-antibody complex that will precipitate. However, if the antigen concentration is low (often the case in cell and tissue extracts), the assembly of antigen-antibody complexes into precipitates can take hours, even days, and it is difficult to isolate the small amount of immunoprecipitate that forms. When used in conjugation with biosynthetic radioisotope labelling, immunoprecipitation can also be used to determine whether a particular antigen is actually synthesized by a cell or tissue. 100
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Immunofluorescence In 1944, Albert Coons showed that antibodies could be
labeled with molecules that have the property to fluorescence. If antibody molecules are tagged with fluorescent dye, or fluorochrome, immune complexes containing these fluorescently labeled antibodies can be detected by colored light emission when excited by light of appropriate wavelength. Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized. The emitted light could be viewed with fluorescence microscope, which is equipped with UV light source. 102
In this technique (immunofluorescence), various
fluorescent compounds in use include Fluorescein, Rhodamine, Phycoerythrin. Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect. In direct staining, the specific antibody (the primary Ab) is directly conjugated with fluorescein. In indirect staining, the primary Ab is unlabeled and is detected with an additional flurochrome-labeled reagent.
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Immunoelectron microscopy Specificity of antibody has made them powerful tools
for visualizing specific intracellular tissue components by immunoelectron microscopy. In this technique,
Electron-dense label conjugated to Fc portion of a specific antibody for direct staining or conjugated to an anti-immunoglobulin reagent for indirect staining Electron dense label(commonly used are ferritin and colloidal gold) absorbs electrons it can be visualised with electron microscope as small black dots. 104
Monoclonal Antibodies Monoclonal Antibodies
are usually produced from
hybridoma clones. Each hybridoma clone is derived by the fusion of a myeloma cell and an antibody producing lymphocyte, and the hybridoma clone producing the desired antibody is identified and isolated. Hybridoma cells are mass-cultured for the production of monoclonal antibodies either (1) in vivo in the peritoneal cavity of mice or (2) in vitro in large scale culture vessels.
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Application When Mabs are used to detect the presence of a
specific antigen or of antibodies specific to an antigen in a sample or samples, this constitutes a diagnosic application. Antibodies specific to a cell type, say, tumor cells, can be linked with a toxin polypeptide to yield a conjugate molecule called immunotoxin. This immunotoxin will bind to tumor cell and kills it. Immunopurification.
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ANTIGENS AND HAPTENS The two terminologies viz., antigens and haptens are intimately associated with immunology ; and, hence one may understand and have a clear concept about them as far as possible.
Antigens An antigen is either a cell or molecule which will bind with preexiting antibody but will not definitely cause induction of antibody production. Antigen may also be defined as — ‘a macromolecular entity that essentially elicits an immune response via the formation of specific antibodies in the body of the host’. In a broader perspective the antigen (or immunogen) is invariably regarded as the afferent branch of the prevailing immune system, and is any cell or molecule which would provoke an immune response very much in an immunologically viable and competent individual. Generally, immunogens (antigens) must fulfill the following two characteristic features, namely: (a) should be larger than 2000 in molecular weight, e.g., protein, glycoprotein and carbohydrates, and (b) must be absolutely foreign to the individual into whom they have been introduced appropriately.
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Example : The best example of an ‘antigen’ is ones own
erythrocytes. Because, they will not induce antibody formation in oneself but will definitely react with an antibody essentially contained in an improperly matched blood transfusion. Quite often an antigen is a protein, but it could also be a polysaccharide or nucleic acid or any other substance. Importantly, it may also be possible that a foreign substance (e.g., protein)-not necessarily belonging to a pathogenic microorganism, may act as an antigen so that on being injected into a host, it may induce antibody formation. Besides, they may turn out to be antigenic and thereby cause stimulation of antibody production, incase they are intimately and lightly get bound to certain macromolecules, for instance : proteins, carbohydrates and nucleic acids. 108
Haptens In usual practice, the relatively smaller, less rigid or rather less
complex molecules usually are not immunogenetic in their purest form, but may be made so by simply linking them strategically to either larger or more complex structures. Consequently, the smaller molecules are invariably termed as haptens ; whereas, the larger molecules or cells are known as carriers. Hapten may also be defined—‘as a substance that normally does not act as an antigen or stimulate an immune response but that can be combined with an antigen and, at a later time, initiate a specific antibody response on its own’. Furthermore, small molecules (micromolecular), such as : drug substances, that may serve as ‘haptens’ and can normally be made antigenic by coupling them chemically to a macromolecular substance e.g., protein, polysaccharide, carbohydrate etc. The hapten is obtained from a non-antigenic compound (micromolecule) e.g., morphine, carteolol etc., which is ultimately conjugated, covalently to a carrier macromolecule to render it antigenic.
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One of the good example is of gastrin (hapten) which is duly
coupled to albumin (i.e., protein carrier) by treatment with carbodiimides (CCD), which couple functional carboxyl, amino, alcohol, phosphate or thiol moieties. Importantly, the hapten-conjugate thus obtained is normally subjected to emulsification in a highly refined ‘mineral oil’ preparation containing-killed Mycobacterium (Complete Freund’s Adjuvant), and subsequently injected intradermally either in healthy rabbits or guinea pigs on several occasions at intervals. Evidently, the serum antibody should have not only high degree of specificity but also a reasonably strong affinity for the prevailing antigens.
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Hypersensitivity Immune system mobilizes variety of effector molecules that act to
remove antigen by various mechanisms. Generally, these effector molecules induce a localized inflammatory
response that eliminates antigen without extensively damaging the host’s tissue. Under certain circumstances, however, this inflammatory response can have deleterious effects, resulting in significant tissue damage or even death. This inappropriate immune response is termed hypersensitivity or allergy. Although the word hypersensitivity implies an increased response, the
response is not always heightened but may, instead, be an inappropriate immune response to an antigen. Hypersensitive reactions may develop in the course of either humoral or cell-mediated responses. Hypersensitivity may be defined as — ‘an abnormal sensitivity to a stimulus of any kind’. 111
There are four types of hypersensitivity reaction:
Type I hypersensitivity (IgE Mediated Hypersensitivity) 2. Type II (IgG Mediated Hypersensitivity) 3. Type III (Immune complex mediated hypersensitivity) 4. Type IV (Cell Mediated Hypersensitivity) 1.
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1. IgE mediated Hypersensitivity A type I hypersensitive reaction is induced by certain types of antigens (such as foreign serum, vaccine, penicillin, rye grass, ant venom, bee venom, etc) referred to as allergens, and has all the characteristics of a normal humoral response.
That is, an allergen induces a humoral antibody response by the same mechanisms as for other soluble antigens, resulting in the generation of antibody-secreting plasma cells and memory cells.
What distinguishes a type I hypersensitive response from a normal humoral response is that the plasma cells secrete IgE. This class of antibody binds with high affinity to Fc receptors on the surface of tissue mast cells and blood basophils.
Mast cells and basophils coated by IgE are said to be sensitized. A later exposure to the same allergen cross-links the membrane-bound IgE on sensitized mast cells and basophils, causing degranulation of these cells. The pharmacologically active mediators released from the granules act on the surrounding tissues. The principal effects—vasodilation and smooth-muscle contraction—may be either systemic or localized, depending on the extent of mediator release. The clinical manifestations of type I reactions can range from life-threatening conditions, such as systemic anaphylaxis and asthma, to hay fever and eczema, which are merely annoying 113
General mechanism underlying a type I hypersensitive reaction. Exposure to an allergen activates B cells to form IgE secreting plasma cells. The secreted IgE molecules bind to IgE specific Fc receptors on mast cells and blood basophils. (Many molecules of IgE with various specificities can bind to the IgE-Fc receptor.) Second exposure to the allergen leads to crosslinking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, from mast cells and basophils. The mediators cause smooth-muscle contraction, increased vascular permeability, and vasodilation. 114
2. Antibody Mediated cytotoxic Hypersensitivity Type II hypersensitive reactions involve antibody-mediated destruction of cells. Antibody can activate the complement system, creating pores in the membrane of a foreign cell, or it can mediate cell destruction by antibody dependent cell-
mediated cytotoxicity (ADCC). In this process, cytotoxic cells with Fc receptors bind to the Fc region of antibodies on target cells and promote killing of the cells .Antibody bound to a foreign cell also can serve as an opsonin, enabling phagocytic cells with Fc or C3b receptors to bind and phagocytose the antibody-coated cell. Examples : The various examples are as stated below : (i) Transfusion reactions i.e., when blood groups are not matched properly, (ii) Haemolytic disease concerning the newly born babies via Rhesus incompatibility, (iii) Graft destruction or rejection i.e., antibody-mediated ‘graft’ destruction or rejection. (iv) Autoimmune reactions usually directed against the formed elements of the blood, and the kidney glomerular basement membrances, etc.
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Example 1: Transfusion Reactions Are Type II Reactions A large number of proteins and glycoproteins on the membrane of red blood cells
are encoded by different genes, each of which has a number of alternative alleles. An individual possessing one allelic form of a blood-group antigen can recognize other allelic forms on transfused blood as foreign and mount an antibody response. In some cases, the antibodies have already been induced by natural exposure to similar antigenic determinants on a variety of microorganisms present in the normal flora of the gut. This is the case with the ABO blood-group antigens. Antibodies to the A, B, and O antigens, called isohemagglutinins, are usually of the IgM class. An individual with blood type A, for example, recognizes B-like epitopes on intestinal microorganisms and produces isohemagglutinins to the B-like epitopes. If a type A individual is transfused with blood containing type B cells, a transfusion reaction occurs in which the anti-B iso-hemagglutinins bind to the B blood cells and mediate their destruction by means of complement-mediated lysis. Antibodies to other blood-group antigens may result from repeated blood transfusions because minor allelic differences in these antigens can stimulate antibody production. These antibodies are usually of the IgG class. The clinical manifestations of transfusion reactions result from massive intravascular hemolysis of the transfused red blood cells by antibody plus complement. These manifestations may be either immediate or delayed. 116
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Hemolytic Disease of the Newborn Is Caused by Type II Reactions Hemolytic disease of the newborn develops when maternal IgG antibodies specific for fetal blood-group antigens cross the placenta and destroy fetal red blood cells. The consequences of such transfer can be minor, serious, or lethal. Severe hemolytic disease of the newborn, called erythroblastosis fetalis, most commonly develops when an Rh+ fetus expresses an Rh antigen on its blood cells that the Rh– mother does not express. This most commonly happens when a woman with Rh negative blood becomes pregnant by a man with Rh positive blood and conceives a baby with Rh positive blood. Red blood cells from the baby can leak across the placenta into the woman's bloodstream during pregnancy or delivery. This causes the mother's body to make antibodies against the Rh factor. If the mother becomes pregnant again with an Rh-positive baby, it is possible for her antibodies to cross the placenta and attack the baby's red blood cells. After birth, an affected newborn may develop kernicterus. This happens when bile pigments are deposited in the cells of the brain and spinal cord and nerve cells are degenerated. Incompatibilities between ABO blood types can also cause this condition. These are less common than those of the Rh factor and tend to be less severe.
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3. Immune Complex–Mediated (Type III) Hypersensitivity The reaction of antibody with antigen generates immune complexes. Generally this complexing of antigen with antibody facilitates the clearance of antigen by phagocytic cells. In some cases, however, large amounts of immune complexes can lead to tissue-damaging type III hypersensitive reactions. The magnitude of the reaction depends on the quantity of immune complexes as well as their distribution within the body. When the complexes are deposited in tissue very near the site of antigen entry, a localized reaction develops. When the complexes are formed in the blood, a reaction can develop wherever the complexes are deposited. In particular, complex deposition is frequently observed on blood-vessel walls, in the synovial membrane of joints, on the glomerular basement membrane of the kidney, and on the choroid plexus of the brain. The deposition of these complexes initiates a reaction that results in the recruitment of neutrophils to the site. The tissue there is injured as a consequence of granular release of lytic enzymes from the neutrophil.
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Type III hypersensitive reactions develop when immune complexes activate the complement system’s array of immune effector molecules. The C3a, C4a, and C5a complement split products are anaphylatoxins that cause localized mastcell degranulation and consequent increase in local vascular permeability. C3a, C5a, and C5b67 are also chemotactic factors for neutrophils, which can accumulate in large numbers at the site of immune-complex deposition. Larger immune complexes are deposited on the basement membrane of blood vessel walls or kidney glomeruli, whereas smaller complexes may pass through the basement membrane and be deposited in the subepithelium. The type of lesion that results depends on the site of deposition of the complexes. Much of the tissue damage in type III reactions stems from release of lytic enzymes by neutrophils as they attempt to phagocytose immune complexes. The C3b complement component acts as an opsonin, coating immune complexes. A neutrophil binds to a C3b-coated immune complex by means of the type I complement receptor, which is specific for C3b. Because the complex is deposited on the basement- membrane surface, phagocytosis is impeded, so that lytic enzymes are released during the unsuccessful attempts of the neutrophil to ingest the adhering immune complex. Further activation of the membrane-attack mechanism of the complement system can also contribute to the destruction of tissue. In addition, the activation of complement can induce aggregation of platelets, and the resulting release of clotting factors can lead to formation of microthrombi.
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Type IV or Delayed-Type Hypersensitivity (DTH) When some subpopulations of activated TH cells encounter certain types
of antigens, they secrete cytokines that induce a localized inflammatory reaction called delayed-type hyper- sensitivity (DTH). The reaction is characterized by large influxes of nonspecific inflammatory cells, in particular, macrophages. This type of reaction was first described in 1890 by Robert Koch, who observed that individuals infected with Mycobacterium tuberculosis developed a localized inflammatory response when injected intradermally with a filtrate derived from a mycobacterial culture. He called this localized skin reaction a “tuberculin reaction.” The characteristic of a type IV reaction are the delay in time required for the reaction to develop and the recruitment of macrophages as opposed to neutrophils, as found in a type III reaction. In this type, sensitized TH1 cells release cytokines that activate macrophages or TC cells which mediate direct cellular damage. Macrophages are the major component of the infiltrate that surrounds the site of inflammation.
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Antibody Production (Immunogen Preparation) The production of specific antibody probes is a
relatively straightforward process involving immunization of animals and reliance upon their immune systems to raise responses that result in biosynthesis of antibodies against the injected molecule. Even so, several factors affect the probability of inducing an immunized animal to produce useful amounts of target-specific antibodies. Antigens must be prepared and delivered in a form and manner that maximizes production of a specific immune response by the animal. This is called immunogen preparation. 128
Antibody production is conceptually simple. However, because it
depends upon such a complex biological system (immunity of a living organism), results are not entirely predictable. Individual animals – even those that are genetically identical – will respond uniquely to the same immunization scheme, generating different suites of specific antibodies against an injected antigen. Even so, equipped with a basic understanding of how the immune system responds to injection of a foreign substance and a knowledge of available tools for preparing a sample for injection, researchers can greatly increase the probability of obtaining a useful antibody product. For example, small compounds (drugs or peptides) are not sufficiently complex by themselves to induce an immune response or be processed in a manner that elicits production of specific antibodies. For antibody production to be successful with small antigens, they must be chemically conjugated with immunogenic carrier proteins such as keyhole limpet hemocyanin (KLH). Adjuvants can be mixed and injected with an immunogen to increase the intensity of the immune response. 129
