Microbiology Immunity and Serology Notes Study Material
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Microbiology Immunity and Serology Notes Study Material
The word immune is derived from the Latin stem Immuno, meaning safe or free from. In a general sense, the term implies a condition under which an individual is protected from disease (not all but some specific diseases).
There are two general types of immunity:
(i) Innate immunity, and (ii) Acquired immunity. Innate Immunity is an inborn capacity for resisting disease. It begins at birth and depends on genetic factors. Examples of this type of immunity are species and racial immunities. Acquired immunity, by contrast, begins after birth. It depends on the presence of antibodies and other factors originating from the immune system. Four types of acquired immunity are generally recognized, which are described here. It may be seen that the emphasis will be on antibodies and humoral immunity. Cellular immunity is also important in the total spectrum of disease resistance.
[I] Naturally acquired active immunity
Active immunity develops after antigens enter the body and the individual’s immune system responds with antibodies. The exposure to antigens may be unintentional or intentional. When former, the immunity that develops is called naturally acquired active immunity. Naturally acquired active immunity usually follows a bout of illness.
However, this may not be always and subclinical diseases may also induce this immunity. For example, many have acquired immunity from subclinical cases of mumps (viral) or cryptococcosis (fungal). Memory cells in the lymphoid tissue are responsible for the production of antibodies that yield this immunity. The cells remain active for many years and produce IgG immediately upon later entry of the parasite (antigen) into the host. Such an antibody response is also called a secondary anamnestic response.
[II] Artificially acquire active immunity
This immunity develops after intentional exposure to the antigens. The antigens are usually contained in an immunizing agent such as a vaccine or toxoid. (Microbiology Immunity and Serology Notes Study Material)
Viral vaccines consist either of inactivated or killed viruses (i.e. the viruses incapable of multiplying in the body) or attenuated viruses (i.e, live but multiplying at low rates in the body amount of disease). The Salk polio vaccine is an example of the former, while the Sabin oral polio vaccine that of the latter type. Bacterial vaccines are also of similar categories: the whooping cough vaccine has dead cells whereas the tuberculosis vaccine has live, attenuated bacteria. Whole microorganism, viral and bacterial, vaccines are commonly called first-generation vaccines. Attenuated organisms provide a higher level of immune response than that obtained by a single dose of inactivated organisms.
This is due to the reason that attenuated organisms multiply in the body for some time thus increasing the dose of antigen. This is an advantage of vaccines made with attenuated organisms. But, attenuated organisms may cause health hazards due to this ability of multiplication. (Microbiology Immunity and Serology Notes Study Material)
Expert for whooping cough (pertussis) vaccine, which gives long-term immunity, bacterial vaccines are used for temporary protection (several months). Rickettsial vaccines for Rocky Mountain spotted fever, Q fever, and typhus are also available.
Toxoids are immunizing agents consisting of chemically altered toxins. They are used to stimulate immunity to toxins. Toxoids are currently available for protection against diphtheria and tetanus, the two diseases whose major effects are due to toxins. The toxoids are prepared by incubating toxins with a chemical such as formaldehyde until the toxicity is lost. To avoid multiple injections of immunizing agents, vaccines may be combined into a single dose. This is possible for the diphtheria-pertussis-tetanus vaccine (DPT), the measles-mumps-rubella vaccine (MMR), and the trivalent oral polio vaccine (TOP). (Microbiology Immunity and Serology Notes Study Material)
It is likely that in near future the whole organism vaccines will be completely replaced by subunit vaccines or second-generation vaccines. For example, pili from bacteria can be extracted and purified for stimulatory antipili antibodies. This would inhibit the attachment of bacteria to the host surface. Another vaccine of this type is that pneumococcal pneumonia. This vaccine contains 23 different polysaccharides from the capsules of strains of Streptococcus pneumoniae. A third example is hepatitis B. This vaccine contains fragments of hepatitis virus surface antigens isolated from the blood of hepatitis carriers.
Another form of vaccine is synthetic vaccines or third-generation vaccines. Their preparation is based on the application of recombinant DNA technology. Briefly, the whole procedure involves: (i) the immune-stimulating antigen must be identified, (ii) live cells must be re-engineered to produce the antigens; and (iii) the size of the antigens must be increased to promote phagocytosis and the immune response. A vaccine of this type is licensed in 1981. This is a vaccine for foot-and-mouth disease. For other vaccines, work is in progress.
Viral Vaccines
A vaccine (an immunogen used against infectious disease) may be either infectious (live) or non-infectious (killed). Upon administration, all vaccines should fulfill the following requirements.
(1) It causes less severe disease than the natural infection. The process of producing a virus strain which causes a reduced amount of disease attenuation. The disease-causing virus is called virulent strain and the attenuated strain is avirulent.
(2) It provides effective and long-lasting immunity.
(3) It is genetically stable.
Live vs killed vaccines
Formaldehyde and Beta-propiolactone are usually used for the inactivation of viruses. These agents inactivate viral nucleic acid function. Generally, a small fraction of the population (approx. 1,000,000,000 particles make one dose) is inactivated far more slowly than the majority, so the whole virus population has to be kept in contact with the inactivating agent for a much longer period.
There is a danger of the destruction of immunogenicity during this period. Live vaccines present no such problems. Jonas Salk faced this type of problem when he presented the first poliovirus vaccine (killed) in 1953 to the public. Albert Sabin 1957 could produce a successful live vaccine for polio. (Microbiology Immunity and Serology Notes Study Material)
A killed vaccine is injected twice to build up a sufficient immune response since they are not able to multiply. Live vaccines may multiply and reach places where the infectious virus is present. (Microbiology Immunity and Serology Notes Study Material)
Live vaccines are cheap to produce compared with killed vaccines. The killed vaccines are needed more in quantity. In multiple vaccinations, killed vaccines are preferred by our live ones. It is possible to immunize with a vaccine cocktail, that minimizes the number of injections and inconvenience. Results are satisfactory with multiple killed vaccines. Live vaccines may have problems due to mutual interference in multiplication possibly as a result of the induction of interferon. However, a triple live vaccine of measles, mumps, and rubella viruses has been successfully used. (Microbiology Immunity and Serology Notes Study Material)
New approaches to vaccines
Vaccine manufacture is an immensely difficult process. The central problem is safety. One must be sure that a killed vaccine contains no residual virulent particles or that a live attenuated vaccine does not revert to virulence. Neither can be guaranteed absolutely. The work is on along the following lines in search of effective vaccine therapy. (Microbiology Immunity and Serology Notes Study Material)
1. Understanding the antigenic determinants. In killed vaccines (that consist of whole particles) only a selected surface structure of the virus stimulates immunity, the rest is non-immunogenic or the immune response is ineffective. Studies have been made to find these selected structures. Why then not have a vaccine that has only these structures and not the whole particle? In the influenza virus, for example, only four regions of the molecule are involved in building neutralizing antibodies. A vaccine should consist solely of oligopeptides in the correct conformation. (Microbiology Immunity and Serology Notes Study Material)
2. Prediction of antigenic determinants. Antigenic determinants are likely to be parts of the molecule which are rich in hydrophilic amino acids. There are computer programmes to look for hydrophilic regions. Hydrophilic oligopeptides are then synthesized chemically, covalently linked to a carrier molecule, and used to raise antibodies. (Microbiology Immunity and Serology Notes Study Material)
3. Hepatitis B virus vaccine. HBV is a worldwide cause of the commonest type of cancer, primary hepatic carcinoma, transmitted through contact with infected blood and salvia. The problem is that HBV cannot be grown in culture and infects only man and higher primates like chimpanzees. It has partly double-stranded DNA. The purified virus formed the basis of a unique (literally manmade) killed vaccine that provided the first public health measure against this serious disease.
Genetically engineered vaccines
These are also called synthetic vaccines or third-generation vaccines. While opting for a killed vaccine one must ensure that (i) enough material could be produced cheaply and (ii) no infectious virus survives the inactivation procedure. The recently developed DNA technology could identify the part of the viral genome that encoded the particular virus protein against which protective immunity (usually antibody) was directed. Viral DNA, or a DNA copy of the virus that had an RNA genome, can be excised and inserted into an appropriate expression vector together with control (promoter, stop, and polyadenylation) signals.
Thus we have a small part of the viral genome, by definition non-infectious, which by insertion into host cells growing on an industrial scale will produce very large amounts of protein very cheaply. One even need not grow the virus in culture, a great advantage for viruses like Hepatitis B.
What are the plus and minus points of genetically engineered vaccines? Well, the advantages are that they are non-infectious, their large-scale production methods are available, are cheap, to produce, and can use genes from non-cultivable viruses. At the same time, the problems likely to arise are identification of neutralization antigen, need for proper co-and post-translational modifications of the viral polypeptide, need for proper assembly of viral protein to avoid poor immunogenicity, and separation of viral protein from cell constituents. Genetic engineers are likely to utilize bacterial expression systems.
However, the important human and veterinary diseases are caused by viruses of eukaryotes whose newly synthesized polypeptides undergo co-translational and post-translational modifications such as glycosylation and proteolytic cleavage which prokaryotic cells cannot accomplish, therefore we have to use eukaryotic cells. However, technology is well-developed for cultured cells of higher animals (including monoclonal antibody production) and bulk culture of yeast.
Experimental vaccines composed of influenza virus haemagglutinin, hepatitis B virus surface (S), and foot-and-mouth disease virus have been produced. But it has been found that genetically engineered proteins are poorly immunogenic as compared with the same antigens assembled into natural virus particles. (Microbiology Immunity and Serology Notes Study Material)
Genetically engineering a virus as a vaccine
(Recombinant virus as a vaccine)
Live vaccines evoke the most effective immunity and are the cheapest to produce but in practice are very difficult to make. The idea of inserting the gene for a foreign neutralization antigen into a pre-existing live vaccine so that it is expressed naturally as the virus multiplies is indeed attractive. This has already been achieved experimentally for antigens of influenza, rabies, herpes simplex type 1, and hepatitis B viruses using the vaccinia virus as the live vaccine. The virus is administered intradermally by scratching the skin.
The virus multiplies at that site causing a reaction about the size of a small boil and stimulating both antibody and cell-mediated immunity to viral antigens i.e. to those viruses whose genes have been cloned to make recombinant vaccinia virus. Thus vaccinating with a recombinant vaccinia virus having cloned haemagglutinin gene for, say, influenza virus will protect against influenza virus antigens.
The mechanics of producing a recombinant virus vaccine. There is inserted a cloned gene into a plasmid under the control of the promoter of the vaccinia thymidine kinase (TK) gene. Upon transfection into vaccinia virus-infected cells, recombination takes place because of the homologous TK sequences which both possess. The TK gene is inactivated by insertion of the plasmid which means that recombinants can be selected because they are unable to utilize (and hence be inhibited by) the DNA synthesis inhibitor bromodeoxyuridine. Consequently, only recombinant virus produces plaques. (Microbiology Immunity and Serology Notes Study Material)
Animals infected with recombinant vaccinia carrying genes for hepatitis B S-antigen or influenza virus haemagglutinin respond with excellent antibody and cell-mediated immune responses. So vaccines of this type are an exciting prospect. The original virus vaccine, obtained from fluid exuded from localized intradermal infections of sheep, costs only $ 0.31 per dose compared with $ 1.00 for a live poliovirus vaccine or $ 100 for the currently killed hepatitis B virus vaccine. A recombinant vaccinia virus vaccine would be produced in tissue culture and would cost about the same as the poliovirus vaccine.
We do not know yet how effective and long-lived could be anti-vaccinial immunity. The interesting point is that the vaccinia genome can accommodate around 25 kilobases of DNA without losing infectivity. This means that the recombinant virus could express 10 to 20 foreign antigens and serve as a one-off polyvalent spectrum of virus diseases. (Immunity and Serology Notes Study Material)
Immunization may be administered by injection, oral consumption, or nasal spray as currently used for some respiratory viral diseases. Booster immunization is commonly followed as a way of raising the antibody level by stimulating the memory cells to induce the secondary anamnestic response. Some substances are called adjuvants to increase the efficiency of a vaccine or toxoid by increasing the availability of the antigen in the lymphatic system. Common adjuvants include aluminum sulphate and aluminum hydroxide in toxoid preparations as well as mineral oils or peanut oil in viral vaccines. (Microbiology Immunity and Serology Notes Study Material)
[III] Naturally acquired passive immunity
Passive immunity develops when antibodies enter the body from an outside source. The exposure to antibodies may be unintentional or intentional. When unintentional, the immunity that develops is called naturally acquired passive immunity.
Naturally acquired passive immunity, also called congenital immunity develops when antibodies pass into the fetal circulation from the mother’s bloodstream via the placenta and umbilical cord. These antibodies, called maternal antibodies, remain with the child for about three to six months after birth. Certain antibodies such as measles antibodies remain for 12 to 15 months. Maternal antibodies are important in providing resistance to whooping cough, staphylococcal infections, and viral respiratory diseases during the first few months of the child. The predominant antibody is IgG. (Microbiology Immunity and Serology Notes Study Material)
Maternal antibodies may also pass to the newborn through the first milk or colostrums. In this case, IgA is the predominant antibody although IgG and IgM have also been found. These antibodies provide resistance to diseases of the respiratory and gastrointestinal tracts.
[IV] Artificially acquired passive immunity
This arises from the intentional injection of antibody-rich serum into the circulation. Before the development of vaccines and antibodies, this was an important therapeutic device for disease treatment. This method is still used for viral diseases such as Lassa fever, hepatitis, and arthropod-borne encephalitis and for bacterial diseases where toxins are involved. For example, established cases of botulism, diphtheria, and tetanus are treated with serum containing the respective antitoxins.
Since the diseases are very dangerous and fatal, no risk is taken for the introduction of antigens. Instead, already-made antibodies are introduced into the blood. Various terms are used for the serum that renders artificially acquired passive immunity. Antiserum is one such term.
Another term is a hyperimmune serum which indicates that the serum has a higher-than-normal level of a particular antibody. If the serum is used to protect against a disease, it is called prophylactic serum, if the serum is used in the therapy of an established disease it is called therapeutic serum. A common term is gamma globulin. Gamma globulin usually consists of a pool of sera from human donors, which should contain a mixture of antibodies including those for the disease to be treated. In some individuals, the immune system may recognize foreign serum proteins as antigens and may form antibodies against them in an allergic reaction.
When the antibodies interact with the proteins, a series of chemical molecules called immune complexes may form, and with activation of complement, the person may develop a type of disease called serum sickness. This type of immunity provides substantial and immediate protection against disease but is only a temporary measure. This goes off after a few days. Examples are the serum preparations for chickenpox, hepatitis B, etc. (Microbiology Immunity and Serology Notes Study Material)
Serological Reactions (Serology)
Antigen-antibody reactions studied under laboratory conditions are known as serological reactions, so named because they commonly involve serum from a patient. In the late 1800s, serological reactions were first adapted to laboratory tests used in the diagnosis of disease. An abnormal level of a specific antibody in the serum of the patient indicates the presence of the agent of the particular disease.
Today, serological reactions have diagnostic and many other applications. For a successful reaction, the antigen or antibody solutions are to be set at the concentration at which the reaction is most favorable. The titer is the most dilute concentration of serum antibody that yields a detectable reaction with its specific antigen. (Microbiology Immunity and Serology Notes Study Material)
Serology has become a highly sophisticated and often automated branch of immunology. The serological reactions have direct application to the laboratory. The following are the common reactions.
[I] Neutralisation
This is a reaction in which antigens and antibodies neutralize each other. The reaction is used to identify toxins and antitoxins, as well as viruses and viral antibodies. An example is the detection of botulism toxin in food.
[II] Precipitation
This reaction involves thousands of antigen and antibody molecules cross-linked at multiple sites to form a structure called a lattice. These become so large that precipitate form can be easily seen. Precipitation tests are performed either in fluid-fluid precipitation or gel-gel precipitation. In the former, the antibody and antigen solutions are layered over each other in a thin tube. In the latter, the diffusion of antigen and antibody takes place through a semisolid gel as agarose.
There are used the Oudin tube technique or Ouchteriony plate technique. In a procedure, called immunoelectrophoresis, the two techniques of electrophoresis and diffusion are combined for the detection of antigens.
[III] Agglutination
In this reaction, antibodies react with antigens on the surface of particulate objects and cause the objects to clump together, or agglutinate. These reactions were the earliest to be adapted to a diagnostic laboratory. The Widal test is used for the diagnosis of typhoid fever. This test, developed by Georges Fernand I. Widal (French physician) in 1896, is now supplemented by more sophisticated procedures. A polyvalent serum (a serum containing a mixture of antibodies) is prepared. (Microbiology Immunity and Serology Notes Study Material)
A passive agglutination is a modern approach to this procedure, where antigens are adsorbed onto the surface of latex spheres, polystyrene particles, red blood cells, bacteria, or other carriers. Serum antibodies are then detected by observing the agglutination of the carrier particle. Haemagglutination is the agglutination of red blood cells. This process is very important in the determination of blood types before the transfusion process. Some viruses as those of mums at measles that agglutinate red blood cells may be detected in patients’ serum by this test. (Microbiology Immunity and Serology Notes Study Material)
[IV] Flocculation
This test combines the principles of precipitation and agglutination. The antigen exists in a non-cellular particulate form that reacts with antibodies to form large, visible aggregates. An example of this test is Veneral Disease Research Laboratory (VDRL) test used for the rapid screening of patients to detect syphilis. The antigen is an alcoholic extract of beef heart-called cardiolipin. This reacts with syphilis antibodies in the patient’s serum to form aggregates. (Microbiology Immunity and Serology Notes Study Material)
Besides the abovementioned tests, there are some other reactions used serology. These are as follows:
[I] Complement fixation
The complement fixation test was developed by Jules Bordet Octave Gengou in 1907. It was later adopted for syphilis by August von Wassermann in 1906 and for about 75 years it was a mainstay for syphilis diagnosis. These days, technologists use it for the detection of antibodies against a variety of viruses, bacteria, and fungi. (Microbiology Immunity and Serology Notes Study Material)
The test is done in two parts: (i) the test system, which utilizes the patient’s serum, a preparation of antigen and complement derived from guinea pigs (ii) the indicator system, which requires sheep red blood cells and hemolysin (antibodies against sheep red blood cells). Haemolysins cause lysis of red blood cells in the presence of complement.
[II] Fluorescent antibody technique
This technique is a slide test performed by combining particles containing antigens with antibodies and a fluorescent dye. When these three complements react, the dye causes the complex to glow an illumination with UV light under a fluorescent microscope. Two commonly used dyes are fluorescein, which emits an apple-green glow, and rhodamine which gives off orange-red light.
These techniques may be direct or indirect. In the direct method, the dye is linked to known antibody molecules. The antibodies are then combined with particles (maybe bacteria) that may contain complementary antigens. The tagged antibodies accumulate on the particle surface and the particle glows under the microscope. The indirect method is illustrated by the FTA-ABS diagnostic procedure used for syphilis antibodies in the blood of the patient. (Microbiology Immunity and Serology Notes Study Material)
[III] Radioimmunoassay (RIA)
This is an extremely sensitive serological method used to measure the concentration of low molecular weight antigens, like haptens. It was developed in the 1960s and since then has been adapted for quantification of hepatitis antigens, reproductive hormones, insulin, and some drugs. This test also is useful for the detection of tumor viruses in the body before the appearance of tumors. This test can detect trillionths of a gram of substances. (Microbiology Immunity and Serology Notes Study Material)
[IV] Radioallergosorbent test (RAST)
This test is an extension of the radioimmunoassay. This may be used to detect IgE or other antibodies as well as a variety of small antigens. RAST is commonly known as a “sandwich” technique. To detect IgE, specific antigens for this antibody are attached to a matrix particle. Serum suspected to contain IgE is added. An antibody, if present combines on the surface of the particle.
Now another antibody, one that reacts with human antibodies, is added. This antiglobulin antibody carries a radioactive label. The entire complex will, therefore, be radioactive if the antiglobulin antibody combines with the IgE. If IgE is not present, the particles will not show radioactivity. (Microbiology Immunity and Serology Notes Study Material)
[V] Enzyme-linked immunosorbent assay (ELISA)
ELISA has actually the same sensitivity as RAST and RIA. However, it does not require radioactivity or expensive equipment. Antigens or antibodies are attached to a solid surface, and the coated surfaces are combined (immunosorbed) with the test material. An enzyme system is then linked to the complex, the remaining enzyme system is washed away and the extent of enzyme activity is measured. This indicates the presence of antigens or antibodies in the material. (Microbiology Immunity and Serology Notes Study Material)
An application of ELISA is found in the Gonozyme test used to detect gonorrhea agent (Neisseria gonorrhoeae) antigens in the patient.
Microbiology Immunity and Serology Notes Study Material
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