Practical tasks

1. Perform the ELISA to detect the Hbs Ag in the patient’s blood serum. Estimate the results. Make a conclusion. Draw the scheme of the test. Write down and fill in Table 14-1 in your protocol notebook.

2. Familiarize yourself the schemes of direct and indirect IF-test. For these schemes see wall display in the classroom.

LESSON 15

MEDICAL IMMUNOBIOLOGICAL PREPARATIONS

Prelab conference. Topics for discussion:

1. Acquired active and passive immunity.

2. Preparations to induce active artificial immunity.

3. Vaccines. Classification, application.

4. Vaccine strains. Requirements and preparation.

5. Toxoids. Preparation, titration, application.

6. Associated vaccines. Adjuvants.

7. Preparations to form artificial passive immunity.

8. Antibody-containing antisera for immunotherapy and immunoprophylaxis.

9. Serotherapy and seroprophylaxis complications. Mechanism, protection from complications.

10. Allergens. Their application.

Immunobiological preparations used for diagnosis, treatment and immunoprophylaxis of infectious diseases are listed in Table 15-1.

Table 15-1

Medical Immunobiological Preparations

MIBP for (immuno)therapy and (immuno)prophylaxis	MIBP for infectious disease diagnosing
Antigen-containing preparations (vaccines, toxoids) Antibody-containing preparations (antisera, immunoglobulins, etc.) Immunomodifiers and adaptogens Eubiotics Bacteriophages	Components of serological reactions (diagnosticums, diagnostic immune antisera, complement, monoclonal antibodies, labeled antibodies, etc.) Diagnostic reagents for microbiological examination In vivo (allergens) In vitro (bacteriophages - for phagotyping)

• ACTIVE and PASSIVE IMMUNITY

Besides innate immunity, immunity may be acquired either actively or passively, depending on whether the immune person produces his own protective antibodies or T cells, or receives presynthesized antibodies or sensitized lymphocytes from another individual. Both active and passive immunity can be acquired either naturally or artificially (see Table 15-2).

Table 15-2

Categories of Acquired Humoral Immunity

Active immunity	Immunized individual produces antibodies	Natural active immunity follows natural infection Artificial active immunity follows vaccination (immunization)
Passive immunity	Immunized individual receives antibody produced by another individual	Natural passive immunity is due to transfer of maternal antibodies to fetus or suckling newborns. Artificial passive immunity is induced by injecting presynthesized antibodies into recipient

• Active immunity

Active immunity is the production of antibodies or specialized lymphocytes by the host as a result of exposure to a foreign antigen. It is characterized by:

1. a latent period of at least 2 weeks between initial exposure to the antigen and development of protective immunity

2. an extended duration of immunity that often (but not always) lasts for years.

Natural active immunity develops following exposure to an antigen as a result of natural infection where an immune memory develops. Immunity is directed against the specific infectious agent as well as its toxic by-products. Immunologic recall in both cell-mediated and humoral responses maintains protection for months and often for years. The protective efficiency of natural active immunity is demonstrated by the infrequency of second attacks of chickenpox, mumps, and measles. Even in the absence of overt symptoms, these infections may stimulate lifelong immunity.

Artificial active immunity is similar to natural active immunity except for the nature of the antigen and the method of introduction into the host. Instead of natural infection by a potentially virulent microbe, a vaccine is intentionally introduced into the body. Induction of active immunity against disease by introducing a vaccine into the individual is called vaccination.

A vaccine is preparation of microbes or toxoid that can no longer induce severe disease but can still stimulate immunity against the corresponding pathogen or toxin. An effective vaccine retains the pathogen antigens, so it is antigenically similar to a corresponding pathogen or to its toxic by-products and is highly immunogenic. Vaccine has been treated so that it does not have pathogen's ability to damage the host and it can be administered to people with little danger of disease.

A vaccine should contain some (or at least one) of the protective antigens of the microbe. A vaccine should as far as possible be effective, safe, stable and of low cost. This is accomplished in a number of ways, including the use of killed and attenuated live organisms, purified antigen fractions, toxoids, synthetic peptides, etc. (see Table 15-6).

The vaccine sensitizes the immune system to the corresponding pathogen, inducing immunity without the danger of infectious disease developing during the lag period. In immunized people, natural exposure to the corresponding virulent pathogen triggers a protective anamnestic or secondary immune response (based on the immune memory) that eradicates the pathogen or neutralizes toxins before symptoms of disease develop due to very intensive and increased antibody production.

Each type of vaccines has its merits and drawbacks.

Problems with vaccine safety

• Live attenuated vaccines:

  • insufficient attenuation

  • reversion to wild type

  • no safe administration to immunodeficient patient

  • persistent infection

  • contamination by other microbes

  • fetal damage

• Non-living vaccines:

  • contamination by live organisms

  • contamination by toxins

  • allergic reactions

  • autoimmunity

• Genetically engineered vaccines:

  • ?inclusion of oncogenes

• LIVE ATTENUATED VACCINES. The most effective vaccines are those that actually multiply in the host, mimicking the early stages of natural infection. In these vaccines, the pathogens are attenuated—alive but weakened in their capacity to cause severe disease.

These attenuated microbes are called vaccine strains. Such strains are produced by different methods of attenuation:

• Serial passages in cells cultured in vitro

• Serial passage in nutrient culture media

• Adaptation to low temperatures

• Selection of spontaneous mutants

• Chemical mutagenesis

• Required genetic changes (by gene engineering)

It is rather difficult to achieve satisfactory attenuation of a “wild” microbial strain and to prepare a safe and stable vaccine strain. Because of that, there are not many effective live vaccines. Furthermore, attenuated microbes have been known to regain virulence and have caused serious disease in vaccinated persons. Only those attenuated organisms unlikely to revert can be safely used in vaccines. In general, living vaccines induce stronger and more lasting immunity than non-living vaccines.

NOTE: Vaccines containing attenuated organisms are especially effective if they can be introduced by the same routes the corresponding pathogen uses for entering the body. The attenuated microbes harmlessly propagate in the body to provide a greater and more prolonged antigenic stimulation. Furthermore, the attenuated pathogens may be shed by the immunized individuals and vaccinate other susceptible persons.

The mostly important live vaccines are as follows:

• Sabin poliovaccine. Attenuated trivalent preparation of poliovirus administered orally to induce immunity against poliomyelitis.

The attenuated Sabin polio vaccine is administered orally. It harmlessly replicates in the intestine, similar to the early stages of poliomyelitis. Unlike virulent poliovirus, the attenuated variant causes no paralysis. The Sabin vaccine stimulates the formation of secretory IgA, antibodies that neutralize viruses in the intestine before they invade the bloodstream.

Sabin vaccine virus is shed in the feces of vaccinated persons. Since polioviruses are naturally transmitted by the fecal-oral route, people may be inadvertently vaccinated by ingesting attenuated viruses shed by vaccinated individuals.

• BCG vaccine (The Bacille Calmette-Guérin). Vaccine to provide immunity against tuberculosis. It is made from attenuated bovine tubercle bacillus. The attenuation have been achieved by passage for 10 years in glycerol-bile-potato medium.

• Other vaccines that use attenuated versions of pathogens include those that help protect against influenza, yellow fever, measles, mumps, rubella, plaque, tularemia, brucellosis, etc.

• KILLED (inactivated) VACCINES. A pure culture of the highly immunogenic and virulent pathogen is exposed to an agent that will kill the microbe without altering the surface antigens. Injection of these killed organisms into a host provides safe initial exposure to the pathogen and subsequent resistance to disease.

A variety of methods are available for inactivation: formaldehyde inactivation, phenol, acetone, ultraviolet light, simple heating, β-propiolactone and various ethylenimines, prosalens, etc.

Killed vaccines have the advantage of non-infectivity and therefore relative safety, but disadvantage of generally lower immunogenicity, high risk of hypersensitivity complications and the consequent need for several doses. Most of these vaccines require several booster shots before protection is adequate.

NOTE: Booster – is an immunization given to enhance the memory response to an antigen

This group of vaccines includes the following:

• Typhoid fever vaccine, is a formalin-killed preparation of the pathogen Salmonella typhi.

• Salk poliovaccine. Formalin -inactivated poliovirus preparation administered by injection to induce immunity against poliomyelitis. The inactivated Salk vaccine must be injected into muscle, inducing little protection in the intestine.

• Vaccines containing killed organisms or inactivated viruses have been used to prevent cholera, influenza, whooping cough, rabies, epidemic typhus, etc.

• SUBUNIT VACCINES (Purified Antigen Fractions). Some vaccines contain only the antigens against which the protective immune reactions are directed (such antigens are called protective). Removal of all live infectious material is obviously a vital element in ensuring the safety of such vaccines.

Antigen fractions are too small to activate the adequate immune response (for example, polysaccharide antigens fail to stimulate T cells and therefore induce only primary responses). Many adjuvants will enhance the immune response when administered with antigen. Adjuvants are such substances that were shown to be safe, convenient, and effective to enhance the consequent immune response when administered simultaneously with antigen. A variety of foreign and endogenous substances can act as adjuvants, but only aluminum and calcium salts are routinely used in clinical practice (aluminum hydroxide, aluminum phosphate, and calcium phosphate). Alum-precipitated antigens are especially effective at inducing antibody responses, and help to induce cell-mediated immunity.

The subunit vaccines are as follows:

• Pneumovax. Purified extracts of the capsular antigens from virulent Streptococcus pneumoniae stimulate the host to produce opsonizing antibodies that effectively protect most immunized persons. This vaccine contains capsular antigens of the 14 most common virulent strains of S. pneumoniae. About 80 percent of all cases of pneumococcal pneumonia are caused by one of these 14 strains.

• Typhim Vi. Polysaccharide typhoid vaccine that is composed of purified Vi capsular polysaccharide of Salmonella typhi.

• Antigen subunits are also used in vaccines against influenza, cholera, some types of meningitis, etc.

• TOXOIDS. Active immunity against toxemic diseases can be induced by injecting a toxoid, an inactivated form of bacterial exotoxin that remains antigenically unaltered but has been chemically treated to destroy its poisonous properties. Exotoxin inactivation is usually by formaldehyde (0,4 per cent solution) for 4 weeks at 40˚C. Toxoid antigens are then purified and adsorbed on the adjuvants. Toxoid stimulates production of antitoxic antibodies (antitoxins), thereby inducing immunity against the corresponding disease.

The commonly used toxoids are the following:

• Tetanus toxoid. Prior immunization with a toxoid prepared from the toxin that the bacterium Clostridium tetani releases into the bloodstream from an infected wound site protects against the post-wound disease tetanus. After three boosters, protection against tetanus lasts at least 10 years, explaining why physicians treating injuries always inquire about the patient’s last tetanus shot. If it has been more than 10 years, the injury victim gets a booster shot.

• Toxoids have also been made against the neurotoxin of Clostridium botulinum and some other toxins of Clostridium sp.

There are also some combined preparations where toxoids are utilized with another types of vaccines:

• DPT vaccine (diphtheria, pertussis, tetanus toxoids). Triple vaccine which is combination of two toxoids –tetanus and diphtheria- and killed Bordetella pertussis. This vaccine contains two different types of vaccines - toxoid and killed vaccines.

• Cholera combined vaccine. This vaccine contains two types of vaccines – toxoid plus killed Vibrio cholerae.

The newest types of vaccines are:

• SYNTHETIC PEPTIDES. Through the use of sophisticated laboratory technology, the amino acid sequence of purified protein antigens can be precisely determined. Once the sequence is known, it is possible to synthesize peptides of approximately 20 amino acids that may represent antigenic determinants against which protective immunity is directed.

Vaccines have been created using peptides of hepatitis В virus surface antigen, Streptococcus pyogenes M protein, diphtheria toxin, influenza virus, and a number of other proteins. The advantage of synthetic vaccines is that they ensure exclusion of contaminating materials that might harm the host. The widespread use of synthetic vaccines awaits the development of high-yield production systems.

• GENETICALLY ENGINEERED VACCINES. Several vaccine components are manufactured through genetic engineering using easily cultured bacteria or yeast to synthesize the protein antigen. Genetically engineered vaccines against influenza and hepatitis В are cheaper and safer than conventional whole-virus vaccines.

Another technological advance in vaccine production is the creation of piggyback vaccines. The genes for the desired antigens are inserted into the genome of an infectious, but harmless, virus. The engineered virus is inoculated into the host. As the virus replicates, it produces the "vaccine" protein along with its own products, The host then launches immune responses against the viral products and the extra antigen as well. Vaccinia virus, successfully used in smallpox eradication, is the most commonly used vector for piggyback vaccines.

• ANTI-IDIOTYPE VACCINES. Antibodies themselves can be used as antigens. They stimulate production of other antibodies targeted at their variable regions, the binding sites specific for the corresponding antigenic determinant. Each antibody for a distinct epitope is called an idiotype.

Anti-antibody antibodies recognize and bind to the antigen binding sites of the antibody idiotype that stimulated their production. These antibodies are aptly known as anti-idiotype antibodies. Because the original antigen and the anti-idiotype antibody have identical antigenic determinants, both can bind to the same antibody molecule, so anti-idiotype antibodies can replace a potentially harmful pathogen or toxin as the antigen in a vaccine. The advantage of anti-idiotype vaccines is that there is no pathogen present and therefore no possibility of reactivating the disease. Currently several such vaccines are under investigation.

Active immunoprophylaxis can be considered under two headings:

• Routine immunization of children which forms part of basic health care of communities

• Immunization of individuals or selected groups exposed to risk of particular infections.

Routine immunization schedules have been developed for different countries, based on the prevalence of infectious diseases, their public health importance, availability of suitable vaccines, their adverse reactions, etc.

The duration of response is of prime importance. For short-term protection (e.g. a tourist about to visit a disease area), the presence of antibody arising from the vaccine itself may be perfectly adequate and memory cells may not be strictly necessary. On the other hand, for protection against exposure at some time in future, the induction of memory is essential. Memory is often naturally boosted by periodic outbreaks of disease in the community.