Improvement of ELISA for the diagnosis of animal diseases

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Currently, in improving serological diagnosis of infectious diseases are widely used methods based on the use of ELISA. This is understandable: ELISA is a highly sensitive test, allowing for a short time to examine a large number of serum samples. However, the high sensitivity of the test requires the use of antigen-specific pathogen. Otherwise, the high sensitivity of the ELISA is harmful for its diagnostic value, showing false positive results due to the presence of common antigenic determinants between pathogen and some microorganisms.

Animal brucellosis remains a major problem for veterinary of Kazakhstan. In the ELISA kits which are used recently for the serological diagnosis of infection lipopolysaccharide (LPS) molecules of Brucella are served as antigen. It is known that LPS Brucella almost similar to antigens derived from Yersinia enterocolitica 0:9, and also have homologous epitopes with many Gram-negative microorganisms to which there are antibodies in blood of animals. In addition, LPS located superficially are more accessible to immune system of the vaccinated organism, and therefore antibodies are produced mainly against determinants of the polysaccharide molecule. In this regard, there is no doubt that using LPS antigen in ELISA generates unnecessary livestock slaughter of certain healthy animals which produce antibodies as a result of immunizing subinfection or vaccination.

At the Research Institute of Biotechnology S.Seifullin KazATU (A.K.Bulashev et al.) investigation on the definition of the diagnostic value of outer membrane proteins (OMP) Brucella abortus 19 were carried out. The choice of the antigen based on following features. First, the gram-negative bacteria, although they have great similarities among themselves by LPS antigens but they significantly differ in OMP. Secondly, vaccine strains, usually delayed in immunized organism for a short time (1-3 months). Therefore, LPS, located on the cell surface are more available for the immune system of vaccinated animals than that of protein antigens "masked" by complex surface structure. In this context vaccinated body has time to develop antibodies primarily to LPS. In the body of an infected animal there is long-term persistence of Brucella, accompanied by a reaction of the immune system of the agent, who is trying to localize in their beloved organs. This leads to accumulation in tissue of a large number of decay products of Brucella including OMP which were previously not available to the immune system.

ELISA test system for the detection of Brucella specific antibodies in the serum of cattle developed by Seifullin KazATU is based on using OMP of pathogen. As the results of research, the test proved to be more specific than the well-known serological reactions. Statement of ELISA was carried out in two ways. In the indirect ELISA OMP were nonspecifically immobilized to the solid phase, and in "sandwich" version with the help of monoclonal antibodies (Mab) specific for the protein epitope with a molecular mass of 50 kDa. Comparing the diagnostic value of two methods of ELISA researchers gave preference to "sandwich" option. First, in the latter case, the protein antigen which included an epitope of monoclonal antibody is fixed to a solid phase specifically. While other protein components of Brucella OMP are removed by washing of wells. Thus, during the staging "sandwich" ELISA first antibodies carries out selection of antigens from OMP which are peculiar to Brucella. This certainly increases specificity of the test. Seifullin KazAU has the scientific and technical documentation and prototypes of ELISA kit for diagnosing Brucellosis (registered in the State Register of veterinary products for the number of RK-MR-2-0126-04 dated by 27.01.2004). All rights reserved by the patent of RK № 14230 "Method of detection of antibodies against the causative agent of brucellosis" (Bulletin № 4, published 15.04.2008). The high specificity of protein antigens of Leptospira, serogroup Hebdomadis in ELISA for diagnosis of cattle leptospirosis in comparison with other immunological methods was established by our researchers (A.K.Bulashev, 2005). Diagnosticum significantly exceeded microagglutination test and lysis test in sensitivity as well as in specificity and elicited seropositive animals more effectively than its foreign analogues "ELISA-leptospirosis-BPX" (M.A.Kuybagarov, 2006). In the literature there are reports about perspectives of OMP in diagnosis of other infectious diseases. For example, on the basis of OMP Yersinia pseudotuberculosis Polymer Agglutination Test was designed on the basis of species-specific antigen. Sensitivity of this diagnosticum was equal to 100%, specificity - 85,7-89,5% (D.I.Simakova et al, 2011).

Methods of detection of infectious agents antigens proposed by scientists of S.Seifullin KazAU are of great practical significance. Thus, diagnostic kit which is based on Mab of hybridome strains BAMA and BIMA can detect Brucella abortus in pathological material, namely in parenchymal organs and lymph nodes, as well as in stomach contents of the abortion fetuses (A.K.Bulashev, 1992). Mab of mentioned strains were used by N.A.Byzovoy et al. (2011) for the development of express chromatographic immunoassay (CIA) for the detection of Brucella abortus antigens and antibodies against the pathogen.

Tests for determining Brucella abortus antigen allow to detect LPS in biomaterial at concentrations up to 5 ng / ml within 10-15 minutes and Standard brucellosis antigen up to 1 mln.cell/ml. Serologic research results using IHT coincided with data of ELISA.
Express test based on Mab has been also tested by us for the detection of Mycobacterium tuberculosis in biological material. It is established that «dot» ELISA using antibodies to Mycobacterium bovis in its "sandwich" and in «direct» versions exceeded on sensitivity and specificity, and, of course, by the time of staging (3.5-4 hours) Classical methods of analysis (microscopic, bacteriological) and comparable with PCR. This test, in contrast to others, made it possible to differentiate Mycobacterium bovis from other species, including representatives of the atypical mycobacteria (A.K.Bulashev et al., 2010, 2011). Immunoenzyme diagnostic kits designed for serological screening of cattle for tuberculosis also deserves attention (AKBulashev, 2003). According to the results of testing all animals that showed positive results for both ELISA and allergy tests had lesions in lung and lymph nodes that are typical of tuberculosis, while in the internal organs of cows only reacted to PPD tuberculin visible changes were not observed. In connection with the unfavorable epizootic situation on anthrax, foot and mouth disease and rabies veterinary service of Kazakhstan needs effective rapid tests to monitor the health condition of the environment objects. Researchers from S.Seifullin KazAU and the National Center for Biotechnology of RK developed highly specific method for indicating B.anthracis in soil, based on the use of two types of the Mab in «dot» ELISA on nitrocellulose paper (A.K.Bulashev et al. , 2005). The sensitivity of the test was equal to 4500 m.cell/ml. In addition, it can be also used for detecting anthrax bacilli in other objects of the environment (water, food), as well as raw materials of animal origin.

Three producer strains of Mab to FMD virus and 4 hybridomas synthesizing antibodies to rabies virus were created by scientists of named academic institutions, and proposed ELISA methods for laboratory diagnosis of the mentioned viral diseases (A.K.Bulashev et al., 2005). Test system for identification of rabies virus antigen in ELISA was also developed by Kazakh Research Institute of Biological Safety (E.S.Zhilin et al., 2011). Research results have shown that in 13 of the 15 investigated field materials (brain and saliva) which were taken from cows, sheep, and foxes with clinical signs of rabies antigen of pathogenic virus is revealed. These results were confirmed by testing samples of a similar panel of field materials using a commercial ELISA kit for the diagnosis of rabies (Scientific Research Veterinary Institute, Kazan).

E.coli strain producing recombinant non-structural proteins of FMD virus 3A was obtained by National Center for Biotechnology of RK and Research Institute of Physico-Chemical Medicine (Moscow). ELISA based on mentioned antigen having high specificity detected antibodies in blood serum of cows in titer of 1:320 (K.N.Mukantaev et al., 2011).
One of the promising approach of increasing specificity of ELISA is the use of anti-idiotype antibodies (AIab), ie "Internal image" of the original antigen in this test.

In this case, once obtained Mab to a specific epitope of certain pathogen could be used for the production of monoclonal AIab which subsequently replaced the original antigen and thus eliminate necessity of working with pathogenic microorganisms. AIab can mimic any, including the weak and poorly immunogenic antigenic determinants of haptens, polysaccharides, lipids, which are often isolated in degraded form and therefore deprived of the epitopes specific to the native molecule, or preserving the high toxicity due extraction features. AIab acting as protein "substitute" of certain bacterial antigen eliminates these obstacles and effectively represent non-toxic determinants to immune system cells. That's why AIab now successfully used by many researchers as a "surrogate" antigen or specific determinants for the induction of a protective immune response against bacterial LPS, a toxin, a number of intracellular parasites - of malaria, leishmaniasis, trypanosomiasis, bacterial and viral infections, etc. Real benefit of this approach is the absence of necessity to pre-isolation of antigenic substance. Absolute specificity of Mab used to obtain AIab predetermines their homology to the original antigen.

AIab to idiotype AT2-beta of Mab directed to poly-B antigen of Brucella (S.G.Ospanova et al, 2009) were received by researchers of S.Seifullin KazAU. One of the characteristics of AT2-beta specificity of monoclonal AIab is their ability to interact with xenogenetic antisera specific for the original antigen, because once this idiotype bears an "internal image" of the antigen it can bind specific antibodies. Therefore interaction of monoclonal AIab with rabbit polyclonal monospecific polyclonal antiserum and positive serum of cattle in ELISA was studied. Mab produced by hybrid strains have been active with respect to indicated xenogeneic sera in the titers of 1:1600-1:3200. One of the important properties of the AT2-beta is their ability to induce synthesis of the third order of homologous antibodies (anti-AIab) against the original antigen. For this purpose, we conducted a series of immunizations of linear mice with Aiab preparations.

Sera of mice were investigated in indirect ELISA for antibodies of the third order, capable of interacting with the original poly-B antigen of Brucella. According to the results AIab have been able to stimulate the synthesis of antibodies of third order (Am3) against the original antigen when they were administered to animals. Thus, the researchers have shown the possibility of using AIAT as antigen in analysis of blood serum of animals for brucellosis in ELISA.
Antiidiotipii phenomenon in diagnosing blood parasite diseases (piroplasmosis, anaplasmosis, nuttaliozov, trypanosomiasis) has been also studied by GS Shabdarbayev et al. (2009). They established that AIAT to antigens of various blood parasites obtained as a result of double immunization of rabbits are quite informative, specific, and they can be used as diagnostic tools in the serological tests, as IHA and ELISA.

Immunosorbent assay using F(ab)2 fragments of anti-idiotype antibodies instead of the native antigen was proposed by BB Gnedenko et al. (2006). They determined serum autoantibodies to the three groups of proteins in normal and schizophrenic patients using standard methods and test systems with immobilized F(ab)2 fragments of AIab. The levels of antibodies to all the proteins in patients with schizophrenia were higher than in the control group.
E.S.Zhilin et al. (2011) tested a polyclonal anti-idiotypic immunoglobulins and their conjugates with horseradish peroxidase ELISA for the detection of antirabies antibodies in the sera of animals. In this case, positive results were obtained with all rabies specific sera, regardless of species of antibodies producers while normal and heterologous sera shown negative findings. The sensitivity of test system was 2-4 times higher than that of indirect and competitive ELISA.

Investigations devoted to problems of adaptation ELISA tests under practical conditions by extending the shelf life of immunoenzymatic conjugate are of great interest. In this case the possibility of using immobilized enzymes artificially associated with an insoluble carrier while maintaining their properties are studied. Immobilized enzymes are thousands times more stable than the free ones. All this ensures high efficiency and competitive technologies using immobilized preparations. One of the important factors limiting the widespread use of ELISA in practice is the short expiry date of immunoassay conjugate (6-12 months when properly stored in the refrigerator). Immobilized enzymes were obtained by A.S.Geogdzhayan et al. (2011) and used them in the development of diagnostic kits for detection of infectious agents. Technology for producing immobilized immunoperoxidase conjugate consisted of several stages: activation of horseradish peroxidase, inclusion of enzyme in liposomes (or immobilization on silica-alumina), immobilization of obtained preparations with immunoglobulins, cleaning of unrelated components, lyophilization and control. As the carrier had to choose the one that would not prevent penetration of substrate to it and did not deprive the enzyme mobility which is necessary to perform its inherent function. Immobilized material in liposomes is protected from the effects of environmental factors by lipid membrane, thereby increasing the expiration date. Optimal conditions for obtaining highly stable and specific immobilized immunoperoxidase conjugates using as carriers liposomes and aluminosilicate were determined by researchers. They proved immobilization perspectivity of carriers with enzyme and immunoglobulins. Immunoperoxidase conjugates were tested by ELISA for the homo-and heterologous strains of tularemia and leptospirosis. Storage life of developed preparations increased up to 5 years for the liposome and up to 3 years for aluminosilicate immunoperoxidase conjugates without reducing sensitivity and specificity.

Methods of diagnosing infectious diseases based on immunochromatography also deserve attention of practices. So N.A.Byzovoy et al. (2010) proposed immunochromatographic detection of Mycobacterium tuberculosis. Different variants of competitive and "sandwich" immunochromatographic analysis were developed in which contact of sample and test strips with applied immunoreagents directly initiate fluid motion on the membrane components of the test strip. In this case immunochemical interaction is found in the form of visually detectable colored zones. It is shown that competitive and "sandwich" options of immunochromatographic analysis allow to detect Mycobacterium tuberculosis at a concentration of up to 10 thousand cells/ ml (50-100 cells in the sample). The advantage of the 'sandwich' analysis is the possibility of quality visual diagnosis when staining analytical zone regardless of its intensity indicates a positive result.

Zherdev et al. (2010) developed immunoassay method for detection of antibodies to the causative agent of tuberculosis. For this purpose were used two antigenic reagent - immobilized in the analytical area of the test strip Mycobacterium tuberculosis and antigen conjugated to colloidal gold. Due to the presence in antibodies at least two antigen-binding sites during contact test strip with the sample immune complexes consisting of immobilized molecules on the membrane of antigen contained in the sample of antibodies to Mycobacterium tuberculosis antigen and conjugate agent with colloidal gold is formed in analytical area. Complexes is determined visually or using an optical detector.

ELISA, especially its «dot» variant is widely used in the diagnosis of parasitic diseases such as amebiosis, babesiosis, fascioliasis, leishmaniasis, gipodermatoz, echinococcosis and toxoplasmosis. This test has obvious advantages over other options of immunoassay. Above all, it gives possibility to carry out large number of analyzes in much smaller amounts of the sample, reagents, consumables, materials and immunoreagents. Antibodies or antigens in immunodote are applied in minimum quantities for a limited portion on polymeric sheet material with a 80-120 times greater sorption capacity than that of carriers with a smooth surface used in panel ELISA. On the other hand, «dot» ELISA does not require electric equipment, which is important during mass investigations in the field. V.G.Berezhko et al. (2009) tested «dot» ELISA in diagnostics of trichinellosis and echinococcosis of pigs. Specificity of this test at the first invasion was 97.4%. The greatest number of false-positive responses were recorded in pigs with natural invasion of E.granulosis. According to the authors, on the one hand, this result can be explained by the presence of common protein epitopes between Trichinella and Echinococci, and on the other - it could be provoked by a variety of liver diseases, in which the false positive response may be observed to various antigens of pathogens. Specificity of «dot» ELISA for echinococcosis made up 68%. The main cause of false-positive results with the sera of pigs infested by C.tenuicollis was a close antigenic relationship of this parasite with E.granulosis proven by immunochemical method.

At the last three years actual problems of veterinary science are studied by our researchers under the two Republican scientific-technical programs. From the names of research projects it follows that they are devoted to infectious immunology, namely to improvement of diagnostic methods of infectious diseases such as Bovine Leukemia, Echinococcosis, and Opisthorchiasis.
The choice of these diseases is not accidental. Analysis of the epizootic situation shows that the Bovine Leukemia recorded almost in all countries with highly developed dairy cattle. Infection caused great damage to breeding and pedigree work, reduces service life of animals and milk production, as well as milk and meat quality.

It is established that in breeding farms in Kazakhstan the share of cattle reacting positively to the leukemia comes to an average of 6%.Eradication activities are based on the early detection and isolation of infected animals from the herd. However, RID and ELISA, the most frequently used for this purpose, do not always give objective results. The first test has low specificity, and the second because of its high sensitivity may give false-positive reactions in the case of insufficiently purified antigen. In this regard, we aimed at developing ELISA test system, which allows in the course of analysis to get rid of unwanted components of virus-containing material, and thus contribute to sensitization of the solid phase with antigens specific to the pathogen of leukemia. To give the test system such selective properties, ie property to separate specific antigen from ballast substances Mab have been tested as one of the popular "tools" of immunologists.

The strains producing Mab were obtained with the help of Hybrodoma technique. Creation of Hybrodoma strains with desirable properties - is not easy job, but, as world practice shows, once obtained strains can more than recoup the costs by giving researchers a very valuable preparations - homogeneous antibodies with strict specificity.

Immunization of mice was performed with glycoprotein gp51 and polypeptide p24 antigens of Leukemia virus. Following the fusion of immune splenocytes with myeloma cells 3 strains of hybrid cells synthesizing Mab to polypeptide antigen and 6 strains of hybridomas producing antibodies to glycoprotein antigen of the virus were obtained. Mab resulting from the cultivation of Hybridoma producers in the peritoneal cavity of syngeneic mice were characterized by high activity and affinity for the original antigens. In our studies, Mab were tested as ligands for the fixation of virus-specific antigen to the solid phase in staging of "sandwich" ELISA.

At first, active centers of polystyrene wells of ELISA plate sensitized with Mab were blocked by BSA, and then virus-containing material were applied . In this case antibody binds selectively to those antigens, which in its composition have specific epitopes to them. After washing wells from unbound components samples of serum or milk were applied. In the presence of antiviral antibodies in test samples, they linked with other epitopes of the antigen, which is fixed to the solid phase by means of Mab forming an immune complex. This complex were detected by using anti bovine conjugate. As a result of numerous experiments we have determined the optimal parameters and conditions of setting ELISA for the diagnosis of Bovine Leukemia.

By its diagnostic value, our method was not inferior to commercial ELISA test systems. These results formed the basis of laboratory regulations of manufacturing components of Diagnostic "Kit ..." . Method of screening of cattle for markers leukemia virus by identifying integrated genome of the pathogen in the blood has been developed by the staff of NCB of RK (E.B.Evtyhova et al., 2011). Samples of DNA isolated from blood were tested for the presence of provirus by multiplex PCR using a mixture of primers targeting env gene and the gene fragment of actin (internal amplification control). Detection of amplifiers was carried by electrophoresis in 2% agarose gel. Researches of the same organization (K.K.Mukanov et al., 2011) developed ELISA test based on the recombinant p24 antigen for serological diagnosis of leukemia.

Among the parasitic diseases of animals, the greatest concern is the Echinococcosis because this disease causing not only economic but also social damage. People become infected by direct contact with dogs – affected by mature Helminth - Echinococcus granulosis, as well as eating vegetables and fruits contaminated with helminth eggs. At this invasion in the liver, sometimes in the lungs and other organs of intermediate hosts (domestic animals and people) multiple cysts containing larvae of the parasite are formed. Treatment of infected animals has not been developed. In rare cases, selfrecovery of animals with the formation of calcified hydatid cysts is observed. Diagnosis of disease is very difficult on the clinical manifestations. Sometimes serological reactions, based on the detection of antibodies in the serum of animals, such as skolexprecipitation test, indirect hemagglutination test, PCFT, as well as allergic method are used. However, none of them have found application in veterinary practice because of low specificity. This is understandable – in these tests complex mixture of helminth antigens are used. In short, for the eradication of Echinococcosis accurate methods of diagnostics are needed which allowing identify and discard diseased animals in good time with subsequent disposal of infected organs under the control of veterinary specialists.

In our research we used four types of antigen: contents of the hydatid bladder; protoskolexes (a product of the germinal membrane, floating freely in the cavity of the capsule), the membrane of the bladder and the excretory-secretory antigen (ES-AG), secreted by protoskolexes into the nutrient medium in the course of Modelling invasion in vitro.
As you can see from the slide, all the antigens in RID and ELISA actively interact with serum antibodies obtained from animals with specific lesions for Echinococcosis. For future study, ES-AG were selected as antigen.

Protoskolexes were obtained from echinococcal bladders of animals during slaughter. ES-AG was isolated from protoskolexes culturing in liquid nutrient medium. The antigen consisted of 11 protein fractions with molecular weights in the range of 19-63 kDa. By the method hybridoma technology strains of cells producing antibodies to the epytope of the excretory protein with a molecular mass of 21 kDa were obtained.
The principle of detection of serum antibodies specific to the parasite, was based on the use of Mab as the first antibody in "sandwich" ELISA. These antibodies selectively interact with the ES-AG and contribute to the sensitization of the solid phase with helminth metabolites. The results of testing showed that in all cases of detecting postmortem changes typical to Echinococcosis specific antibodies in serum samples are identified.

The results of laboratory and practical testing were used in the development of normative-technical documentation for the diagnostic "Kit ...". An experimental party of this Kit submited to the Ministry of Agriculture for registration in the state register veterinary medicines.
ELISA based on the use as antigens - metabolic products of helminth was also tested by us for Immunodiagnosing of the other infective diseases - Opisthorchiasis - widespread natural focal parasitic human disease caused by helminth Opisthorchis felineus.

Helminths localized in the bile ducts of the liver, gallbladder and pancreas. Infection of human and carnivores - definitive hosts of this parasite occurs by eating cyprinids infested by larvae of the helminth. The disease runs a chronic, without marked clinical signs, and in the fecal samples of patients with opisthorchiasis, helminth eggs are not always found. Proposed method is allergic, which, like other tests based on the delayed-type hypersensitivity, often gives false-results.

To extract ES-AG of Opisthorchis felineus at first Metacercariae were isolated from the superficial and deep spinal muscles of fish living in waters of Lake Tengiz and the Irtysh River. It should be noted that the degree of contamination of fish was quite high, and in certain species, it reached 26%. Then laboratory animals (dogs, rabbits and hamsters) were infected with Metacercariae. After 60 days the animals were killed, sexually mature helminth were isolated and cultured in incomplete Igla medium for the accumulation of metabolites.
By Hybridoma technology 2 strains producing Mab were obtained which showed high activity with respect to the original antigen.

In developing ELISA diagnosis we determined the possibility of using Mab as an agent fixing Opisthorchis antigen to the solid phase, as well as antibodies capable of interacting with blood serum Circulating immune complexes (CIC) consisting of molecules of specific immunoglobulin and the antigen. According to the literature, in the later stages of the disease active centers of antibodies are occupied by the parasite antigens, that is why they can not be detected by serological tests. These immune complexes can be determined by other antibodies specific to free epitopes of antigen. The proposed tests were tested on samples of blood serum of people known as opisthorchiasis patients in comparison with commercial ELISA-Best (Novosibirsk, Russia). Both variance of ELISA test systems gave very comparable results both between each other and with analogue being compared.

In connection with forthcoming accession to the WTO, examination of products of animal origin for the presence of residues of antibiotics and other biologically active substances has become one of the most important tasks of veterinary practice. The presence of contaminants cause allergic reactions in humans and other pathologies. To determine the antibiotics in animal products a number of methods are offered. Microbiological method which is widely used for this purpose, despite its reliability, has some major drawbacks. These include low sensitivity and reproducibility of results, the need for continued cultivation and storage of test strains of micorganisms. In the European Union, including Italy ELISA is the recommended method for the determination of residues of veterinary drugs in foods of animal origin. Antibody production to antibiotics is a difficult task because of their chemical nature, they are haptens and do not cause an immune response. In this regard, our research antibiotics were conjugated with high-bearers (BSA, ovalbumin). These conjugates served as an antigen in the hybridoma technique. By carrying out seven fusion of immune splenocytes with a myeloma cell line, we managed to get 3 strain-producer of antibodies to hydroxyl group of a molecule of streptomycin, and 2 strains that synthesize immunoglobulins to antigenic determinants of chloramphenicol and oxytetracycline.

The method proposed by us to determine the residual amounts of antibiotics, based on free competition, of antibiotic contained in the sample and the antibiotic immobilized on a solid phase as part of protein conjugate for the active sites of antibodies. Diagnostic value of tests for determining residues of antibiotics in livestock production has been tested on 1050 samples of milk and meat in comparison with commercial counterparts. Test results indicate the possibility of using it for rapid detection of antibiotics in food. In conclusion, I want to note that at present ABRI is working on the commercialization of diagnostic test kits.

Test questions: 1. Tell us about the main results of Agricultural Biotechnology Research Institute of S.Seifullin KazAU (ABRI) in the field of improving diagnosis of infectious diseases 2. Highlights of ABRI on improving methods of diagnosing invasive disease, 3. What explains the prospects of application of anti-idiotype antibodies in the immunodiagnosis of infectious diseases?

Lecture №4

Biotechnology of vaccines

In medicine and veterinary variety of vaccines against infectious diseases of humans and animals are widely used. Unfortunately, a number of vaccines weakly immunogenic or have side effects, or require a high production costs.
Recombinant bacterial vaccine. Immunogenic properties of pathogens are often determined by specific protein or polysaccharide molecule of the pathogen, which is encoded by a single gene. Current achievements of genetic engineering provide an opportunity to force prokaryotic or eukaryotic cells synthesize specific antigen of the pathogen, which will serve as the basis for creating genetically engineered vaccine.
The principle of a genetically engineered vaccine is that in the structure of weakened viruses, bacteria, yeast, or the cells of higher organisms the gene is incorporated that is responsible for the formation of an antigen of pathogen against which vaccine will be directed. This eliminates the need for a killed or weakened bacteria and viruses, to provide security for the workers during production of vaccines, there is no toxic or infectious material contaminating microbial antigens derived from cell cultures, and finally it improves ecological conditions. Anti-viral vaccines have become the main object of the application for genetic engineering due to simplicity of the organization of viral genomes. A more complicated structure of the bacterial cells and relatively low cost of antibacterial vaccines are factors hindering the development of genetic engineering. In the future, vectors, in which are integrated not only the genes that control the synthesis of pathogen antigens, but also genes encoding various mediators of (proteins) immune response (interferons, interleukins, etc.) will be used.
Treponema pallidum - causative agent of syphilis, was the first   bacterium which attracted attention of researchers involved in the creation of genetically engineered vaccines. And this is no accident. First, despite the fact that at the disposal of modern medicine there are effective methods of diagnosis and therapy, syphilis acquired epidemic spread in both developed and developing countries, and secondly, obtaining pure cultures of Treponema pallidum is very difficult, because it is not growing in artificial medium and, thirdly, it is impossible to get the vaccine against it by conventional methods based on the extraction and purification of antigens. Lovett and colleagues (University of California) in 1982, DNA of this spirochete cloned into E. coli cells using bacteriophage as a vector. The genetic material for the experiment was isolated from the testicles of specifically infected rabbits. They got the strain E.coli, which contained at least seven specific antigens of treponemy. These studies were designed to develop more specific diagnostic tests for syphilis and the production of effective vaccine (cited A.Sasson, 1987).
In veterinary medicine, the first genetically engineered antibacterial vaccine which found application in practice was a vaccine against colibacillosis (Escherichiosis) of pigs and calves caused by pathogenic strains of E.coli. The developer of the vaccine is Dutch veterinary pharmaceutical company "Intervet international". In order to isolate the protein in sufficient quantities for a preparation of a vaccine, they cloned the gene responsible for the synthesis of adhesion antigens of Escherichia coli K88 and K99, in a strain of E. coli K-12. These antigens in combination with an adjuvant were used to obtain the vaccine. Immunization of cows and pigs with vaccine causes the formation of protective antibodies, which are then transmitted to the newborn with colostrum and milk. Similar vaccines have been also developed by company "Cetus" together with "Norden laboratories" (USA) and "Tech America Group".
   For the construction of live genetically engineered (recombinant) vaccine three of components are required: bacterial vector - the carrier of heterologous protective antigens, gene synthesising of heterologous antigen and genetic structures, providing a stable and controlled expression of protective antigens that in its turn can induce an effective protection of the immunized organism.
Nowadays Salmonella, Escherichia, Mycobacterium, Bacillus, Listeria, yersinii, Corynebacterium and Lactobacillus frantsiselly are found applications as bacterial vectors. For today vaccine strains of S.typhimurium, S.choleraesuis, S.dublin, S. enteritidis, S. abortusovis, Mycobacterium bovis (sht.BCG), Bac.subtilis, Francisella tularensis (E.A.Svetoch et al., 2000) are most suitable to create of recombinant vaccines for veterinary medicine. Most researchers are using genetically characterized strains of Salmonella as a bacterial vector, putting forward in favor the following arguments: Salmonella can be used both orally and parenterally, stimulating local and systemic immunity, including the production of serum antibodies and mucosal secretory antibodies, cell-mediated immunity and antibody cytotoxicity. For example, to antigens, providing full protection can be attributed adhesion antigens of pathogenic Escherichia coli and heat labile enterotoxin O-and Vi-antigens of Salmonella, cholera and diphtheria toxins, pathogens’ toxins of tetanus, botulism, gas gangrene, malignant edema, capsular antigens plague and others. Especially actual for veterinary medicine is the development of combination vaccine against anthrax and brucellosis. Genetic structure in which the genes of anthrax’s lethal toxin as well as surface protein of Brucella abortus with mol. mass of 31 kD is designed by Shumilov KV et al.   Genetically engineered strains with the cloned gene pag, encoding synthesis of protective antigen - the main immunogen of anthrax were created by Russian SRI "Microbe".

Vaccines containing only the individual components of the pathogen are also called as "subunit". Subunit vaccines have their own advantages and disadvantages. Advantages are that the preparation containing the purified protein is immunogenic, stable and secure, its chemical properties are known, there are no additional proteins and nucleic acids, which could cause undesired side effects in the host organism. The disadvantages are that cleaning of a specific protein is expensive, and conformation isolated protein can be different from the one he has in situ (ie, In the composition of viral capsid or envelope), which may lead to a change in its antigenic properties. Decision on the production of subunit vaccines are made taking into account all relevant to the case of biological and economic factors. In veterinary science, some progress has been made in the development of subunit vaccines against FMD. Vaccine containing a virus inactivated with formalin is used to protect animals against this infection. World annual production of the vaccine is approximately 1 billion doses.
The main antigenic determinant inducing antibody formation, is a viral capsid protein 1 (VP1, viral protein 1). This is a weaker antigen than intact viral particles, but it induces the formation of antibodies and protect animals against infection. Therefore, attempts to clone VPl-gene were made.

Recently heat shock proteins of Mycobacterium tuberculosis, as the basis for a subunit TB vaccine have been intensively studied. Using ELISA and monoclonal antibodies to HSP65 the presence of heat shock proteins of Mycobacterium tuberculosis in sera of patients with confirmed tuberculosis and in sera of patients with suspected tuberculosis was defined (I.A.Basnakyan et al, 2010). HSP65 Mycobacterium tuberculosis was detected in cerebrospinal fluid of patients with tuberculous meningitis, and the presence of this antigen may be a diagnostic marker for tuberculous meningitis. The presence of serum antibodies to HSP70, HSP65 and HSP 16 Mycobacterium tuberculosis in tuberculosis and sarcoidosis were iInvestigated. Significantly higher levels of antibodies were found in the sera of patients with tuberculosis and sarcoidosis than in blood serum of healthy people, thus proving important role of heat shock proteins in the stimulation of immunity.

The methods for production of recombinant heat shock protein HSP70M of Mycobacterium tuberculosis, its properties and analysis on conformity to requirements of preclinical trials were studied by A.Sharapovoy et al. (2009). Szewczyk YS et al. (2009) found that recombinant HSP70M Mycobacterium tuberculosis increases the activation of innate and adaptive immunity in the cause of combined administration with bacterial antigens.
In an effort to create a safer and more effective TB subunit vaccine were studied immune protective properties of extracellular proteins of purified M. tuberculosis. From liquid bacterial cultures six major secreted proteins out of 100 were isolated and purified. Each of them separately, and then in various combinations were used for immunization of guinea pigs. Animals were administered by aerosol approximately with 200 living cells of M. tuberculosis, that is extremely high dose for them. After 9-10 weeks animals were killed and their lungs and spleen were examined for the presence of pathogenic bacteria. At the introduction of some combination of purified proteins weight loss, lung and spleen damages, mortality rate were the same as in the cause of vaccination with live BCG vaccine. Now it is necessary to compare the efficiency of protein M. tuberculosis, obtained by recombinant DNA technology with the efficiency of secretory proteins, and to develop a safe and effective vaccine for prevention of tuberculosis in humans.

Synthetic peptide vaccines. Then the next question is: can a small part of the protein molecule (domain) serve as an effective subunit vaccine and induce the production of antibodies? Intuitively, it seems that those domains that are available for the antibodies (ie, those that are on the surface of the virus), have immunogenic properties, and internal domains are negligible, unless they affect the conformation of the immunogenic domain. If this assumption is true, the short peptides that mimic epitopes (antigenic determinants) can be used to create vaccines. The idea of using synthetic peptides as vaccines was born when studying cellular and molecular mechanisms of immunity. In 1974 M.Sela first described artificially derived peptide that causes formation of antibodies to protein lysozyme. Nowadays polysaccharides analogous to natural-antigens, for example, Salmonella polysaccharides are synthesized and tested. Producing vaccines by Recombinant DNA technology opened new perspectives in the development of synthetic vaccines. Production of the latter can replace the existing bacterial and viral vaccines with extraneous antigenic determinants, proteins and other substances that cause side effects. Odibert et al. (1981) used a synthetic antigen of diphtheria toxin for active immunization. This toxin is a polypeptide chain with a molecular mass of 62 kDa and has two disulfide bonds. Toxicity and immunogenicity of the protein due to a loop on the N-terminus of the molecule, consisting of 14 amino acids that are hold the by disulfide bridge. Synthetic peptide associated with two different carriers initiates the synthesis of antibodies that bind to the toxin and prevent its dermonecrotic and lethal effect. Formation of immunity succeeded also by injection of synthetic peptide Streptococcus pyogenes M protein just 20 amino acids in length. Such immunogenic oligopeptides can be the basis of safe vaccines against streptococcal infections that cause rheumatic fever and related heart disease. At Scripps Clinic Research Institute and at the Institute of Virology of Animals (USA) polypeptides corresponding to several areas of the protein VP1 of FMDV were synthesized. In further studies, they found that one of the polypeptides including the area from 141 th to 160 th amino acid of VP1 by injection with adjuvant and in combination with keyhole hemocyanin (KLH) causes antibody synthesis in guinea pigs to the virus and rabbits. Still, there are some restrictions on the use of short peptides as vaccines: an epitope that is used to create an effective peptide vaccines should be a short but continuous portion of the protein molecule, and this does not always happen, the conformation of the peptide must be the same as that of the epitope in the intact virus particle, isolated epitope may not be sufficiently immunogenic. In the future, synthetic peptide vaccines can be highly specific, relatively inexpensive, safe, and effective alternative to traditional vaccines, although it is necessary to conduct a lot of research. Other antigens aimed at creating immunity against viral infections are in development and for example, anti-idiotype antibodies obtained in response to immunization with antiviral antibodies (Lerner et al., 1985; Dreesman et al., 1985). Antibodies against synthetic antibodies that capable of recognizing specific antigenic epitopes, in its turn retains the basic structure of these determinants. As it is known, the antigen and antibody fit together like a key and lock. In other words, the active site of antibody is like a replica of the determinants of the immunizing antigen. As a result of consecutive immunizations with original antigen and then with antibodies of first and second generations can get the third generation, essentially repeating original antibodies of first generation obtained by immunization with direct viral antigen.

The possibility of using monoclonal antibodies for anti-idiotype antibodies to the "image" of certain antigens opens new perspectives in the design of a new type of vaccine. This is particularly important against viral infection pathogens of which is difficult to obtain in sufficient quantities for the development of vaccines, as well as working with the antigenic epitopes of the conformational nature, for which it is impossible or difficult to develop chemical or genetically engineered vaccines.

Idiotype vaccines have several advantages over traditional prophylactic preparations. First of all, immunoglobulin nature of AIAT prevents reversion of live attenuated microorganism in virulent form. Imitation by CDR-area of (Complementarity determining region) AIAT certain protective epitope of antigen provides targeted induction of protective antibodies by macroorganism, eliminating antibody synthesis against a poorly immunogenic and non-immunogenic determinant of the microbe or chemical components of vaccines, which significantly affects the efficiency of immunization. Furthermore, as it is known, synthetic peptides corresponding to parts of the primary amino acid sequence, created by chemical synthesis or molecular cloning is not always able to maintain a native three-dimensional structure necessary for the induction of antibodies desired specificity and immunogenicity, which was confirmed on the model plague pathogen in contrast to AIAT, selected precisely for conformational specificity as "mirrors" of the antigenic epitope.

Prospects for creation of experimental anti-idiotype vaccine against plague studied by Fedorov VA and Devdariani (2006). They obtained the mouse AIAT to proteins Y.pestis with mol. mass 72, 54, 25 and 87 kDa encoded by the virulence plasmid of plague pathogen. For this purpose BALB/c mice were immunized with Mab to each of the antigens according to the original scheme. The complementarity to the original antigens was confirmed by using immunochemical, functional, immunological and immunobiological criteria for assessing the nature of "internal image" of the corresponding antibodies. All AIAT reacted with xenogeneic (rabbits, horses, guinea pigs and mice) sera to the original antigens, inducing high levels of specific antibodies with serological properties similar to idiotypic Mab, they showed expressed adjuvant activity and were classified as belonging to AIAT subtype AT2-beta. The most important feature was the ability of AIAT to protect from experimental plague at least 77-80% of mice. Authors suggest that AIAT are promising components in the construction of preventive preparations against plague. One of the significant advantages of anti-idiotype vaccine in comparison with attenuated or recombinant live vaccines is the possibility of unidirectional urgent application with antibacterial preparations in emergency situations of natural, technogenic or bioterrorist character.

DNA vaccines. A new approach for inducing body's immune response without antigen injection is based on inclusion a target gene encoding a protein antigen into animal cells. In the first experiments of this kind E. coli-plasmid containing the cloned gene of protein-antigen, transcription of which was under the control of the promoter of animal viruses were conjugated to gold microparticles and cells of the mouse ear were bombarded with them. It was later revealed that the cloned cDNA can be also introduced into cells by intramuscular injection of a solution with a lots of plasmid carrying the corresponding DNA. To do this it is necessary to have DNA in 103-104 times more than in bombardment of the microparticles.In one experiment, more than 75% of the genes included in mouse cells, and synthesized protein antigen-induced antibody synthesis. This approach avoids the purification of antigen, which requires a lot of time and resources or using DNA technology to create recombinant vaccines. Moreover, proteins obtained with its help are more likely subjected to proper posttranslational modification than proteins synthesized by host organisms. This method, called the Genetic immunization can be used to immunize animals.
Plasmid DNA represents circular molecule covalently closed length of 4.6 base pairs. It has a site responsible for initiating transcription (promoter), gene of protective protein, gene that provides the resistance of cells to antibiotics (ampicillin), and the site of replication of plasmid DNA. Plasmid DNA replication occurs only in bacterial cells, whereas transcription of the gene of protective protein is carried out only in mammalian cells. In the cells of E. coli plasmid DNA is replicated in the presence of ampicillin, and then is purified and injected intramuscularly to animals at a dose of 10 to 12 billion molecules. Plasmid DNA is absorbed by the cells of animals in a small amount (0.01-1.0%), and most of it is quickly destroyed. DNA penetrated into the cell is transported to the nucleus of the cell and transcribed by DNA-dependent RNA polymerase 2 with the formation of messenger RNA, which in the cytoplasm provides a synthesis of protective protein. Plasmid DNA functions in cells for a long time (up to 3-6 months.). In the body of the animal, plasmid DNA does not replicate and embedded in the chromosomes, and does not formed antibodies. Protective protein synthesized in the cells is cleaved in the cytoplasmic proteasome into short peptides (8-10 amino acids).

The last associated with the molecules of major histocompatibility complex (MHC) class 1 and are transported to the cell surface. Synthesized protective proteins can be transported out of the cell into the extracellular space in the free unsplit state. It binds to the antigen-presenting cells (macrophages), penetrates in it by endocytosis and are cleaved in endosomes into short fragments (10-20 amino acids). Fragments of proteins are combined with the molecules MHC class 2 and integrated into the surface membrane of the cell. On the cell surface antigen + MHC 2 complex is recognized by T-helper cells. B cells are transformed to antibody producing cells under the influence of protective protein and antigen-activated T-helper cells. The fate of DNA introduced into the cell is not known exactly. In principle, it can integrate into the host genome with very serious consequences, if this touched some important gene, or malignant transformation of cells takes place. However, this development is highly unlikely. Most likely such DNA will survive for a while in the cell in the form of unreplicating extrachromosomal element and then will be destroyed. Today Genetic immunization is used to build immunity against some of pathogens (influenza A virus, human immunodeficiency virus type I, bovine herpes virus, rabies virus, Plasmodium sp., caused by malaria, hepatitis B virus of animals, as well as pathogens of mycoplasma, tuberculosis and salmonellosis. In experiments on laboratory animals the possibility of using DNA vaccination to protect against AIDS and other diseases have been shown. Research on using DNA vaccines in veterinary medicine is also being carried out. The possibility of using vector plasmids for immunization against the virus of infectious bovine rhinotracheitis was established by PJLewis (1997). W.Jiang (1998) demonstrated the efficiency of DNA vaccination for parvovirus infection of dogs. Protective immunity was observed in animals vaccinated with a plasmid encoding glycoprotein of Aujeszky's disease virus (V.Gerdts et al., 1997). It is known the results on obtaining DNA vaccine against viral diarrhea of calves (S.Harpin et al., 1997). Plasmid encoding Hbs-antigen of ducks’s hepatitis B virus protected them from infection after contamination (M.Triyatni et al., 1998).

Modified strain of Shigella flexneri was created to facilitate the delivery of DNA into animal cells during genetic immunization. This bacterium enters the epithelial cells of animals by phagocytosis, and plasmid DNA which is presented in it enters the host cell cytoplasm, where transcription and translation of gene under the control of a eukaryotic promoter. Shigella is pathogen, that is why it can not be used to deliver DNA. Its non-pathogenic strain can be obtained by entering a deletion in the gene of asd, encoding the enzyme aspartate-beta-semi-aldehyde dehydrogenase, which is involved in the synthesis of cell wall components namely diaminopimelic acid. Strains with mutations in the gene asd grow only in the presence of diaminopimelic acid and can be used to deliver plasmid DNA in the epithelial cells of animals, as they are not proliferating. At present current trends in the development of recombinant vaccines is the construction of various DNA vaccines based on a single plasmid vector. In this case, only the gene encoding a protective protein changes. It should be noted that DNA vaccines have the safety of inactivated vaccines and efficacy of alive ones. Protective protein genes of several pathogens and cytokine genes - regulators of the immune response can be integrated in one plasmid DNA. Experimental studies of DNA vaccine were made from human immunodeficiency virus, influenza, rabies, hepatitis B and C, herpes simplex, warts, and tuberculosis pathogens and parasitic diseases (malaria and leishmaniasis).

Effectiveness of immunization of DNA vaccines is obvious, but it will take a lot of effort for the practical implementation of a new approach to prevention of infectious diseases of animals. However, safety issues of vaccines from plasmid DNA for human remains unsolved. The risk of mutagenic effects and immunopathological reactions in response to the DNA vaccine is also not defined. There is no clear idea of the side effects of the resulting antigens and immune response mediators.

Vector" vaccine. A live cowpox virus (CPV) belonging to the genus of Poxviruses was used as an effective smallpox vaccine. The genome of the virus completely sequenced and it is a double-stranded DNA of 187 pairs of nucleotides encoding about 200 different proteins. DNA of CPV replicates in the cytoplasm of infected cells, but not in nucleus due to the presence in virus of genes of DNA polymerase, RNA polymerase and enzymes performing capping, methylation and polyadenylation of mRNA. Therefore, if in genome of CPV integrate foreign gene so that it will be under the control of CPV -promoter, it will be expressed independently of the regulatory and enzyme systems of the host. CPV has a wide range of hosts (vertebrates and invertebrates) and it remains viable for many years after lyophilization (evaporation of water by freezing), does not possess oncogenic properties, and therefore can be used to create so-called vector vaccines. Delivery and expression of cloned genes encoding antigenic proteins that induce the production of protective antibodies in a host organism are carried out with the help of CPV. Gene of CPV is large and does not contain unique restriction sites, which allow to insert in it additional nucleotide sequence. However, the required genes can be introduced into the genome of CPV by homologous recombination in vivo as follows. DNA segment encoding a specific antigen (eg, HBcAg), inserted into a plasmid vector directly after cloned CPV-promoter which is included in some non-essential gene for CPV, for example, gene of thymidine kinase. Further, the culture of chick embryo fibroblasts deficient in thymidine kinase, is transformed by this plasmid. Previously given culture is infected with wild-type of CPV, which synthesizes a functional thymidine kinase. As a result of recombination between the nucleotide sequences flanking the promoter and the gene for protective antigen and homologous sequences of the viral genome integration of the cloned gene into viral DNA is occurred . The frequency of such recombination is low, but the population of cells containing recombinant CPV can be enriched by using selective medium with bromodeoxyuridine. This toxic analogue of thymidine in the absence of thymidine kinase is not included in synthesized DNA and has no toxic effect.

Thymidine kinase-defective host cells that contain normal CPV in the presence of bromodeoxyuridine are killed and cells carrying the recombinant CPV with a break in the gene for thymidine kinase, become resistant to its toxic effects. Finally, final selection is conducted by DNA probe which is hybridising with gene of antigenic protein. In the genome of CPV have already managed to integrate and express in the culture of animal cells several genes of antigenic proteins: G-protein of the rabies virus, hepatitis B surface antigen, surface proteins of Sindbis virus, NP-and HA proteins of influenza virus, N-and G-proteins of the virus of vesicular stomatitis, herpes simplex virus glycoproteins. CPV -vector vaccine allow to immunize from several diseases. This can be done by recombinant CPV, which carries several genes encoding different antigens.
Live recombinant virus vaccine has several advantages over non-living virus and subunit vaccines: 1) presentation of authentic antigen does not differ from that in normal infection, 2) virus can replicate in the host cell and increase the amount of antigen that activates the production of antibodies by B-lymphocytes (humoral immunity) and stimulates the production of T-cells (cellular immunity), and 3) integration of genes of antigenic proteins in a single or greater number of genome sites of CPV even greater reduces its virulence.
The lack of a live recombinant virus vaccine is that in vaccinated individuals with reduced immune status (such as AIDS) severe viral infection may occur. Gene, encoding human interleukin-2, which stimulates T-cell response and limits the proliferation of the virus, may be inserted into a viral vector to solve this issue.

Bacteria as antigen delivery system. Antigens located on the outer surface of bacterial cells are more highly immunogenic than which are localized in the cytoplasm. Therefore, one of the approaches used for the development of vaccines, is to place the protective antigen of pathogenic bacteria on the surface of living non-pathogenic bacteria. Many bacteria have flagella composed of flagellin protein, under a microscope, they look like threads extending from the bacterial cell. If do so that flagella of nonpathogenic microorganism will bear a specific epitope of a pathogen, it can induce the production of protective antibodies. This is the approach used when creating vaccines. Synthetic oligonucleotide encoding the epitope of subunits B cholera toxin was inserted into hypervariable region of Salmonella flagellin gene and the resulting structure introduced in flagellin-defective strain of Salmonella. It was known that the epitope comprising 50-64th amino acid residues of B cholera toxin, induces the production of antibodies to intact cholera toxin. Chimeric flagellin functioned normally, and cholera toxin epitope was located on the surface of flagella. Immunization of mice with intraperitoneal injection of approximately 5 mln. live or killed bacteria with modified flagellin induced the production of large amounts of antibodies to a peptide (amino acid residues 50-64) and to the molecule intact cholera toxin. Similarly it is possible to integrate two or even three different epitope in one flagellin gene of Salmonella and create antibacterial polyvalent vaccine.
With the help of oral administration with attenuated Salmonella strains it is possible to deliver to a host many of bacterial, viral and parasitic antigens. Important role here plays the choice of the promoter that controls the transcription of the foreign gene. If too strong promoter is used, it may be a metabolic "overload", restraining the proliferation of bacteria. In contrast to fermenter, the body of the host animal is not a closed system and expression of a foreign gene can not be controlled by changing temperature or addition of specific metabolites. Regulatory role can play only promoter that responds to certain signals. For example, the activity of the promoter nirB E. coli can be adjusted by varying the content of nitrite and oxygen in the environment, and it is most active in anaerobic conditions. In one experiment, the promoter nirB was used to control expression of gene of non-toxic immunogenic C fragment of tetanus toxin in attenuated strains of Salmonella. In developing countries, infection Clostridium tetani kills more than 1 million lives each year. If a genetically modified strain of Salmonella is grown under aerobic conditions, C-fragment of tetanus toxin is not synthesized. In oral administration of mice with the same bacteria C-fragment was synthesized, and animals developed antibodies to it. Thus, strain of Salmonella with gene of C-fragment of tetanus toxin which is under control of the promoter nirB can be used as a living oral tetanus vaccine. Further studies are necessary to find out how effective this approach to human vaccination.

Edible vaccines. Advances in genetic engineering have opened up new opportunities for the production of recombinant proteins. For this purpose bacterial cells, yeast, mammals and insects are widely used. However, they have several drawbacks. In the cells of prokaryotes posttranslational modification and correct folding of polypeptide chains of many eukaryotic proteins does not occur. Mammalian and insect cells are deprived of such shortcomings, but the use is limited by high production costs of recombinant proteins yield. In comparison to aforementioned plant expression systems have a number of features and benefits. First of all, it should be noted that in higher plants glycosylation and protein folding occurs, similar to those in mammalian cells. Cultivation of plants does not require expensive equipment, in contrast to animals, plant cells do not contain viruses and prions pathogenic for human and thus serve as a safe source of recombinant proteins. In addition, transfer of exogenous DNA fragments into the plant genome and the regeneration of plants is much easier as compared to animals.
Revolutionary trends in modern vaccinology is the development of vaccines based on transgenic plants in genome of which corresponding fragment of pathogenic microorganism’s genome is inserted. Transgenic plants-producers of epitopes of disease agents are called "edible vaccines". The mechanism of immunization by Vaccines is based on antigen-presenting ability of peritoneal macrophages of the small intestine of mammals. Secretory immunoglobulin IgM transported to the surface of the mucous membrane, where they bind to the foreign agent and prevent their penetration into the body.

It should be noted that the mucosal vaccine stimulates an immune response of mucous membranes (the first protective barrier to pathogenic objects) and as well as overall immune response. The first such vaccine was received in 1992: a transgenic tobacco plant became producing "Australian" antigen. Derived from plants and partially purified antigen injected into mice caused a powerful immune response similar to hepatitis B vaccine. In 1998, using potatoes , producing B-subunit of cholera toxoid severe protection of mice from cholera was obtained. In the same year, 10 of the 11 volunteers who received 100 g of raw potato, producing antigens of enterotoxigenic Escherichia coli, began to develop antibodies to this pathogen in intestinal mucosal.Potato" vaccine to the pathogen of diarrhea and hepatitis B is experiencing nowadays with promising results. Vaccines against rabies, foot and mouth disease are tested on animals. The research is conducted on the basis of transgenic potato, lettuce, corn, spinach, alfalfa, etc. Today, transgenic plants-producers of different types of antibodies to several epitopes of antigens (staphylococcus, streptococcus, herpes simplex virus, cancer embryonic antigen) are received. Transgenic plants are considered as a potential source of low-cost human immunoglobulins and animals.

Test questions: 1. Principles for creating genetically engineered vaccines 2. What organisms are used as bacterial vectors to create recombinant vaccine? 3. What vaccines are called subunit, synthetic vector and DNA?

Lecture №5

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