Genetically engineered vaccines are a new “meat grinder” for children. Vaccines obtained using genetic engineering methods Methodology for creating genetically engineered vaccines
Genetically engineered vaccines- these are drugs obtained using biotechnology, which essentially comes down to genetic recombination.
Genetic engineering vaccines were developed in the 70s of the twentieth century, since the need for such developments was due to the insufficiency of natural sources of raw materials and the inability to multiply the virus in classical objects.
The principle of creating genetically engineered vaccines consists of the following stages: isolating antigen genes, integrating them into simple biological objects - yeast, bacteria - and obtaining the necessary product during the cultivation process.
Genes encoding protective proteins can be cloned from DNA-containing viruses directly, and from RNA-containing viruses after reverse transcription of their genome. In 1982, an experimental vaccine against the hepatitis B virus was first produced in the United States.
A new approach to creating viral vaccines is the introduction of genes responsible for the synthesis of viral proteins into the genome of another virus. Thus, recombinant viruses are created that provide combined immunity.
Synthetic and semi-synthetic vaccines are obtained through large-scale production of chemical vaccines purified from ballast substances. The main components of such vaccines are an antigen and a polymer carrier - an additive that increases the activity of the antigen. Polyelectrolytes are used as a carrier - PVP, dextran, with which the antigen is mixed. Also, based on the composition of antigens, a distinction is made between mono-vaccines (for example, cholera) - against one disease, divaccine (against typhoid) - for the treatment of 2 infections; associated vaccines - DTP - against whooping cough, diphtheria and tetanus.
Polyvalent vaccines against one infection, but contain several serological types of the causative agent of the disease, for example, a vaccine for immunization against leptospirosis; combination vaccines, that is, the administration of several vaccines simultaneously in
First, a gene is obtained that must be integrated into the recipient's genome. Small genes can be obtained by chemical synthesis. To do this, the number and sequence of amino acids in the protein molecule of the substance are deciphered, then from these data the order of nucleotides in the gene is determined, followed by chemical synthesis of the gene.
Large structures that are quite difficult to synthesize are obtained by isolation (cloning), targeted elimination of these genetic formations using restriction enzymes.
The target gene obtained by one of the methods is fused with enzymes to another gene, which is used as a vector for inserting the hybrid gene into the cell. Plasmids, bacteriophages, human and animal viruses can serve as vectors. The expressed gene is inserted into a bacterial or animal cell, which begins to synthesize a previously unusual substance encoded by the expressed gene.
E. coli, B. subtilis, pseudomonads, yeast, viruses are most often used as recipients of the expressed gene; some strains are able to switch to the synthesis of a foreign substance up to 50% of their synthetic capabilities - these strains are called superproducers.
Sometimes an adjuvant is added to genetically engineered vaccines.
Examples of such vaccines are the vaccine against hepatitis B (Engerix), syphilis, cholera, brucellosis, influenza, and rabies.
There are certain difficulties in development and application:
For a long time, genetically engineered drugs were treated with caution.
Significant amounts of money are spent on developing technology to produce a vaccine.
When obtaining drugs using this method, the question arises about the identity of the resulting material with a natural substance.
Vaccination helps the recipient develop immunity to pathogenic microorganisms and thereby protects him from infection. In response to oral or parenteral administration of the vaccine, the host's body produces antibodies to the pathogenic microorganism, which during subsequent infection lead to its inactivation (neutralization or death), block its proliferation and prevent the development of the disease.
The effect of vaccination was discovered more than 200 years ago - in 1796 - by the doctor Edward Jenner. He proved experimentally that a person who has had cowpox is not very serious illness cattle becomes immune to smallpox. Smallpox is a highly contagious disease with a high mortality rate; even if the patient does not die, he often develops various deformities, mental disorders and blindness. Jenner publicly inoculated an 8-year-old boy, James Phipps, with cowpox, using exudate from a pustule of a cowpox patient, and then after a certain time twice infected the child with pus from a pustule of a smallpox patient. All manifestations of the disease were limited to redness at the injection site, which disappeared after a few days. Vaccines of this type are called Genera vaccines. However, this method of vaccination was not received great development. This is explained by the fact that in nature it is not always possible to find a low-pathogenic analogue pathogen, suitable for vaccine preparation.
The vaccination method proposed by Pasteur turned out to be more promising. Pasteur vaccines are received based on killed (inactivated) pathogenic microorganisms or living, but not virulent ( attenuated) strains. To do this, the wild-type strain is grown in culture, purified, and then inactivated (killed) or weakened (attenuated) so that it produces an immune response that is sufficiently effective against the normal virulent strain.
For immunoprophylaxis of some diseases, such as tetanus or diphtheria, the presence of the bacteria themselves in the vaccine is not necessary. The fact is that main reason These diseases are caused by pathogenic toxins released by these bacteria. Scientists have discovered that these toxins are inactivated by formaldehyde and can then be safely used in vaccines. When the immune system encounters a vaccine containing a safe toxoid, it produces antibodies to fight the real toxin. These vaccines are called toxoids.
Previously such infectious diseases, like tuberculosis, smallpox, cholera, typhoid fever, bubonic plague and polio, were a real scourge for humanity. With the advent of vaccines, antibiotics and the introduction of preventive measures, these epidemic diseases were brought under control. Unfortunately, vaccines against many human and animal diseases still do not exist or are ineffective. Today, more than 2 billion people worldwide suffer from diseases that could be prevented by vaccination. Vaccines can also be useful in preventing “new” diseases that are constantly emerging (for example, AIDS).
Despite significant advances in the creation of vaccines against diseases such as rubella, diphtheria, whooping cough, tetanus and polio, the production and use of classic “Pasteur” vaccines faces a number of limitations.
1. Not all pathogenic microorganisms can be cultivated, so vaccines have not been created for many diseases.
2. To obtain animal and human viruses, an expensive animal cell culture is required.
3. The titer of animal and human viruses in culture and the rate of their reproduction are often very low, which increases the cost of vaccine production.
4. Strict precautions must be taken when producing vaccines from highly pathogenic microorganisms to prevent infection of personnel.
5. If the manufacturing process is disrupted, some batches of the vaccine may contain live or insufficiently weakened virulent microorganisms, which can lead to the unintentional spread of infection.
6. Attenuated strains can revert (restore their virulence), so it is necessary to constantly monitor their virulence.
7. Some diseases (such as AIDS) cannot be prevented by traditional vaccines.
8. Majority modern vaccines have a limited shelf life and remain active only at low temperatures, making their use difficult in developing countries.
In the last decade, with the development of recombinant DNA technology, it has become possible to create a new generation of vaccines that do not have the disadvantages of traditional vaccines. Basic approaches to creating new types of vaccines based on methods genetic engineering are as follows:
1. Modification of the genome of a pathogenic microorganism. Work in this area is carried out in two main directions:
A) A pathogenic microorganism is modified by deleting (removing) from its genome the genes responsible for virulence (genes encoding the synthesis of bacterial toxins). The ability to induce an immune response is retained. Such a microorganism can be safely used as a live vaccine, since growing in a pure culture eliminates the possibility of spontaneous restoration of the deleted gene.
An example of this approach is the recently developed cholera vaccine based on a recombinant strain V.cholerae, in which the nucleotide sequence encoding the synthesis has been removed enterotoxin, responsible for the pathogenic effect. Currently being carried out clinical trials The effectiveness of this form as an anti-cholera vaccine has not yet given a clear result. The vaccine provides almost 90% protection against cholera, but some subjects experienced side effects, so it needs further improvement.
B) Another way to obtain non-pathogenic strains suitable for creating live vaccines based on them is to remove from the genome of pathogenic bacteria the chromosomal regions responsible for some independent vital functions. important functions(metabolic processes), for example the synthesis of certain nitrogenous bases or vitamins. In this case, it is better to delete at least two such areas, since the likelihood of their simultaneous restoration is very small. It is assumed that a strain with a double deletion will have limited proliferative capacity (limited lifespan in the immunized organism) and reduced pathogenicity, but will ensure the development of an immune response. A vaccine against salmonellosis and leishmaniasis has now been created and is undergoing clinical trials using a similar approach.
2. Use of non-pathogenic microorganisms with built-in cell wall specific immunogenic proteins. Using genetic engineering methods, they create living non-pathogenic systems for transferring individual antigenic sites (epitopes) or entire immunogenic proteins of unrelated pathogenic organism
. One of the approaches used to create such vaccines is to place the protein - antigen of a pathogenic bacterium on the surface of a living non-pathogenic bacterium, since in this case it has a higher immunogenicity than when it is localized in the cytoplasm. Many bacteria have flagella made of the protein flagellin; Under a microscope, they look like threads extending from a bacterial cell. If you make the flagella of a non-pathogenic microorganism carry a specific epitope (protein molecule) of the pathogenic microorganism, then it will be possible to induce the production of protective antibodies. A vaccine created on the basis of such recombinant non-pathogenic microorganisms will contribute to the development of a pronounced immune response to the pathogenic microorganism.
This is exactly the approach used to create cholera and tetanus vaccines. If some pathogenic microorganisms do not grow in culture, then it is not possible to create a classical Pasteur vaccine on their basis. However, it is possible to isolate, clone and express in an alternative non-pathogenic host (e.g. E. coli or mammalian cell lines) genes responsible for the production of certain antigenic proteins, and then isolate and use these proteins after purification as “subunit” vaccines.
Subunit vaccines have their advantages and disadvantages. The advantages are that the drug, containing only purified immunogenic protein, is stable and safe, its chemical properties are known, it does not contain additional proteins and nucleic acids, which could cause unwanted side effects in the host. Disadvantages are that purification of a specific protein is expensive and the isolated protein may have a different conformation than it has in situ(i.e., as part 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. Currently in different stages Vaccines against herpes, foot-and-mouth disease and tuberculosis are in development and clinical trials.
4. Creation of “vector vaccines”. These vaccines are fundamentally different from other types of vaccines in that immunogenic proteins are not introduced ready-made into the immunized organism with vaccine components (microorganism cells and products of their destruction), but are synthesized directly in it, due to the expression of the genes encoding them, which in turn are then transferred to the immunized organism using special vectors. The most widely used “vector vaccines” are based on the cowpox virus (VSV), as well as a number of other opportunistic or low pathogenic viruses (adenovirus, poliovirus, chickenpox). VKO has been studied quite well, its genome has been completely sequenced. VKO DNA replicates in the cytoplasm of infected cells, and not in the nucleus, due to the presence of the virus genes for DNA polymerase, RNA polymerase and enzymes that carry out capping, methylation and polyadenylation of mRNA. Therefore, if a foreign gene is inserted into the VKO genome so that it is under the control of the VKO promoter, then it will be expressed independently of the host’s regulatory and enzymatic systems.
East Kazakhstan region has wide range hosts (vertebrates and invertebrates), remains viable for many years after lyophilization (evaporation of water by freezing) and does not have oncogenic properties, and therefore is very convenient for creating vector vaccines.
Vector VKO vaccines allow immunization against several diseases at once. To do this, you can use recombinant VKO, which carries several genes encoding different antigens.
Depending on the VKO promoter used, the foreign protein can be synthesized in the early or late phase of the infectious cycle, and its amount is determined by the strength of the promoter. When several foreign genes are inserted into one VKO DNA, each of them is placed under the control of a separate VKO promoter to prevent homologous recombination between different parts of the viral DNA, which can lead to the loss of the inserted genes.
A live recombinant vector vaccine has a number of advantages over non-live viral and subunit vaccines:
1) the formation and activity of the authentic antigen is practically no different from that during a normal infection;
2) the virus can replicate in the host cell and increase the amount of antigen, which activates the production of antibodies by B cells (humoral immunity) and stimulates the production of T cells ( cellular immunity);
3) the integration of several genes of antigenic proteins into the genome of VKO further reduces its virulence.
The disadvantage of a live recombinant viral vaccine is that when vaccinating individuals with reduced immune status(for example, people with AIDS) they may develop a severe viral infection. To solve this problem, a gene encoding human interleukin-2 can be inserted into the viral vector, which stimulates the T-cell response and limits the proliferation of the virus.
Undesirable side effects of VKO proliferation can be prevented by inactivating the virus after vaccination. For this purpose, a virus sensitive to interferon was created (wild-type VKO is relatively resistant to its action), the proliferation of which can be regulated in the event of complications arising during vaccination.
A vector based on live attenuated poliovirus (its research is just beginning) is attractive because it allows for oral vaccination. Such “mucus” vaccines (vaccines whose components bind to receptors located in the lungs or gastrointestinal tract) are suitable for the prevention of the most various diseases: cholera, typhoid fever, flu, pneumonia, mononucleosis, rabies, AIDS, Lyme disease. But before any clinical trials of any seemingly harmless virus as a delivery system and expression of the corresponding gene, it is necessary to ensure that it is truly safe. For example, the commonly used VKO causes complications in humans with a frequency of approximately 3.0-10 -6. Therefore, it is desirable to remove sequences responsible for virulence from the genome of a recombinant virus that is intended to be used for human vaccination.
Vaccines for animals have less stringent requirements, so the first vaccines obtained using recombinant DNA technology were vaccines against foot-and-mouth disease, rabies, dysentery and piglet diarrhea. Other vaccines for animals are being created, and recombinant vaccines intended for humans will soon appear.
One more promising direction in the creation of new generation vaccines is the use of specially created transgenic plants. If genes encoding the synthesis of immunogenic proteins or individual antigenic epitopes of various pathogenic microorganisms are inserted into the genome of these plant viruses, then the plants will begin to express them. After eating such plants, the corresponding antibodies (so-called mucosal antibodies) will be produced in the mucous membrane of the human stomach and intestines. In bananas, for example, it was possible to express the Vibrio cholerae antigen and hepatitis B virus antigens, and such vaccines are already undergoing clinical trials. Glutamic acid decarboxylase antigens are expressed in potatoes and have an antidiabetic effect in animal experiments. It is assumed that such “banana vaccines” in the near future can seriously compete with both traditional and genetically engineered vaccines.
No. 43 Genetic engineering vaccines. Principles of obtaining, application.
Genetically engineered vaccines are drugs obtained using biotechnology, which essentially comes down to genetic recombination.
First, a gene is obtained that must be integrated into the recipient's genome. Small genes can be obtained by chemical synthesis. To do this, the number and sequence of amino acids in the protein molecule of the substance are deciphered, then from these data the order of nucleotides in the gene is determined, followed by chemical synthesis of the gene.
Large structures that are quite difficult to synthesize are obtained by isolation (cloning), targeted elimination of these genetic formations using restriction enzymes.
The target gene obtained by one of the methods is fused with enzymes to another gene, which is used as a vector for inserting the hybrid gene into the cell. Plasmids, bacteriophages, human and animal viruses can serve as vectors. The expressed gene is integrated into a bacterial or animal cell, which begins to synthesize a previously unusual substance encoded by the expressed gene.
E. coli, B. subtilis, pseudomonads, yeast, viruses are most often used as recipients of the expressed gene; some strains are able to switch to the synthesis of a foreign substance up to 50% of their synthetic capabilities - these strains are called superproducers.
Sometimes an adjuvant is added to genetically engineered vaccines.
Examples of such vaccines are the vaccine against hepatitis B (Engerix), syphilis, cholera, brucellosis, influenza, and rabies.
There are certain difficulties in development and application:
- long time genetically engineered drugs were treated with caution.
- significant funds are spent on developing technology for obtaining a vaccine
- when obtaining drugs using this method, the question arises about the identity of the resulting material with a natural substance.
Associated and combined vaccine preparations. Advantages. Vaccine therapy.
Associated vaccines are preparations that include several different antigens and allow immunization against several infections simultaneously. If the drug contains homogeneous antigens, then such an associated vaccine is called a polyvaccine. If the associated drug consists of dissimilar antigens, then it is advisable to call it a combined vaccine.
Combination immunization is also possible, when several vaccines are simultaneously administered to different parts of the body, for example, against smallpox (cutaneously) and plague (subcutaneously).
An example of a polyvaccine is a live polio vaccine containing attenuated strains of polio virus types I, II, III. An example of a combination vaccine is DPT, which includes inactivated corpuscular pertussis vaccine, diphtheria and tetanus toxoid.
Combined vaccines are used in difficult anti-epidemic situations. Their action is based on the ability of the immune system to respond to several antigens simultaneously.
The essence of the method: the genes of a virulent microorganism responsible for the synthesis of protective antigens are inserted into the genome of a harmless microorganism, which, when cultivated, produces and accumulates the corresponding antigen. An example would be recombinant vaccine against viral hepatitis B, vaccine against rotavirus infection. Finally, there are positive results use of the so-called vector vaccines, when the surface proteins of two viruses are applied to the carrier - a live recombinant vaccinia virus (vector): glycoprotein D of the virus herpes simplex and hemagglutinin of influenza A virus. Unlimited replication of the vector occurs and an adequate immune response develops against both types of viral infection.
Recombinant vaccines - These vaccines use recombinant technology to produce the vaccine by inserting the genetic material of a microorganism into the yeast cells that produce the antigen. After cultivating the yeast, the desired antigen is isolated from it, purified, and a vaccine is prepared. An example of such vaccines is the hepatitis B vaccine (Euvax B).
Ribosomal vaccines
To obtain this type of vaccine, ribosomes found in every cell are used. Ribosomes are organelles that produce protein using a matrix - mRNA. The isolated ribosomes with the matrix in their pure form represent the vaccine. An example is bronchial and dysentery vaccines (for example, IRS - 19, Broncho-munal, Ribomunil).
Another issue that should be kept in mind in any mass immunization program is the relationship between vaccine safety and effectiveness. In childhood immunization programs against infectious diseases, there is a conflict between the interest of the individual (the vaccine must be safe and effective) and the interest of society (the vaccine must induce sufficient protective immunity). Unfortunately, today, in most cases, the higher the frequency of vaccination complications, the higher its effectiveness.
The use of new technologies has made it possible to create second generation vaccines.
Let's take a closer look at some of them:
Conjugated
Some bacteria that cause such dangerous diseases, like meningitis or pneumonia (hemophilus influenza, pneumococci), have antigens that are difficult to recognize by immature immune system newborns and infants. Conjugate vaccines use the principle of binding such antigens with proteins or toxoids of another type of microorganism that is well recognized by the child’s immune system. Protective immunity is developed against conjugated antigens.
Using the example of vaccines against Hemophilus influenzae (Hib-b), effectiveness in reducing the incidence of Hib meningitis in children under 5 years of age in the United States for the period from 1989 to 1994 was shown. from 35 to 5 cases.
Subunit vaccines
Subunit vaccines consist of antigen fragments that can provide an adequate immune response. These vaccines can be presented either as microbial particles or obtained in laboratory conditions using genetic engineering technology.
Examples of subunit vaccines that use fragments of microorganisms are the Streptococcus pneumoniae vaccine and the meningococcus type A vaccine.
Recombinant subunit vaccines (for example, against hepatitis B) are produced by introducing part of the genetic material of the hepatitis B virus into baker's yeast cells. As a result of viral gene expression, antigenic material is produced, which is then purified and bound to an adjuvant. The result is an effective and safe vaccine.
Recombinant vector vaccines
A vector, or carrier, is a weakened virus or bacteria into which genetic material from another microorganism that is causally significant for the development of a disease to which it is necessary to create protective immunity can be inserted. Vaccinia virus is used to create recombinant vector vaccines, in particular against HIV infection. Similar studies are being conducted with weakened bacteria, in particular salmonella, as carriers of hepatitis B virus particles.
Currently, vector vaccines have not been widely used.
In the 70s our century, the successes of genetic cell engineering have made it possible to develop new technology obtaining antiviral vaccines, called genetically engineered vaccines. The need for such developments was dictated for the following reasons: 1) lack of natural sources of raw materials/suitable animals; 2) the inability to reproduce the virus in classical objects/tissue culture, etc. The principle of creating genetically engineered vaccines includes: a) isolation of natural antigen genes or their active fragments; b) integration of these genes into simple biological objects - bacteria, yeast; c) obtaining the necessary product during the cultivation of a biological object - an antigen producer. Virus genomes are negligibly small in size compared to the genome of a cell (prokaryotic or eukaryotic). Genes encoding protective proteins can be cloned directly from DNA-containing viruses, or from RNA-containing viruses after reverse transcription of their genome (for viruses with a continuous genome) or even individual genes (for viruses with a fragmented genome). At the first stage of the development of new biotechnology, scientists were primarily engaged in cloning viral genes encoding the synthesis of proteins carrying the main antigenic determinants. Soon, recombinant bacterial plasmids carrying the genes or genomes of hepatitis B, influenza, and polymyolitis viruses were obtained. The next step was to obtain the antigen. The question turned out to be difficult, because the expression of viral genes in the prokaryotic system was negligible. This can be explained by the fact that viruses, in the course of evolution, have adapted to parasitize the human body. However, over time, antigen expressions were obtained. And one of the most typical examples showing the need to create genetically engineered vaccines is hepatitis B. The problem is that cell or animal cultures sensitive to the virus have not yet been found. Therefore, the development of a genetic engineering method for producing vaccines has become a necessity. The method is that the genome is cloned in E. coli cells using plasmid and phage vectors. Bacteria carrying recombinant plasmids produce proteins that specifically react with antibodies against the virus itself. In 1982, the first experimental vaccine against hepatitis B was produced in the USA. For the production of virus-specific proteins (antigens), eukaryotic cells(yeast, animals). Work is intensively underway to create other genetically engineered vaccines, in particular against influenza, herpes, foot-and-mouth disease, tick-borne encephalitis and others viral infections. The newest approach in the creation of viral vaccines is the inclusion of genes responsible for the synthesis of viral proteins into the genome of another virus. In this way, recombinant viruses are created that provide combined immunity.