Theme 12

Theme 12. Serological reactions, which are used in virology.

Theme 13. Vaccines and immune serums.

 

Serological / Immunological Methods

Let’s continue with the alternative methods, which are serological/ immunological methods. These methods consist of :

·         Haemagglutination (HA)

·         Haemagglutination Inhibition (HI)

·         Virus neutralisation

·         Complement fixation

·         Immunostaining

·         Immunoprecipitation/ Immunoblot

·         ELISA

Unfortunately yes, there are many methods to know about. Let’s go through one by one slowly..

Haemagglutination

Haemagglutination is visible macroscopically and is the basis of haemagglutination tests to detect the presence of viral particles. The test does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of red blood cells.

Influenza and other viruses

-Two spike proteins: NEURAMINIDASE , HAEMAGGLUTININ ( Binds specifically to red blood cells)

Steps to haemagglutination:

1. Dispense diluent.

2. Add red blood cells and mix by gently shaking.
3. Allow the red blood cells to settle and observe the pattern.
4. Observe if the cells have a normal settling pattern and there is no auto-agglutination. This will be a distinct button of cells in the micro test and an even suspension with no signs of clumping in the rapid test.

 

 

2. Haemagglutinin Inhibition

Serologic tests in which a known quantity of antigen is added to the serum prior to the addition of a red cell suspension. Reaction result is expressed as the smallest amount of antigen which causes complete inhibition of haemagglutination.

It is possible to carry out rapid haemagglutination tests and haemagglutination-inhibition tests on a plate, just as for the bacterial agglutination tests described above. However HA and HI are generally only used in this way to confirm the presence and identity of a haemagglutinating antigen.

 

·         HA conducted in the presence of antibody

·         Neutralisation of virus inhibits agglutination

Haemagglutination inhibition is based on blocking viral haemagglutinin by antibodies. The test is performed on plexiglass plates and interpreted as positive if erythrocytes fail to agglutinate on adding them to mixture of the  virus and specific serum. In order to remove or destroy non-specific haemagglutinaton inhibitors, test sera are pretreated with potassium periodate, kaolin, bentonite, acetone, or other agents. Then, the  sera are diluted two-fold in isotonic podi­um chloride solution, and every dilution is supplemented with an equal amount of virus-containing fluid which has four haemagglutinating units. The  mixture is incubated for 30-60 min at a tempera­ture optimal for a given virus (0°, 4°. 20°, 37 °C), and an equal volume of 0.5-1 per cent erythrocyte suspension is added. The mixture is reincubated for 30-45 min, and the results of the test are read. The  serum titre is defined as the  greatest serum dilution at which haemaglutination is inhibited.

Microhaemagglutination inhibition test using Takata’s micro-panel and loop is also widely employed.

 

 

 

According to the nature of the material to be tested and the proce­dures utilized, the methods for diagnosing viral infections may be categorized into rapid, viroscopic, virological, and serological (Table).

Table

 Methods of the Diagnosis of Viral Infections

 

Most of the relevant diagnostic techniques rely on the interaction between virus antigens and homologous antibodies in a fluid medium (complement-fixation (CF) test, haemagglutination inhibition (HAI) test, indirect haemagglutination (IHA) test, reversed indirect haem­agglutination (RIHA) test, reversed indirect hemagglutination inhibition (RIHAI) test, radioimmunoassay (RIA), or in gel (the test of precipitation in gel (PG), radial hemolysis (RH) test, immunoelectrophoresis (IEP) test, or during fixation of any ingre­dient in a solid medium (enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), haemadsorption on a solid-medium (HadsSM) test, immunofluorescence (IF) test, haemadsorp­tion (Hads) test, and haemadsorption inhibition (Hadsl) test). In order to improve test sensitivity, antigens or antibodies are adsorbed on erythrocytes (IHA, RIHA, RIHAI, HadsSM, RH) or linked to enzymes (ELISA), isotopes (RIA, PG), and fluorochromes (IF); an alternative principle is erythrocyte lysis induced by the antigen-antibody interaction in the presence of complement (CF, RH).

The appropriate test procedures are described in detail in chapters dealing with serological diagnosis and with virus detection and identification in cell cultures. This chapter is devoted to the specific features of these tests and modifications which are used in the diagno­sis of viral infections.

Haemadsorption inhibition test is used for identifying haemadsorbing viruses and determining serum antibody titres. Specific serum (0.2 ml) diluted 1:5 is placed in test tubes with a culture of virus-infected tissue and following its incubation for 30-60 min. 0.2 ml of 0.5 per cent erythrocyte suspension is added. Nonimmune serum from the same animal species and erythrocytes are instilled in the control test tubes. The tubes are incubated for 20-30 min at a temperature which is optimal for the haemadsorption of the virus to be isolated. A conclusion about a species of the virus is based oil the  absence of erythrocyte adsorption in the test tubes in the presence of typical haemadsorption in the  control test tubes.

Hemadsorbtion test

Neutralization test for an infective and cytopathic effect of viruses is performed in virus-sensitive live systems. A virus-containing specimen is serially diluted, and specific serum, diluted to a titre indicated on the ampoule label, is added. The mixture is incubated for 30-60 min at 37 °C and is used to infect tissue culture, chicken embryos, or laboratory animals. A sensitive system inoculated with the virus treated iormal serum serves as control.

Neutralization test is considered positive if the cell culture displays no CPE, chicken embryos show no changes, and the animals live without exhibiting any signs of disease. The findings obtained are used to determine a neutralization index which is a ratio of the virus titre in the control (where CPE is observed) to the test titre. The test is considered negative if the neutralization index is below 10, am­biguous if it varies from 11 to 49, and positive with an index of 50 or higher (significant virus-antiserum correlation).

The most sensitive version of the N test is inhibition of virus plaque formation by virus-specific antiserum (virus plaque reduction test). For this test, a virus-containing specimen (50-100 plaque-forming units) is supplemented with antiserum (diluted to a speci­fied titre), and, after 30-60-min incubation in a heating block, the mixture is applied onto monolayers of sensitive cell cultures. Match­ing of the virus to the employed antiserum is expressed in reduced plaque formation as compared with control. The N test helps to ascertain the  virus species and type (variant).

Neutralization of a virus is defined as the loss of infectivity through reaction of the virus with specific antibody. Virus and serum are mixed under appropriate condition and then inoculated into cell culture, eggs or animals. The presence of unneutralized virus may be detected by reactions such as CPE, haemadsorption/haemagglutination, plaque formation, disease in animals. The loss of infectivity is bought about by interference by the bound Ab with any one o the steps leading to the release of the viral genome into the host cells. There are two types of neutralization;-

Reversible neutralization – The neutralization process can be reversed by diluting the Ab-Ag mixture within a short time of the formation of the Ag-Ab complexes (30 mins). It is thought that reversible neutralization is due to the interference with attachment of virions to the cellular receptors eg. the attachment of the HA protein of influenza viruses to sialic acid. The process requires the saturation of the surface of the virus with Abs.

Stable neutralization – with time, Ag-Ab complexes usually become more stable (several hours) and the process cannot be reversed by dilution. Neither the virions or the Abs are permanently changed in stable neutralization, for the unchanged components can be recovered. The neutralized virus can be reactivated by proteolytic cleavage. Stable neutralization has a different mechanism to that of reversible neutralization. It had been shown that neutralized virus can attach and that already attached virions can be neutralized. The number of Ab molecules required for stable neutralization is considerably smaller than that of reversible neutralization, Kinetic evidence shows that even a single Ab molecule caeutralize a virion. Such neutralization is generally produced by Ab molecules that establish contact with 2 antigenic sites on different monomers of a virion, greatly increasing the stability of the complexes. An example of stable neutralization is the neutralization of polioviruses, whereby, the attachment of the antibody to the viral capsid stabilizes the capsid and inhibits the uncoating and release of viral nucleic acid.

Viral evolution must tend to select for mutations that change the antigenic determinants involved ieutralization. In contrast, other antigenic sites would tend to remain unchanged because mutations affecting them would not be selected for and could even be detrimental. A virus would thus evolve from an original type to a variety of types, different ieutralization (and sometimes in HI) tests, but retaining some of the original mosaic of antigenic determinants recognizable by CFTs. Because of its high immunological specificity, the neutralization test is often the standard against which the specificity of the other serological techniques is evaluated.

Before the neutralization test is carried out, the known components that are to be used must be standardized. To identify a virus isolate, a known pretitred antiserum is used. Conversely, to measure the antibody response of an individual to a virus, a known pretitred virus is used. To titrate a known virus, serial tenfold dilutions of the isolate is prepared and inoculated into a susceptible host system such as cell culture or animal. The virus endpoint titre is the reciprocal of the highest dilution of virus that infects 50% of the host system eg. 50% of cell cultures develop CPE, or 50% of animals develop disease. This endpoint dilution contains one 50% tissue culture infecting dose (TCID₅₀) or one 50% lethal dose (LD₅₀) of virus per unit volume. The concentration of virus generally used in the neutralization test is 100 TCID₅₀ or 100 LD₅₀ per unit volume.

The antiserum is titrated in the neutralization test against its homologus virus. Serial twofold dilutions of serum is prepared and mixed with an equal volume containing 100TCID₅₀ of virus. The virus and serum mixtures are incubated for 1 hour at 37oC. The time and temperature for incubation varies with different viruses. The mixtures are then inoculated into a susceptible host system. The endpoint titration contains one antibody unit and is the reciprocal of the highest dilution of the antiserum protecting against the virus. Generally 20 antibody units of antiserum is used in the neutralization tests.

 

 

 

Colour test (colorimetric neutralization test). Cell activity in the nutrient medium results in  accumulation of acid products, which induces a corresponding change in the  pH (making the medium or­ange-coloured). Inoculation of the  cell culture with cytopathogenic viruses (enteroviruses, reoviruses, etc.) leads to inhibition of cell metabolism. As a result, the pH of the medium undergoes no change and the medium remains red.

0.25-ml portions of the working virus dilution (100-1000 CPE₅₀) and the respective serum dilution are pipetted into the  test tubes. Let the mixture stand for 30-60 min at room temperature, and, after adding 0.25 ml of the cell suspension into each test tube, stopper them with rubber plugs, or pour sterile vaseline oil into them. The mixture is incubated at 37 °C for 6-8 days. The results are inter­preted colorimetrically: pH equal to or above 7.4 (red-coloured me­dium) indicates virus reproduction, whereas pH of 7.2 or less (orange-coloured medium) suggests virus neutralization by antibodies.

Enzyme-linked immunosorbent assay (ELISA) or the immunoenzymic test relies on the capacity of the enzyme antibody label to break down the substrate with the formation of stained products. Antibodies linked to the enzyme regain their ability to conjugate with antigens. The number of formed enzyme-antigen-antibody complexes corresponds to the intensity of substrate staining.

Peroxidase and alkaline phosphatase are commonly utilized as enzymes while 5-aminosalicylic acid, orthophenylendiamine, and other substances are used as the substrate for peroxidase.

 

 

 

Currently, a solid phase modification of ELISA is most often employed in microbiology. The essence of this variant consists in the fact that at first antigens (or antibodies) are sorbed on a solid material and only after that the remaining ingredients of the serological reaction are added. Plastic plates, beads, films or tubes made of various synthetic inert materials (polystyrene, methacrylate, etc.) are usually used as a solid phase carrier of antibodies or anti­gens. Being adsorbed on the surface of such materials, antibodies or antigens, even in a dry state, retain their immunological specificity and ability to participate in serological reactions for a long time.

 

There are numerous methodological variants of immunoenzymic detection of antigens; in most cases the antigen is caught by anti­bodies bound to the solid phase. Following incubation with the ma­terial, the antigen tested attaches to the antibody and thus to the solid phase. Then the “linked” antigen is demonstrated by means of enzyme-labelled antibodies against this antigen, the direct variant of ELISA- In an indirect variant anti-species (antiglobulin) enzyme-labelled sera are used. The amount of enzyme linked to the solid phase is equal to the amount of the antigen. Activity of the enzyme is determined quantitatively by the intensity of post-incubation staining with the appropriate substrate. This analysis can be made by means of an automatic device, with the results being registered by a special spectrophotometer.

ELISA is distinguished by a fairly high sensitivity and rapidity of obtaining the results (within 2 hours). Improvement in the sensitiv­ity of the solid phase ELISA modification requires the use of anti­bodies with a high degree of specificity. Despite their relatively low-affinity, monoclonal antibodies appear promising in this regard. Hence, the  development of methods for obtaining highly affinitive monoclonal antibodies is one of the top priorities facing modern microbiologists.

The  following buffer solutions are necessary to perform ELISA.

1. Coupling buffer: 0.05 M of sodium-carbonate-bicarbonate buffer (CBB) (pH 9.5-9.7) for sorption of the antigen or antibodies on a solid carrier. The composition of the buffer is as follows: 1.18 g of Na₂CO₃, 3.47 g of NaHCO₃ and 200 mg of NaNO₃. The volume of the  buffer is adjusted to 1 L with distilled water,

2. Incubation buffer: phosphate-salt solution (pH 7.3-7.5) which is used for diluting the components introduced into the reaction after sorption of the first component on the carrier. The composition of the  buffer is as follows:

17.9 g of Na₂HPO₄“H₂O; 0.8 g of NaH₂PO₄“HaO; 42.5 g of NaCI; 2.5 ml of Twin-20. The volume of the buffer is brought to 5 L with distilled water and the buffer is stored at 20-25 °C.

3. Washing buffer: isotonic sodium chloride solution containing 0.05 per cent. of Twin-20. Phosphate-salt solution with 0.05 per cent of Twin-20 may also be utilized as a washing buffer. Orthophenylendiamine or 5-aminosalicylic acid serves as a substrate for peroxidase.

Orthophenylendiamine is prepared ex tempore in the following manner:

10 mg of orthophenylendiamine, 6.1 ml of 0.1 M of citric acid, 6.4 ml of  0.2 M of Na₂HPO₄“12H₂O (for complete solution the mixture is heated on a water bath), 12.5 ml of distilled water, 0.35 ml of 3 per cent H₂O₂.

Dilute 80 mg of 5-aminosalicylic acid in 100 ml of distilled water, adjust the pH of the solution to 6.0 ex tempore with the help of 1 M NaOH. Prior to using, add 1 ml of 0.05 per cent H₂O₂ to each 9 ml of the solution.

To perform ELISA, one should have polystyrene plates with flat-bottom wells and automatic pipettes. To quantitate the results, the spectrophotometer (a registrator of extinction at a 492 nm wave length) is used.

Procedure. The first stage of ELISA is sorption of the corresponding dilution of antibodies or antigen (in concentration of 10-20u.g/ml)on carbonate-bicarbon­ate buffer in a 0.2-ml portion on a solid phase for 1-2 hrs at 37 °C and 10-12 hrs at 4 °C (sensitization). Then, the wells are washed (to remove the antibody or antigen which has not been sorbed on the carrier) with tap water and washing buffer containing 0.05 per cent Twin-20 for 5 min (twice) at room temperature. After that place into each well (solid phase) 0.2 ml of 1 per cent solution of bo­vine serum albumin in CBB and incubate for 1 hr at 37 °C to ensure covering of those sites of the well surface, which have remained free after sensitization, sorption of the first component of the reaction on the solid carrier- Wash the well to remove the unbound bovine serum albumin and introduce the material to be tested (antigen or antibodies) (in 0.2 ml aliquots) diluted with a phosphate-salt solution (pH 7,2) containing 0.05 per cent Twin-20. Each dilution of the material is pipetted into two wells and placed in a 37 °C incubator for 1-3 hrs. Wash off the antigens or antibodies which have not reacted in the immune test and introduce 0.2-ml portions of conjugated antibodies against the test antigen or antibodies in a working dilution on a phosphate-salt solution containing 0.05 per cent Twin-20. Then, incubate the mixture at 37 °C for two hours. The unbound conjugate is washed off with buffer three times for 10 min.

Put 0.1 ml of substrate (chromogen) solution into the well and allow it to stand for 30 min in the dark at mom temperature. In the process of incubation in the presence of peroxidase orthophenylendiamine is stained yellow and aminosalicylic acid, brown.

To stop the reaction of substrate splitting, add 0.1 ml of 1 N H₂SO₄ (or 1 M NaOH) into the well.

Control of the reaction: the test antigen or antibodies are replaced with a ho­mologous component of the reaction.

Control of the conjugate:  0.2 ml of 1 per cent bovine serum albumin per CBB + 0.2 ml of conjugated antibodies in the working dilution.

The results of the reaction are read either visually or instrurnentally. In the first case, one looks for the  greatest dilution of the material tested in which the staining is more intense than in the  control (by bovine serum albumin). In read­ing the results of the  test with the  help of a spectrophotometer, a positive dilu­tion is the greatest dilution of the material tested at which the level of extinc­tion exceeds by at least two times the level of extinction of the corresponding dilution of the  heterologous component of the reaction.

To obtain antibodies, conjugated with the enzyme, one needs highly active precipitating sera against the antigen or against animal or human globulins from which the gamma-globulin fraction is isolated by precipitation with poly­ethylene glycol, ammonium sulphate, and by means of the rivanol-alcohol technique. Immunoglobulins are conjugated by the enzyme with the help of glutaraldehyde. Non-conjugated enzyme is removed by dialysis or chromatography on Sefadex. To prevent bacterial growth, merthiolate in a volume of up to 0.01 per cent of the mixture is added to the conjugates and the latter are kept at 4 °C or in the frozen state.

Radioimniunoassay (RIA). The antigen or antibodies for RIA are labelled with radioactive isotopes, most commonly with ¹²⁵I. RIA is very sensitive and allows the detection of 1-2 ng of the sub­stance tested, or even less. Special radiometric equipment is ne­cessary to perform this assay.

Variable RIA modifications are available, with the solid phase variant being the one most frequently utilized in practice. As in the case of solid phase ELISA, antibodies (antigen) are sorbed on a solid phase carrier [on the surface of plates with wells, beads, and films from polystyrene or other polymer synthetic ma­terials). Adsorbed (immobilized) antigens and antibodies preserve their capacity to participate in serological reactions for a long time.

Figure 1 presents the diagrams of conducting RIA by three methods, viz., competitive, reverse, and indirect.

In the competitive method of RIA antibodies specific in relation to the antigen tested are sorbed on the surface of polystyrene wells. Then, the antigen-containing material to he assayed is placed into the wells and after a definite period of time sufficient for the spe­cific interaction of the antigen with immobilized antibodies to take place, the purified antigen labelled with a radioactive isotope is added. With regard to antigenic specificity, it should correspond to the antibodies immobilized on the surface of wells.

 

 

If the material to be examined contains the antigen correspond­ing to immobilized antibodies, some of the active centres of the latter are blocked. In this case the labelled antigen placed into the -wells will conjugate with immobilized antibodies to a lesser degree (as compared to the  control), the difference being expressed in varying levels of radioactivity in the liquid part of the reacting mixture.

In performing reversed RIA purified unlabelled antigen homologous to the antigen tested is sorbed on the surface of the wells. The antigen-containing material is conjugated in a separate test tube with labelled antibodies specific with regard to the antigen immobilized on the surface of the wells. If the material studied contains the an­tigen capable of interacting with labelled antibodies, the active cen­tres of the latter are blocked either partially or completely. In this case following the introduction of this mixture into the wells with the sorbed antigen, the labelled antibodies will be fixed on their surface in lower amounts (as compared with the control), which can be judged by the degree of radioactivity of the well contents.

 

 

The conduction of solid-phase RIA appears most convenient when an indirect method with anti-species labelled antibodies (the method of double antibodies) is used .

Indirect RIA may be employed for detecting both antibodies (serological diagnosis) and unknown antigens. In both cases an anti-species labelled serum containing the antibodies against gamma globulins is used. To carry out the serological diagnosis by indirect RIA. the antigen is sorbed on the well surface and then the patient’s diluted serum is added. If it contains the corresponding antibodies, the antigen-antibody complex is formed on the well surface. Upon the subsequent introduction into the wells of the anti-species radio-labelled serum, the antibodies present in it are adsorbed on the formed antigen-antibody complex, with human antibodies (gamma globulins) playing the role of an antigen in the given case. The greater the number of antibodies in the patient’s serum, the larger the level of the radioac­tive label linked to the well surface. Measurement of radioactivity in the liquid phase of the well contents gives evidence about the number of antibodies in the patient’s serum.

Complement-fixation test is used in virology for the retrospective diagnosis of numerous viral infections by demonstrating specific antibodies in paired human sera and for the evaluation of various clinical specimens for virus-specific antigens.

Virological application of the complement-fixation test is peculiar in that it is performed in the cold (for 12 hours, at 4 °C) and that an additional control with the so-called normal antigen is used (antigens from cells which are known to have reproduced the virus). This anti­gen is used in the same dilution as the viral one. A working comple­ment dilution is prepared ex tempore. A microcomplement-fixation test is also available

 

Radial haemolysis test involves haemolysis of antigen-sensitized erythrocytes by virus-specific antibodies in the presence of comple­ment in agarose gel. The test is routinely used in the serological diagnosis of influenza, other respiratory infections, rubella, parotitis and arbovirus (togavirus) infections.

Agarose (30 mg) is melted in 2.5 ml of phosphate buffer (pH 7.2), cooled to 42 °C, and mixed with 0.3 ml of sensitized erythrocytes and 0.1 ml of complement. One drop of boric acid is added, the mixture is carefully stirred and spread onto panels or slides with a warm 5-ml pipette. The thickness of the resultant layer should not ex­ceed 2 mm. Three-four minutes after agarose solidincatioa the panel is covered with a lid, inverted, and allowed to stand for 30 min at room temperature. Wells are punched in the solidified agarose and filled with test or control serum. The panel is covered with a lid and placed upturned into a moist chamber (Petpi dishes with a mois­tened piece of cotton wool) at 37 °C for 16-18 hrs.

Table 3

Diagnosis of Viral Infections by Means of the IF Test

 

The results of the test are evaluated by the size of hemolysis areas round the serum-filled wells. The controls should present no evidence of hemolysis.

For this test, sheep erythrocytes are washed with phosphate buffer (pH 7.2) and 0.3 ml of 10 per cent suspension is prepared, with a pH optimally adjusted for a given virus (e.g., 6.2-6.4 for tick-borne encephalitis virus). A 0.1-ml por­tion of undiluted antigen is added to erythrocytes, thoroughly mixed, and left to stand at room temperature for 10 min. Sensitized erythrocytes are precipitated by centrifuging for 10 min at 1000 X g, the pellet is washed with phosphate buffer (pH 7.2), and resuspended in 0.3 ml of borate-phosphate buffer (pH 6.2-6.4).

Test of haemadsorption on solid medium is a modification of en­zyme-linked immunosorbent assay and an indirect haemagglutination test. Because of its high sensitivity, it can be used as a rapid diag­nostic test in viral infections.

The procedure of the test is as follows. Wells of disposable poly­styrene panels are treated with immune globulin (immune serum), and suspension of antigen-containing material is placed in them. Thirty-sixty minutes later the wells are repeatedly washed with buffer, suspension of erythrocytes with adsorbed specific immunoglobulin is added, and haemagglutination is evaluated in 30-60 min.

If a specific antigen is present in the material, it is bound by the serum adsorbed on well surface, and, in turn, binds immunoglobulins on the erythrocyte surface. This results in erythrocyte agglutination (haemagglutination).

The test in the above modification is used for identifying antigens of rotaviruses and other enteroviruses in faeces.

Reversed indirect haemagglutination (RIHA) test is used for detecting bacterial and viral antigens in the  materials to be exam­ined as well as for the rapid diagnosis of a number of infections.

In contrast to IHA. erythrocytes in this test are sensitized not by antigens but by antibodies whose agglutination occurs upon ad­dition of the antigen.

Erythrocytes are first fixed with formalin or glutaraldehyde and bound to gamma-globulin which is isolated from immune sera and purified from other serum proteins. Binding of gamma-globulin with the erythrocyte surface is mediated by chromium chloride. For this purpose, to 8 volumes of distilled water add 1 volume of immunoglobulins obtained from immune serum, 1 volume of 50 per cent sus­pension of formalin-treated red blood .cells, and 1 volume of 0.1-0.2 per cent solution of chromium chloride. Allow the mixture to stand at room temperature for 10-15 min, thetreat the erythro­cytes as in the passive haemagglutination test.

The specificity of the  antibody diagnosticum is checked in the reac­tion of passive haemagglutination inhibition, using a homologous antigen. The reaction should be inhibited by a homologous antigen by at least 16 times and remain unaffected by a heterologous one. Examine the diagnosticum for spontaneous haemagglutination as well.

This test is commonly used to identify the causative agents in post-mortem material taken from the organs of man and animals, for example, from the brain, spleen, liver, and lungs. Prepare 10 per cent suspension of the above organs with isotonic sodium chlo­ride solution, centrifuge it at 10 000 X g for 30-60 min, and use the  supernatant as an antigen.

Procedure. Prepare two-fold dilutions of the material to be studied (antigen) with a stabilizing solution. Place one drop of each dilution of the antigen into 3 neighbouring wells of the micropanel (the reac­tion requires 3 parallel rows of wells). Into each well of the first row add 1 drop of the stabilizing solution, into wells of the second row, 1 drop of homologous immune serum in a 1:10 dilution, those of the third row, 1 drop of heterologous immune serum. The second and third rows serve as controls of reaction specificity. Let the mix­ture stand at room temperature for 20 min.

To all wells add one drop of 1 per cent suspension of sensitized erythrocytes (erythrocyte antibody diagnosticum) and shake the plates well. Read the results of the reaction in 30-40 min. In the presence of the specific antigen, haemagglutination is observed in the first and third rows (with heterologous serum) and is absent in the second row -where the antigen is preliminarily neutralized by homo­logous serum.

To ensure the accuracy of the test, the sensitized red blood cells are checked for spontaneous agglutination.

Reversed indirect haemagglutination inhibition (RIHAI) test makes it possible to detect the presence of antibodies in human and animal sera.

Procedure. Dilute sera by ten-fold with isotonic sodium chloride solution, heat for 20 min at 65 °C to destroy nonspecific inhibitors, then prepare two-fold serum dilutions with stabilizing solution and the working dose of the antigen containing four agglutinating units. Introduce by one drop of each dilution into the wells of the micro-panel and add to each of them one drop of the antigen whose dilution corresponds to the working dose. Allow the mixture to stand for 20 min at room temperature, then place one drop of the erythrocyte antibody diagnosticum into each well, and shake them thoroughly. Read the results of the reaction after 1.5-2 hour-incubation at room temperature. The titre of the serum is its greatest dilution which completely inhibits haemagglutination with four agglutinating units of the antigen.

The test is validated by checking whether sensitized erythrocytes may undergo spontaneous agglutination in the presence of: (a) stabilizing solution; (b) normal antigen (from material free of the virus); (c) the serum tested. Among advantages of the test one can cite its universality and the possibility to use it for finding various antigens.

Assessment of the haemagglutination results. Estimation of the results of IHA, RIHA and RIHAI tests is relied on the degree of erythrocyte agglutination; (++++), complete agglutination; (+++), almost complete agglutination; (++). partial agglutina­tion; (+)» traces of agglutination; (—), no agglutination.

The test is considered positive, if agglutination is complete (++++) or almost complete (+++)i the diagnosticum does not induce spontaneous agglutination in the presence of each compo­nent required for the reaction, and the control test of the specificity of the antigen or antibody is positive.

 

Schematic Representation of Hemagglutination inhibition test for identification of influenza virus

 

Schematic Representation of Hemagglutination inhibition test for serological diagnosis of influenza

 

Schematic Representation of Neutralization test for serological diagnosis of poliomielitis

 

 

 

Agar-Gel Immunodiffusions (AGID)

 

The agar-gel immunodiffusion (AGID), also referred to as an agar gel precipitin (AGP) test, involves the diffusion of virus and antibody through an agar (gela- tin-like substance), which will form a line of identity where the antigen-antibody complexes form.

 

 

 

Specific prophylaxis and treatment of infectious diseases

 

VACCCINES. Of the many diseases that plagued societies for centuries, smallpox was among the most serious. Numerous individuals contracted and died of this viral disease Those who survived, however, no longer seemed to be susceptible They had become resistant to infection with the smallpox virus Even without any knowledge of the immune response, some individuals reasoned that it was possible to acquire immunity (resistance to disease). In China, where many herbs and other substances were long used to treat disease, children inhaled dried scabs from smallpox victims to protect them against serious smallpox infections. Those that developed mild cases of smallpox and survived were subsequently resistant (immune) to this disease. This practice was carried out as early as the thirteenth century and, by the early eighteenth century, individuals throughout the Far East were exposing themselves to smallpox viruses to develop immunity.

In Turkey, elderly women collected material from the sores (pustules) of mild cases of smallpox and placed it into a walnut shell. A small amount of the material was then injected into a vein of an individual to protect her or him from smallpox. The injected individual would become ill within a week, developing fever, sores, and pustules. But in another week, the sores and pustules would heal. There generallywas no permanent scarring and after recovery the individual was resistant to smallpox.

This practice of ingrafting was introduced in England in 1718 by Lady Mary Montagu, whose husband had been the British ambassador to Turkey Lady Mary was a striking English beauty until the age of 26 when she contracted smallpox Although she survived, she was permanently scarred and bemoaned that “my beauty is no more ” It was perhaps because of her experience with smallpox that Lady Mary became interested in ingrafting when she was living in Constantinople In a letter to her family she wrote “The Small Pox, so fatal and general amongst us, is here entirely harmless by the invention of ingrafting. When she returned to England, Lady Mary used her considerable influence in the court of King George I to gain publicity for the increased use of ingrafting She even arranged a test of her idea on prisoners and orphans, then a common practice Today such testing of humans is viewed as unethical Despite her efforts, ingrafting was not accepted by the scientists and physicians of the time as a useful practice for preventing disease Too often, ingrafting resulted in scarring and in some cases it produced fatal cases of smallpox.

It was not until a report by Edward Jenner to the Royal Society of London, 80 years after Lady Mary tried to introduce ingrafting in England, that credence was given in Europe to the practice of immunization to protect against smallpox. Jenner was a middle-class country doctor, whose interest in science was typical of his position: scholarly but amateurish.

 

 

Although it is unclear when he began to develop his ideas about vaccination, Jenner apparently did not develop them from the reports of Lady Mary Montagu about ingrafting in Turkey. He clearly knew of the dairy country’s folk belief that cowpox, which is caused by vaccinia virus, protected its victims from subsequent infections of smallpox. In particular, milkmaids were known for their immunity to smallpox. Jenner also knew that BenjaminJesty, a Dorset farmer, had vaccinated his wife and two children with cowpox material taken from a soreon the udder of an infected cow in 1774. Although smallpox was ravaging the population in the vicinity, it did not affect the Jesty family.

Jenner performed his first inoculation in 1796 on a child with material from the cowpox lesions of a dairymaid. Six weeks later, Jenner inoculated the boy with smallpox virus. The boy did not develop small-pox, indicating that he was resistant to the disease. In this way Jenner experimentally demonstrated the validity of his hypothesis that exposure to cowpox viruses made one resistant to infections with smallpox viruses. Jenner continued his experimental studies, inoculating other children with cowpox viruses. Although friends and other physicians advised him against damaging his reputation by publishing results that were at such variance with accepted knowledge, Jenner decided to present his findings to the Royal Academy. His June 1798 report. An Inquiry into  the Causes and Effects of the Variolae Vaccine, describing the value of vaccination with cowpox as a means of protecting against smallpox established the basis for  the immunological prevention of disease. His work  met the criteria of the scientific method: his hypothesis had been experimentally tested by observations of control and experimental groups, and it was also repeatable by others. Immunization had gained scientific credibility; medical practice and the quest to eliminate smallpox had taken a giant step forward. The work begun with Jenner‘s discovery of the effectiveness of vaccination in preventing smallpox culminated in the 1970s with the eradication of smallpox  from the face of the Earth. Today, vaccines are employed for preventing many diseases, such as  tetanus, diphtheria, measles, and so forth. Research continues to develop new vaccines for preventing many other diseases.

 Immunization (vaccination). The usefulness of immunization rests with its ability to render individuals resistant to a disease without actually producing the disease. This is accomplished by exposing the individual to antigens associated with a pathogen in a form that does not cause disease. This medical process of intentional exposure to antigens is called immunization or vaccination. Implementation of immunization programs has drastically reduced the incidence of several diseases and has greatly increased life expectancies. Many once widespread deadly diseases such as whooping cough and diphtheria are rare today because of immunization programs.

Scientific Basis of Immunization. There are several scientific principles underlying the use of immunization to prevent individuals from contracting specific diseases and for preventing epidemic outbreaks of diseases:

1. Any macromolecule associated with a pathogen can be an antigen—an antigen is not the entire pathogen. Hence, one can use specific target antigens associated with pathogens to elicit the immune response without causing disease.

2. After exposure to an antigen the body may develop an anamnestic (memory) response.  Subsequent exposure to the same antigen can then bring about a rapid and enhanced immune response that can prevent replication of the infecting microorganism and/or the effects of toxins it produces so that disease does not oc- cur In this manner, intentional exposure to an antigen through vaccination can establish an anamnestic response that renders the individual resistant to disease

3. When a sufficiently high proportion of a population is immune to a disease, epidemics do not occur. This is because individuals who are immune are no longer susceptible and thus no longer participate m the chain of disease transmission. When approximately 70% of a population is immune, the entire population generally is protected, a concept known as herd immunity.

 

Herd immunity can be established by artificially stimulating the immune response system through the use of vaccines, rendering more individuals insusceptible to a particular disease and thereby protecting the entire population.

Vaccines. Vaccines are preparations of antigens whose administration artificially establishes a state of immunity without causing disease. Vaccines are designed to stimulate the normal primary immune response. This results in a proliferation of memory cells and the ability to exhibit a secondary memory or anamnestic response on subsequent exposure to the same antigens. The antigens within the vaccine need not be associated with active virulent pathogens. The antigens in the vaccine need only elicit an immune response with the production of antibodies or cytokines. Antibodies and/or cytokine-producing T cells possess the ability to react with the critical antigens associated with the pathogens against which the vaccine is designed to confer protection.

Thus, vaccines are preparations of antigens that stimulate the primary immune response, producing memory cells and the ability to exhibit an anamnestic response to a subsequent exposure to the same antigens without causing disease.

Vaccines may contain antigens prepared by killing or inactivating pathogenic microorganisms; vaccines also may use attenuated or weakened strains that are unable to cause the onset of severe disease symptoms (Table 1).

Comparison of Attenuated and Inactivated Vaccines

 

Some of the vaccines that are useful in preventing diseases caused by various microorganisms are listed in Table 2. Most vaccines are administered to children (Table 3), some are administered to adults (Table 4), and some are used for special purposes. Travellers receive vaccines against pathogens prevalent in the regions they are visiting that do not occur in their home regions (Table 5). In each of these cases the use of the vaccine is prophylactic and aimed at preventing diseases caused by pathogens to which the individual may be exposed.

 

 

TABLE 2

Descriptions of Widely Used Vaccines

 

TABLE 3

Recommended Vaccination Schedule for Normal Children

 

 

 

 

TABLE 4

Recommended Vaccination Schedule for Adults

 

Although vaccines are normally administered before exposure to antigens associated with pathogenic microorganisms, some vaccines are administered after suspected exposure to a given infectious microorganism. In these cases the purpose of vaccination is to elicit an immune response before the onset of disease symptoms. For example, tetanus vaccine is administered after puncture wounds may have introduced Clostridium tetani into deep tissues, and rabies vaccine is administered after animal bites may have introduced rabies virus. The effectiveness of vaccines administered after the introduction of the pathogenic microorganisms depends on the relatively slow development of the infecting pathogen before the onset of disease symptoms. It also depends on the ability of the vaccine to initiate antibody production before active toxins are produced and released to the site where they can cause serious disease symptoms.

TABLE 5

Recommended Vaccinations for Travellers

 

Attenuated Vaccines. Some vaccines consist of living strains of microorganisms that do not cause disease. Such strains of pathogens are said to be attenuated because they have weakened virulence (smallpox, anthrax, rabies, tuberculosis, plague, brucellosis, tularaemia, yellow fever, influenza, typhus fever, poliomyelitis, parotitis, measles, etc.). Pathogens can be attenuated, that is, changed into nondisease-causing strains, by various procedures, including moderate use of heat, chemicals, desiccation, and growth in tissues other than the normal host. Vaccines containing viable attenuated strains require relatively low amounts of the antigens because the microorganism is able to replicate after administration of the vaccine, resulting in a large increase in the amount of antigen available within the host to trigger the immune response mechanism. The principle disadvantage of living attenuated vaccines is the possible reversion to virulence through mutation or recombination. Also, even strains may cause disease in individuals who lack adequate immune responses, such as those with AIDS.

Live, Attenuated Vaccines

Attenuated vaccines can be made in several different ways. Some of the most common methods involve passing the disease-causing virus through a series of cell cultures or animal embryos (typically chick embryos). Using chick embryos as an example, the virus is grown in different embryos in a series. With each passage, the virus becomes better at replicating in chick cells, but loses its ability to replicate in human cells. A virus targeted for use in a vaccine may be grown through—“passaged” through—upwards of 200 different embryos or cell cultures. Eventually, the attenuated virus will be unable to replicate well (or at all) in human cells, and can be used in a vaccine. All of the methods that involve passing a virus through a non-human host produce a version of the virus that can still be recognized by the human immune system, but cannot replicate well in a human host.

When the resulting vaccine virus is given to a human, it will be unable to replicate enough to cause illness, but will still provoke an immune response that can protect against future infection.

One concern that must be considered is the potential for the vaccine virus to revert to a form capable of causing disease. Mutations that can occur when the vaccine virus replicates in the body may result in more a virulent strain. This is very unlikely, as the vaccine virus’s ability to replicate at all is limited; however, it is taken into consideration when developing an attenuated vaccine. It is worth noting that mutations are somewhat common with the oral polio vaccine (OPV), a live vaccine that is ingested instead of injected. The vaccine virus can mutate into a virulent form and result in rare cases of paralytic polio. For this reason, OPV is no longer used in the United States, and has been replaced on the Recommended Childhood Immunization Schedule by the inactivated polio vaccine (IPV).

Protection from a live, attenuated vaccine typically outlasts that provided by a killed or inactivated vaccine.

 

 

Figure 2.  Attenuated vaccines can be produced by multiple passage through animal cell tissue culture. Poliovirus for the oral vaccine was developed by passage through monkey kidney cells. Following clinical trials the use of the vaccine greatly reduced the incidence of polio in the United States. Elsewhere, where the vaccine is not used, the incidence of polio remains high.

 

 

The Sabin polio vaccine, for example, uses viable polioviruses attenuated by growth in tissue culture. Three antigenically distinguishable strains of polioviruses are used m the Sabin vaccine.

The Sabin polio vaccine, for example, uses viable polioviruses attenuated by growth in tissue culture. Three antigenically distinguishable strains of polioviruses are used m the Sabin vaccine.

 These viruses are capable of multiplication within the digestive tract and salivary glands but are unable to invade nerve tissues and thus do not produce the symptoms of the disease polio The vaccines for measles, mumps, rubella, and yellow fever similarly use viable but attenuated viral strains. Attenuated strains of rabies viruses can be prepared by desiccating the virus after growth m the central nervous system tissues of a rabbit or following growth m a chick or duck embryo.

The BCG (bacille Calmette-Guerm) vaccine is an example of an attenuated bacterial vaccine. This vaccine is administered in Britain to children 10 to 14 years old to protect against tuberculosis. It is used in the United States only for high-risk individuals This mycobactenal strain was developed from a case of bovine tuberculosis. It was cultured for over 10 years in the laboratory on a medium containing glycerol, bile, and potatoes. During that time it accumulated mutations so that it no longer was a virulent pathogen. In over 70 years of laboratory culture the BCG mycobacterial strain has not reverted to a virulent form.

Louis Pasteur furthered the development of vaccines when in 1880 he reported that attenuated microorganisms could be used to develop vaccines against chicken cholera. The production of these vaccine depended on prolonging the time between transfers of the cultures. This fact was accidentally discovered through an error by Charles Chamberland, who used an old culture during one of the experiments he was conducting with Pasteur. The old culture contained attenuated microorganisms, that is, weakened or altered microorganisms that were less virulent. Following his work on chicken cholera, Pasteur directed his attention to the study of anthrax. Because he enjoyed being the center of attention and controversy, Pasteur staged a dramatic public demonstration to test the effectiveness of his anthrax vaccine. Witnesses were amazed to see that the 24 sheep, 1 goat, and 6 cows that had received the attenuated vaccine were in good health, whereas all of the animals in this experiment feat had not been vaccinated were dead of anthrax.

In 1885, Pasteur announced to the French Academy of Sciences that he had developed a vaccine for preventing another dread disease, rabies (see Figure). Although he did not understand the nature of the causative organism, Pasteur developed a vaccine that worked. Pasteur’s motto was “Seek the microbe”, but the microorganism responsible for rabies is a virus, which could not be seen under the microscopes of the 1880s. Pasteur, none the less, was able to weaken the rabies virus by drying the spinal cords of infected rabbits and allowing oxygen to penetrate the cords. Thirteen inoculation of  successively more virulent pieces of rabbit spinal cord were injected over a period of 2 weeks during the summer of 1885 into Joseph Meister, a 9-year-old boy who had been bitten by a rabid dog.

“Since the death of the child was almost certain, I decided in spite of my deep concern to try on Joseph Meister the method which had served me so well with dogs… I  decided to give a total of 13 inoculations in 10 days. Fewer inoculations would have been sufficient, but one will understand that I was extremely cautious in, this first case. Joseph Meister escaped not only the rabies that he might have received from his bites, but also the rabies which I inoculated into him”.

 

 

The development of the rabies vaccine crowned Pasteur’s distinguished career.

Killed / lnactivated Vaccines. Some vaccines are prepared by killing or inactivating microorganisms so they cannot reproduce or replicate within the body and are not capable of causing disease (enteric fever, paratyphoid, cholera, whooping cough, poliomyelitis and leptospirosis vaccines, etc.).. When microorganisms are killed  or inactivated by treatment with chemicals, radiation, or heat, the antigenic properties of the pathogen are retained. Killed/inactivated vaccines generally can be used without the risk of causing the onset of the disease associated with the virulent live pathogens. The vaccines used for the prevention of whooping cough (pertussis) and influenza are representative of the preparations containing antigens that are prepared by inactivating pathogenic microorganisms.

One alternative to attenuated vaccines is a killed or inactivated vaccine. Vaccines of this type are created by inactivating a pathogen, typically using heat or chemicals such as formaldehyde or formalin. This destroys the pathogen’s ability to replicate, but keeps it “intact” so that the immune system can still recognize it. (“Inactivated” is generally used rather than “killed” to refer to viral vaccines of this type, as viruses are generally not considered to be alive.)

Because killed or inactivated pathogens can’t replicate at all, they can’t revert to a more virulent form capable of causing disease (as discussed above with live, attenuated vaccines). However, they tend to provide a shorter length of protection than live vaccines, and are more likely to require boosters to create long-term immunity. Killed or inactivated vaccines on the U.S. Recommended Childhood Immunization Schedule include the inactivated polio vaccine and the seasonal influenza vaccine (in shot form).

 

Even when the vaccines are killed cells, problems can occur in some cases. A small percentage of children, for example, have allergic-type reactions to the pertussis component of the standard DPT (diphtheria-pertussis-tetanus) vaccine Some people are now questioning the wisdom of government-mandated administration of this vaccine. Most manufacturers of this vaccine ceased its production rather than face liability lawsuits associated with such reactions. The supply of DPT vaccine is currently dangerously short. Enhanced quality control programs by the major remaining producer and the development of a new form of the vaccine promise to reduce the incidence of adverse reactions.

There have been several problems with inadequate inactivation of vaccines, leading to disease out-breaks when the vaccines were administered In 1976 there was a scare about an impending outbreak of swine flu Some people given swine flu vaccine actually contracted flu because of the inadequate inactivation of the viruses in hastily prepared vaccines. Others developed a neurological disorder called Guillain-Barre syndrome after vaccination against swine flu In the 1950s several tragic cases of polio oc- curred in children given the Salk polio vaccine, an inactivated vaccine prepared from a very virulent strain of poliovirus. This incident occurred because of the failure to fully inactivate some batches of the vaccine prepared by treatment of polioviruses with formaldehyde Because the Slake vaccine is prepared from a particularly virulent strain of poliovirus, replication of the virus in individuals inoculated with the improperly prepared vaccines caused paralytic polio.

The failure of the quality control program for the Slake vaccine, in part, led to the general switch to the “live” attenuated Sanin polio vaccine. The Sanin vaccine is prepared with attenuated viral strains. These strains are not particularly virulent and do not invade the nervous system, causing paralysis. The Sabin vaccine is administered orally and the virus multiplies within the gastrointestinal tract. Although the virus is attenuated, mutations and recombinations are still possible during replication. Some recent cases of polio have been reported with the Sanin vaccine, causing the reevaluation of the relative merits of the Salk versus the Sabin vaccine.

In some cases the toxins responsible for a disease are inactivated and used for vaccination. Some vaccines, for example, are prepared by denaturing microbial exotoxins. The denatured proteins produced are called toxoids. Protein exotoxins, such as those involved in the diseases tetanus and diphtheria, are suitable for toxoid preparation. The vaccines for preventing these diseases employ toxins inactivated by treatment with formaldehyde. These toxoids retain  the antigenicity of the protein molecules. This means that the toxoids elicit the formation of antibody and are reactive with antibody molecules but, because the proteins are denatured, they are unable to initiate the reactions associated with the active toxins that cause disease.

 

Individual Microbiological Components (chemical vacccines). Individual components of microorganisms can be used as antigens for immunization. For example, the polysaccharide capsule from Streptococcus pneumoniae is used to make a vaccine against pneumococcus pneumonia. This vaccine is used in high-risk patients, particularly individuals over 50 years old who have chronic diseases, such as emphysema. Another vaccine has been produced from the capsular polysaccharide of Haemophilus influenzae type b, a bacterium that frequently causes meningitis in children 2 to 5 years old. The Hib vaccine, as it is called, is being widely administered to children in the United States. This vaccine is not always effective in establishing protection in children under 2 years old. It is administered to children between 18 and 24 months old who attend day care centres because they have a greater risk of contracting H. influenzae infections.

A polyvaccine against typhoid fever and tetanus is now manufactured and used. It consists of O- and Vi-antigens of the typhoid fever bacteria and purified concentrated tetanus anatoxin. The bacterial antigens and the tetanus anatoxin are adsorbed on aluminium hydroxide.

The first vaccine to provide active immunization against hepatitis B (Heptavax-B) was prepared from hepatitis B surface antigen (HBsAg). This antigen was purified from the serum of patients with chrome hepatitis B. Immunization with Heptavax-B is about 85% to 95% effective in preventing hepatitis B infection. It was administered predominantly to individuals in high-risk categories such as health care workers. It has been replaced by a newer recombinant vaccine, Recombivax HB. To produce Recombivax HB, a part of the hepatitis B virus gene that codes for HBsAg was cloned into yeast. The vaccine is derived from HBsAg that has been produced in yeast cells by recombinant DNA technology,                 

Attempts were made to make a vaccine against gonorrhea using pili from Neissena gonorrhoeae, the bacterium that causes this disease. The vaccine produced, however, was not successful because longlasting immunity against N. gonorrhoeae does not develop. The military, though, has used this vaccine to achieve short-term immunity. Synthetic proteins are also being considered as potential antigens for protection against various diseases.

Thus Individual components of a microorganism can be used in a vaccine to elicit an immune response.

Subunit and Conjugate Vaccines

Both subunit and conjugate vaccines contain only pieces of the pathogens they protect against.

Subunit vaccines use only part of a target pathogen to provoke a response from the immune system. This may be done by isolating a specific protein from a pathogen and presenting it as an antigen on its own. The acellular pertussis vaccine and influenza vaccine (in shot form) are examples of subunit vaccines.

Another type of subunit vaccine can be created via genetic engineering. A gene coding for a vaccine protein is inserted into another virus, or into producer cells in culture. When the carrier virus reproduces, or when the producer cell metabolizes, the vaccine protein is also created. The end result of this approach is a recombinant vaccine: the immune system will recognize the expressed protein and provide future protection against the target virus. The Hepatitis B vaccine currently used in the United States is a recombinant vaccine.

Another vaccine made using genetic engineering is the human papillomavirus (HPV) vaccine. Two types of HPV vaccine are available—one provides protection against two strains of HPV, the other four—but both are made in the same way: for each strain, a single viral protein is isolated. When these proteins are expressed, virus-like particles (VLPs) are created. These VLPs contaio genetic material from the viruses and can’t cause illness, but prompt an immune response that provides future protection against HPV.

Conjugate vaccines are somewhat similar to recombinant vaccines: they’re made using a combination of two different components. Conjugate vaccines, however, are made using pieces from the coats of bacteria. These coats are chemically linked to a carrier protein, and the combination is used as a vaccine. Conjugate vaccines are used to create a more powerful, combined immune response: typically the “piece” of bacteria being presented would not generate a strong immune response on its own, while the carrier protein would. The piece of bacteria can’t cause illness, but combined with a carrier protein, it can generate immunity against future infection. The vaccines currently in use for children against pneumococcal bacterial infections are made using this technique.

 

 

 

 

Besides the above mentioned preparations associated vaccines are used for specific prophylaxis of infectious diseases: whooping cough-diphtheria-tetanus vaccine, diphtheria-tetanus associated  anatoxin, whooping cough-diphtheria.

Methods of preparing other associated vaccines are being devised which will provide for the production of antibacterial, antitoxic and antivirus immunity.

Idiotypes. Because of the high degree of variability in the aminoterminal regions of heavy and light chains of an Ab, the Ab combining site and adjacent variable regions often are unique to that Ab (or at least found infrequently in other Abs). Such V region associated structures are called idiotypes. It may help to think of idiotypes as being analogous to fingerprint patterns—few of either are the same.

The idiotype on Ab from one individual can be seen as foreign by another individual of the same species who has not, or cannot, form the same structure on his or her own Abs. If it is seen as foreign, this second individual can make an Ab that binds to the idiotype structure on the Ab from the first individual. The Ab made in the second individual is called an anti idiotype Ab.

Many different ammo acid sequence and structural combinations can form an Ab to a single site on an Ag. Each of these combinations results in a unique Ab binding site, and each unique Ab binding site can give rise to a unique idiotype. Thus, an Ab response to a single site on an Ag can give rise to many different Abs with many different binding sites and, thus, many different idiotypes. Although Abs can see the same site on an Ag in a variety of ways, there are limits to the variability that will allow the binding to occur. Therefore, among those Abs that do bind, some similarity in binding sites and, as a result, some similarity in idiotypes might be expected. These similar, or shared, idiotypes are called public idiotypes.

There also are idiotypes that are unique to a single Ab. These are called private idiotypes. For example, an Ab response to site 1 on an Ag molecule contains Abs with 10 different combining sites. Seven of the 10 binding sites, although different, are similar enough that antiidiotype Ab made to one reacts with all 7. These 7 Abs share a public idiotype. The other 3 Abs also are similar to each other, but are different from the first 7 Abs. These 3 Abs share an idiotype (public idiotype number 2) that is different from the idiotype (public idiotype number 1) shared by the other 7 Abs.

In addition, 1 or more (and perhaps each) of the 10 Abs can have a second V-region-associated structure that is not shared with any of the other 9 Abs. This would be a private idiotype

This lengthy explanation of allotypes and idiotypes is given because both are used as genetic markers in human genetic studies and as markers of variable and constant region genes in studies of regulation of immune responses. In addition, an understanding of the nature of idiotypes is required to appreciate how they can complicate efforts to use monoclonal Abs to suppress transplant rejection and to kill malignant lymphoid cells.

The antiidiotipic antibodies are “mirror reflection” of antigens and thus can induce antibodies formation an cytotoxic cells which interact with antigens. There are many experimental vaccines against different bacteria, viral, protozoan diseases. But interest to antiidiotipic vaccines are decreased, because it is difficult to receive necessary titre of  specific antibodies which caeutralize causing agents and durable immunity against them. Besides that  antiidiotipic vaccine cause allergic reactions.

Monoclonal Antibodies. Most proteins possess many different antigenic determinants. As a result, serum from an animal or human producing Abs to a protein or cellular constituent contains a complex mixture of Abs. This mixture contains Abs to all determinants as well as Abs that are heterogeneous with respect to heavy chain isotype, light chain type, allotype, variable region sequence, and idiotype. A long held dream of biomedical scientists was to isolate a single Ab producing cell and grow it in vitro to provide a source of homogenous Abs that  would bind to only a single antigenic determinant.

Unfortunately, normal Ab-producing cells do not grow indefinitely in tissue culture. In 1975, Georges Kohler and Cesar Milstein overcame this difficulty by fusing normal cells producing the desired Ab from an immunized animal  with myeloma cells (malignant lymphocytes that can be propagated easily in vitro) in the presence of a chemical that promotes cell fusion (polyethylene glycol or Sendai virus). This caused the cell membranes of some Ab producing spleen cells to fuse with the myeloma cells. Such fused cells, called hybridomas, have the Ab producing capability of the normal cell parent and the in vitro growth properties of the malignant myeloma parent. The normal, nonfused spleen cells cannot survive in culture, whereas the unfused myeloma cells, which can grow in vitro, carry a mutant gene in a critical biosynthetic pathway (ie, a drug marker). The presence of this mutant gene allows the unfused myeloma cell to be killed by adding the appropriate drug in culture. The fused cell is protected from this drug, because the normal spleen cell provides the normal biosynthetic gene. The procedure used to produce hybndoma cell lines secreting monoclonal Abs is shown in Figure.

FIGURE . Production or hybridoma cell lines secreting monoclonal antibodies .The procedure for producing monoclonal antibodies is shown. Activated B cells from an immunized individual (eg, spleen cells from an immunized mouse) are fused with malignant plasma cells isolated from plasmacytomas and adapted to tissue culture. The myeloma cell has a mutant gene that renders it sensitive to the drug aminopterin. The activated B cells, although resistant to aminopterin, have a limited lifetime in culture  and die naturally. The B cell—myeloma cell hybrid is resistant to aminopterin because the B cell provides the missing genes. Therefore, the B cell-myeloma cell hybrid (the hybridoma) is the only fusion product that can survive in the hypoxanthine, aminopterin, thymidine (HAT) selective culture medium used. The hybrids are distributed into many culture wells in the multiwell culture plates and are allowed to grow for a short period. The culture supernatant or these wells then is tested for the desired antibody. Those cultures that are positive are cloned, and the hybridoma cell producing the desired antibody is propagated and used as a source of the monoclonal antibody.

Monoclonal Abs are available for thousands of different determinants and are being used widely as research tools to study protein structure and virus and toxieutralization, and to isolate specific proteins from complex mixtures. Moreover, many commercially available monoclonal Abs are being used in extremely sensitive and specific techniques for the diagnosis of various diseases and, as mentioned earlier, for the experimental treatment of several human diseases.

SYNTHETIC VACCINES. Another approach to the study of Ag structure has been to synthesize peptides with exactly the same sequence as portions of the Ag of interest and to determine whether Ab made to the intact protein will react with these peptides. Similarly, such peptides have been conjugated to a carrier protein (as described earlier for haptens) and used to induce Abs to the peptide Frequently, these latter Abs have been found to react with the native protein molecule as well.

This has led to attempts to predict which ammo acids are involved in the formation of an antigenic determinant. For example, it is known that epitopes recognized by Ab are located on the surface of the Ag molecule. Therefore, it one could predict which segments of the linear sequence exist sequentially on the surface, one could possibly synthesize a peptide with that sequence and use it to induce Ab that would react with the native molecule. Such studies have been conducted, and the results generally bear out the predictions of the ammo acids that contribute to the structure of an antigenic determinant. Other algorithms for predicting antigenic structure have been suggested, but no single one is universally applicable. Much of the problem lies in determining which peptides will invoke an immune response that will neutralize a virus or promote removal and destruction of the bacterium causing the disease Nevertheless, such studies have led to multiple attempts to produce synthetic Ags that could be used to immunize individuals against disease caused by bacteria or viruses. For example, synthetic vaccines have been used experimentally to immunize against diseases such as hepatitis and hoof and mouth disease.

Vector Vaccines. The antigenic structure of most antigens of clinical interest has not been determined, either because we do not have the monoclonal antibodies necessary or because our knowledge of the three dimensional structure is limited or lacking. However, recombinant DNA technology has allowed us to infer the amino acid sequence of several viral proteins from their DNA coding sequences.

This knowledge of the primary structure of viral proteins, combined with the algorithms for predicting antigenicity, may enable us to produce synthetic vaccines in situations in which the production of safe, effective vaccines by current methods is not yet possible.

Recombinant DNA technology is being used to create vaccines containing the genes for the surface antigens for various pathogens. Such vector vaccines act as carriers for antigens associated with pathogens other than the one from which the vaccine was derived. The attenuated virus used to eliminate smallpox is a likely vector for simultaneously introducing multiple antigens associated with different pathogens, such as the chicken pox virus. Several prototype vaccines using the smallpox vaccine as a vector have been made (FIG.).

New vaccines can be formed by using recombinant DNA technology to form vector vaccines, for example, using vaccinia virus as a carrier.

 

Other methods for producing novel vaccines also have been developed. For example, a recombinant vaccnia virus that contains a gene for the immunogenic glycoprotein of rabies virus has been made. This recombinant virus expresses the rabies glycoprotein on its viral envelope in addition to its own  glycoprotein. Immunization of experimental animals with this recombinant virus has led to complete protection against disease following intracerebral injection of rabies virus.

About 1 millioew cases of polio are reported annually, most in undeveloped countries. With the use of Ab resistant mutants of the poliovirus, several ammo acids that constitute epitopes on this virus have been identified Karen Burke and her colleagues in England have used recombinant DNA techniques to construct a hybrid virus containing the epitopes of the three serotypes of poliovirus. Experimental animals immunized with this hybrid vaccine have been shown to produce Ab reactive with all three serotypes. Similar methods can be used to produce improved vaccines against many picornaviruses, including hepatitis A.

More recently, the gene encoding pl20, the surface glycoprotein of the human immunodeficiency virus, has been cloned, and the protein has been expressed in insect cells. The use of this recombinant protein as a vaccine for acquired immunodeficiency syndrome is undergoing clinical trials.

These differ from recombinant DNA vaccines in that the transfection of exogenous DNA occurs in vivo rather than in vitro, and no autologous tumor cells are required. The biologic response modifiers or other proteins to be expressed are inserted in a plasmid vector capable of expressing exogenous proteins in mammalian cells. These plasmids are introduced into a skeletal muscle bed or subcutaneous tissue with the intent of transfecting local host cells with the recombinant plasmid, thus creating a local deposit of ectopically expressed protein. The adjuvants introduced with the plasmid will then help stimulate an immune response to the components of the vaccine. The protein coded in the plasmid could be an antigenic response epitope or a series of them. Alternatively, this could be a way to administer sustained release of the ectopically expressed biologic response modifier known to be useful in stimulating innate immune responses to the tumor, such as IL-2. In the latter role they could be used as an adjunct to other vaccines or treatment strategies. Conceivably they can be used for both, either in different plasmids injected into different sites, in different plasmids co-injected into the same sites, or even in one plasmid encoding one or more proteins encoded from the same plasmid.

A major advantage of this type of vaccine is that it does not require autologous tumor cells or individualized manipulations of DNA. As such they would also be amenable to mass production with minimal technical requirements for administration.

Although they have great potential, DNA vaccines are the most experimental vaccine strategy of those discussed here. A major potential drawback of this approach that has been encountered in other gene therapy studies using exogenous DNA vectors of various types is the difficulty maintaining tissue expression levels of the plasmid-encoded protein(s) over time. This may or may not be a significant problem for antigen (peptide) delivery, although it may be for the continuous delivery of ectopic cytokines and biologic response modifiers.

 

 

Booster Vaccines. Multiple exposures to antigens are sometimes needed to ensure the establishment and continuance of a memory response. Several administrations of the Sabin vaccine are needed during childhood to establish immunity against poliomyelitis. A second vaccination is necessary to ensure immunity against measles. Only a single vaccination, though, is needed to establish permanent immunity against mumps and rubella.

In some cases, vaccines must be administered every few years to maintain the anamnestic response capability. Periodic booster vaccinations are necessary, for example, to maintain immunity against tetanus. A booster vaccine for tetanus is recommended every 10 years.

Adjuvant. Some chemicals, known as adjuvants, greatly enhance the antigenicity of other chemicals (FIG. ). The inclusion of adjuvants in vaccines therefore can greatly increase the effectiveness of the vaccine. When protein antigens are mixed with aluminum compounds, for example, a precipitate is  formed that is more useful for establishing immunity than the proteins alone. Alum-precipitated antigens are released slowly in the human body, enhancing stimulation of the immune response. The use of adjuvants can eliminate the need for repeated booster doses of the antigen, which increases the intracellular exposure to antigens to establish immunity. It also permits the use of smaller doses of the antigen in the vaccine.

 

Some bacterial cells are effective adjuvants. The killed cells of Bordetella pertussis, used in the DPT vaccine, are adjuvants for the tetanus and diphtheria toxoids used in this vaccine. Similarly, mycobacteria are effective adjuvants. Freund’s adjuvant, which consists of mycobacteria emulsified in oil and water, is especially effective in enhancing cell-mediated immune responses. This adjuvant, however, can induce issue damage and is not used for that reason.

Chemical adjuvants are used in vaccines to increase the antigenicity of other chemical components and hence the effectiveness of the vaccine. They can eliminate the need for booster doses of the antigen.

Routes of Vaccination. The effectiveness of vaccines depends on how they are introduced into the body. Antigens in a vaccine may be given via a number of routes: intradermally (into the skin), subcutaneously (under the skin), intramuscularly (into a muscle), intravenously (into the bloodstream), and into the mucosal cells lining the respiratory tract through inhalation, or orally into the gastrointestinal tracts. Killed / inactivated vaccines normally must be injected into the body, whereas attenuated vaccines often can be administered orally or via inhalation. The effectiveness of a given vaccine depends in part on the normal route of entry for the particular pathogen. For example, polioviruses normally enter via the mucosal cells of the upper respiratory or gastrointestinal tracts.

The Sabin polio vaccine, therefore, is administered orally, enabling the attenuated viruses to enter the mucosal cells of the gastrointestinal tract directly. It is likely that vaccines administered in this way stimulate secretory antibodies of the IgA class in addition to other immunoglobulins. Intramuscular administration of vaccines, like the Salk polio vaccine, is more  likely to stimulate IgM and IgG production. IgG is particularly effective in halting the spread of pathogenic microorganisms and toxins produced by such organisms through the circulatory system.

Vaccination is carried out with due account for the epidemic situation and medical  contraindications. The contraindications include acute fevers, recent recovery from an infectious disease, chronic infections (tuberculosis, malaria), valvular diseases of the heart, severe lesions of the internal organs, the second half of pregnancy, the first period of nursing a baby at the breast, allergic conditions (bronchial asthma, hypersensitivity to any foodstuffs), etc.

Vaccines are stored in a dark and dry place at a constant temperature (+2 to +10 °C). The terms of their fitness are indicated on labels and the method of their administration in special instructions enclosed in the boxes with the flasks or ampoules.

Frustrating efforts to find a vaccine for AIDS. It is not always easy to find antigens associated with pathogens that confer long-term active immunity. Desperate efforts are now underway to formulate a vaccine that will prevent AIDS. Years of research, however, have failed to produce vaccines against other sexually transmitted diseases such as syphilis, as well as other prevalent diseases, including malaria and tooth decay.

Producing an AIDS vaccine will be very difficult because of several properties of HIV. Being a virus, it replicates intracellularly and is released by budding. Hence a method will have to be found for preventing the initial adsorption of the virus to host cells or for detecting and eliminating host cells within which the virus is replicated. Also, the DNA made during replication of this retrovirus is incorporated into me host cell  chromosomes. Use of an attenuated HIV that still permits in corporation of DNA into the chromosomes of the host cell could lead to malignancy. To complicate matters, HIV exhibits variable surface antigens so that multiple antigens may have to be employed to ensure that the  body’s immune system recognizes infecting HIV. So far, the most promising approaches are aimed at blocking the sites on the virus that bind to host cell receptors and are involved m entry of HIV into the host cells. Some research groups are targeting tine CD4 binding protein of HIV, others are exploring CD26 as a site for blocking the  ability of me virus to enter and to infect human host cells.

Vaccinotherapy. For treating patients with protracted infectious diseases (furunculosis, chronic gonorrhoea, brucellosis) vaccines prepared from dead microbes and anatoxins are used. The staphylococcal anatoxin, the polyvalent staphylococcal and streptococcal, the gonococcal and antibrucellosis vaccines and the vaccine against disseminated encephalitis and multiple sclerosis produce a good therapeutic effect.

Combined immuno-antibiotic therapy yields favourable results in typhoid fever, dysentery, brucellosis, ornithosis, and actinomycosis. In some cases autovaccines are used which are prepared from microbes isolated from patients.

Pyrogenal, a preparation from Gram-negative bacteria, is recommended as non-specific therapy in inflammatory processes of the eyes and female genitalia, in syphilis of the nervous system, progressive paralysis, eczema, chronic streptoderma, mycoses, different forms of tuberculosis and in many other diseases for increasing the reactivity of the patient and for activation of the functions of organs and systems of the macro-organism.

The mechanism of its action comprises an increase in the permeability of the capillary walls and the main substance of the connective tissue, a stimulation of the function of the hypophyso-adrenal system, an increase in the protein synthesis in the body, an inhibition of the processes of formation of fibroblasts from young cells of the connective tissue, and a decrease in the development of scar tissue.

Artificial passive immunity. Passive immunity can be used to prevent diseases when there is not sufficient time to develop an acquired immune response through vaccination. The administration of sera, pooled gamma globulin that contains various antibodies, specific immunoglobulins, or specific antitoxins provides immediate protection (Table 6).

Table 6

Substances Used for Passive Immunization

 

Sera are injected in definite doses intramuscularly, subcutaneously, sometimes intravenously, with strict observation of all the rules of asepsis. A preliminary desensitization according to Bezredka’s method is necessary. Sera are employed for treatment and for prophylaxis of tetanus, gas gangrene and  botulism. The earlier the serum is injected, the more marked is its therapeutic and prophylactic action. The length of protective action of sera (passive immunity) is from 8 to 14 days.

At present many institutes of vaccines and sera in the Soviet Union produce therapeutic and prophylactic sera in a purified state. They are treated by precipitating globulins with ammonium sulphate, by fractionation, by the method of ultracentrifugation, electrophoresis and enzymatic hydrolysis which allow the removal of up to 80 per cent of unrequired proteins. These sera have the best therapeutic and prophylactic properties, contain the least amount of unrequired proteins, and have a less distinct toxic and allergic action.

Sera thus produced are subdivided into antitoxic and antimicrobial sera. Antitoxic sera include antidiphtheritic, antitetanic sera and sera effective against botulism, anaerobic infections, and snake bites.

Antimicrobial sera are used against anthrax, encephalitis and influenza in the form of globulins and gamma globulins.

Before the development of antibiotics, passive immunization – often using horse sera – was widely practiced. Unfortunately, precipitation from extensive antigen-antibody complex formation caused kidney damage when horse sera was routinely administered. Today the use of passive  immunity to treat disease is limited to cases of immunodeficiencies and to specific reactions to block the adverse effects of pathogens and toxins.

Antitoxins  special substances (antibodies) formed in the organism of animals or man upon entry into it of toxins—that is, poisons of bacterial or animal origin. Every antitoxin has a strictly specific effect; it renders harmless (neutralizes) only that toxin under the influence of which it was formed (for example, only the toxin secreted by the causative agent of diphtheria is neutralized by diphtheria antitoxin), and it has no neutralizing effect on other toxins. Antitoxins are gamma globulins that are capable of interacting specifically with toxins.

Antitoxins are used in medical practice in the form of antitoxic sera (antidiphtheria, antitetanus, antidysentery, antigangrene, antibotulinum, antiscarlatina, antivenom, and so on), which are obtained by subcutaneous injection of a horse (or other animal) with toxins or anatoxins; antitoxin is thereupon formed in the blood serum of the horse. Blood serum containing antitoxin is widely used in prophylaxis and treatment of diphtheria, tetanus, botulism, and other diseases, and it is also used for treatment of persons bitten by poisonous snakes. The therapeutic and prophylactic properties of immune sera are determined by their strength, which is measured in conventional antitoxic units (AU). Thus, 1 AU of diphtheria antitoxic serum is considered to be that quantity of the serum which neutralizes 100 minimum lethal doses of diphtheria toxin when injected into a guinea pig weighing 200–250 g.

Methods of purifying and concentrating antitoxic sera have been elaborated which permit production of preparations that have high AU content and are free of inert substances.

 

Various antitoxins (antibodies that neutralize toxins) can be used to prevent toxins of microbial or other origin from causing disease symptoms. The administration of antitoxins establishes passive atificial immunity. Antitoxins are used to neutralize the toxins in snake venom, saving the victims of snake bites. The toxins in poisonous mushrooms can also be neutralized by administration of appropriate anti-toxins. The administration of antitoxins and immunoglobulins to prevent disease occurs after exposure to a toxin and/or an infectious microorganism.

Antitoxins are antibodies that neutralize toxins and can be used to prevent toxins from causing disease symptoms.

It is also possible to establish passive immunity by the administration of gamma globulin, which contains mainly IgG and some IgM and IgA This is a widely used treatment in Africa for many diseases. It is important that the gamma globulin used for establishing passive immunity is pooled in order to combine the immune functions from many people. Passive immunity lasts for a limited period of time because IgG molecules have a finite lifetime in the body. The administration of IgG does not establish an anamnestic response capability. The administration of IgG is also

 

particularly useful therapeutically in preventing disease in persons with immunodeficiencies and other high-risk individuals