Modern approaches to the creation of subunit, synthetic, vector and DNA vaccines
The principle of creating genetically engineered vaccines is that a gene is built into the structure of weakened viruses, bacteria, yeast or cells of higher organisms, which is responsible for the formation of the antigen of the pathogen against which the vaccine will be directed. At the same time, there is no need to use killed or weakened bacteria and viruses, the safety of workers in vaccine manufacturing enterprises is ensured, there is no toxic or infectious material that often pollutes the microbial antigen obtained from cell cultures, and the ecological situation improves. The main object of the application for genetic engineering has become antiviral vaccines, which is explained by the simplicity of the organization of the viral genomes. The more complex structure of bacterial cells and the relatively low cost of antibiotic vaccines are factors that inhibit the development of genetically engineered work.
To construct living genetic engineering (recombinant) vaccines, three components are necessary: a bacterial vector, a carrier of heterologous protective antigens, genes for heterologous antigen synthesis, and genetic structures that provide stable and controlled expression of protective antigens that can in turn induce effective protection of the immunized organism. As bacterial vectors, Salmonella, Escherichia, Mycobacteria, Bacillus, Listeria, Yersinia, Corynebacterium lactobacilli and Franciscella are used. When creating recombinant vaccines for veterinary medicine, vaccine strains of S. typhimurium, S. choleraesuis, S.dublin, S.teritidis, S. abortusovis, Mycobacterium bovis (pc. BCG), Bac.subtilis, Francisella tularensis (E A.Svetochko et al., 2000).
Vaccines containing only certain components of a pathogenic microorganism are also called "subunit". Subunit vaccines have their advantages and disadvantages. Advantages are that the preparation containing the purified immunogenic protein is stable and safe, its chemical properties are known, there are no additional proteins and nucleic acids that could cause undesirable side effects in the host organism. The disadvantages are that the purification of a specific protein is expensive, and the conformation of the isolated protein may differ from that which it has in situ (i.e., in the composition of the viral capsid or envelope), which can lead to a change in its antigenic properties. The decision to produce a subunit vaccine is made taking into account all relevant biological and economic factors.
Synthetic peptide vaccines. The idea of using synthetic peptides as vaccines was born in the study of cellular and molecular mechanisms of development of immunity. In 1974, M. Sela first described the artificially produced peptide, which causes the formation of antibodies to the lysozyme protein. Synthesized and tested polysaccharides, similar to natural antigens, for example, salmonella polysaccharides. The technique of recombinant DNA for the production of vaccines has opened new prospects in the development of synthetic vaccines. Production of the latter can replace existing bacterial and viral vaccines, often containing extraneous antigenic determinants, proteins and other substances that cause side effects. Odibert et al. (1981) used synthetic diphtheria toxin antigen for active immunization. This toxin is a polypeptide chain with a molecular weight of 62 kD and has two disulfide bonds. The toxicity and immunogenicity of the protein is caused by a loop at the N-terminus of a molecule consisting of 14 amino acids held by a disulfide bridge. A synthetic peptide associated with two different carriers, initiated the synthesis of antibodies that bind to the toxin and prevent its dermonecrotic and lethal effects. Formation of immunity was also achieved by injecting a synthetic peptide of the protein M Streptococcus pyogenes with a length of only 20 amino acids. Such immunogenic oligopeptides can form the basis of safe vaccines against streptococcal infections that cause rheumatic fever and related heart damage. The creation of vaccines against foot and mouth disease based on synthetic polypeptides could eliminate the problems caused by insufficient inactivation of the virus and instability of inactivated vaccines at pH below 7 and unfavorable temperature conditions of the environment. This approach would allow the use of synthetic peptides corresponding to different serotypes of the virus as immunogens. These results are very promising, but the amount (dose) of peptide material necessary to induce an immune response is about 1000 times higher than in the case of a killed vaccine. To solve this problem, a DNA fragment encoding a peptide from amino acid residues 142-160 of VP1 was ligated to the gene encoding the hepatitis B core protein (HBcAg). When this chimeric gene was expressed in E. coli or in the culture of animal cells, its products, protein molecules, formed stable "27nm particles" in the process of self-assembly, on the surface of which there was a peptide from VP1 of foot-and-mouth disease. These particles possessed high immunogenicity. Thus, HBcAg can be used as an effective molecule carrier of synthetic peptides. A comparison of the immunogenicity of various peptide vaccines containing the domain of 142-160 VP1 protein in guinea pigs showed that the immunogenicity of a chimeric protein consisting of HBcAg and said domain is 10 times lower than that of inactivated viruses, 35 times higher than in a chimeric protein containing E. coli -galactosidase and domain 137-162 of the VP1 foot-and-mouth disease virus, and 500 times higher than the free synthetic peptide consisting of amino acid residues 142-160. Since the synthetic peptide crosslinked with HBcAg forms 27nm particles similar to the hepatitis B virus and they have almost the same immunogenicity as the intact virus on the basis of which the synthetic peptide is obtained, this approach can become the main way to deliver peptide vaccines to the site their actions. Still, there are several limitations on the use of short peptides as vaccines: the epitope used to create an effective peptide vaccine should be a short but continuous region of the protein molecule, and this is not always the case; the conformation of the peptide should be the same as that of the epitope in the intact viral particle; an isolated epitope may not have sufficient immunogenicity. In the future, synthetic peptide vaccines can become a highly specific, relatively inexpensive, safe and effective alternative to traditional vaccines, although more research is needed to achieve this.
DNA vaccine. Currently, the actual direction in the development of genetically engineered vaccines is the design of various DNA vaccines based on a single plasmid vector. In this case, only the gene that codes for the protective protein is changed. It should be noted that DNA vaccines have the safety of inactivated vaccines and the efficacy of live vaccines. In one plasmid DNA, it is possible to incorporate the genes of protective proteins of several pathogens and the cytokine-regulator gene of the immune response. An experimental study of DNA vaccines made from human immunodeficiency viruses, influenza, rabies, hepatitis B and C, herpes simplex, papilloma, and pathogens of tuberculosis and parasitic diseases (malaria and leishmaniasis) is under way. The effectiveness of immunization with DNA vaccines is obvious, but much effort will still be required to implement a new approach to the prevention of infectious animal diseases. However, the safety problems for human vaccines from plasmid DNA remain unresolved. The degree of risk of mutagenic effects and immunopathological responses in response to the introduction of a DNA vaccine has not been determined. There are no clear ideas about the side effects of the formed antigens and mediators of the immune response.
Vector vaccines. As an effective living anti-acute vaccine, the vaccinia virus (VKO), which belongs to the genus Poxviruses, is widely used. The genome of this virus is completely sequenced; it is a double-stranded DNA with a length of 187 tp, coding for about 200 different proteins. The DNA of the EKO replicates in the cytoplasm of infected cells, and not in the nucleus, due to the presence of DNA polymerase, RNA polymerase and enzymes that carry out capping, methylation and polyadenylation of mRNA in the virus. Therefore, if a foreign gene is built into the genome of the EKO, so that it is under the control of the VKPromotora, it will be expressed independently of the regulatory and enzyme systems of the host.
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