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Technology for the preparation of genetically engineered vaccines. Genetically engineered vaccines

Recombinant technology has made a breakthrough in the creation of fundamentally new vaccines. Creation principle genetically engineered vaccines consists in the fact that a gene encoding the formation of a protective antigen of the pathogen against which the vaccine will be directed is inserted into the genome of living attenuated viruses, bacteria, yeast or eukaryotic cells.

Modified microorganisms themselves are used as vaccines. or a protective antigen formed during their cultivation in vitro. In the first case, the immune response is directed not only against the products of the integrated gene, but also against the vector carrier.

An example of a recombinant vaccine consisting of a ready-made antigen is the hepatitis B vaccine, and an example of vector vaccines whose antigens are produced in vivo is the rabies vaccine. It is derived from the vaccinia vaccine and is widely used in the prevention of rabies among wild animals using bait containing this vaccine.

To create vector live virus vaccines, an attenuated DNA-containing virus is used, into the genome of which the necessary pre-cloned gene is inserted. The virus, the carrier of the vector, actively multiplies, and the product of the integrated gene ensures the formation of immunity. The vector may contain several built-in genes responsible for the expression of corresponding foreign antigens. Experimental vector vaccines based on the vaccinia virus have been obtained for chickenpox, influenza A, hepatitis A and B, malaria, and herpes simplex. Unfortunately, vaccines have been tested primarily on animals, which are resistant to most of these infections.

The recombinant product does not always have the same structure as the natural antigen. The immunogenicity of such a product may be reduced. Natural viral antigens in eukaryotic cells undergo glycosylation, which increases the immunogenicity of such antigens. In bacteria, glycosylation is absent or occurs differently than in the cells of higher eukaryotes. In lower eukaryotes (fungi), post-translational processes occupy a middle position.

Developer genetically engineered vaccine must provide data on the stability of the antigen expression system during storage of the working cell bank. If there are changes in the seed culture, which may be accompanied by rearrangement, division or insertion of nucleotides, it is necessary to determine the nucleotide sequence, study peptide maps and the sequence of terminal amino acids of the genetically engineered product. The use of restriction enzyme mapping in combination with the study of markers encoded by the vector (susceptibility to antibiotics, etc.) can indicate the appearance of changes in the structure of the vector.

The principles for creating bacterial recombinant vaccines are similar. An important step is gene cloning and obtaining mutant genes encoding immunogenic, but not toxic forms of the antigen. Genes have been cloned for diphtheria and tetanus toxins, Pseudomonas aeruginosa toxin, anthrax, cholera, pertussis, and shigellosis toxins. Attempts are being made to obtain recombinant vaccines against gonorrhea and meningococcal infection.

BCG, Vibrio cholerae, Escherichia coli, Salmonella tythimurium are used as a bacterial vector carrier. The intestinal group of pathogens is promising for the development of enteral vaccines. Live recombinant vaccines administered orally have a short lifespan, but are capable of inducing lasting immunity during this period. It is possible to create multicomponent vaccines for simultaneous prevention against several diarrheal infections. Bacterial vector vaccines, unlike viral ones, can be controlled with antibiotics. Oral vaccines against hepatitis B and malaria have been experimentally tested.

In the future, it is planned to use vectors that contain not only genes that control the synthesis of protective antigens, but also genes encoding various mediators of the immune response. Recombinant BCG strains have been obtained that secrete interferon, interleukins, and granupocyte-stimulating factor. Preliminary studies indicate that the strains are highly effective against tuberculosis and bladder cancer. It is quite difficult to obtain an effective vector vaccine based on bacteria due to the instability of transfection of the genetic material, the toxicity of the foreign antigen to bacteria, and the small amount of expressed antigen.

RGIV - new products in the prevention of infectious diseases. An example of such a vaccine is the hepatitis B vaccine. Armed with genetic engineering methods, medical biologists have direct access to the genome. It is now possible to insert genes, delete them, or duplicate them. For example, a gene from one organism can be inserted into the genome of another. Such transfer of genetic information is possible even across “the evolutionary distance separating humans and bacteria.” The DNA molecule can be cut into individual fragments using specific enzymes and these fragments can be introduced into other cells. It has become possible to incorporate genes from other organisms into bacterial cells, including genes responsible for protein synthesis. In this way, in modern conditions, a significant amount of interferon, insulin and other biological products is obtained. A vaccine against hepatitis B was obtained in a similar way - the gene of the hepatitis virus is built into the yeast cell.

Like anything new, especially a genetically engineered drug intended for parenteral administration (again, in large quantities and three hours after the birth of a child!), this vaccine requires long-term observations - that is, we are talking about the same “ large-scale trials... on children." From numerous publications it follows: "Observations become more accurate and valuable if they are carried out during mass immunization campaigns. In such campaigns, large numbers of children are vaccinated within a short period of time. The appearance during this period of a group of certain pathological syndromes indicates, as a rule, their causal connection with vaccination.” The concept of a certain pathological syndrome can include short-term fever and cough, as well as complete or partial paralysis or mental retardation.

In addition to the Engerix vaccine against hepatitis B, the South Korean anti-hepatitis vaccine, which is being actively imposed on our country, is declared to be “just as safe and effective.” Genetically engineered vaccines are a “preventive” treatment with many unknowns. Our country is not able to verify the safety of these products due to the lack of appropriate experimental facilities. We can neither qualitatively control the purchased vaccines nor create conditions for the preparation of safe our own vaccines. Testing recombinant drugs is a high-tech experiment that requires enormous costs. Alas, in this regard we are very far from the level of advanced laboratories in the world and are practically completely unfocused on the control of such products. In this regard, in Russia (and Ukraine) everything is registered that has not passed clinical trials with foreign manufacturers of these vaccines, or has passed tests, but in an insufficient volume... Hence the avalanche-like number of vaccines from various well-wishers, “trying to help Russia” and bringing us not tomorrow’s or today’s technologies, but those of the day before yesterday - “essentially, waste from their modern production, or those vaccines that need to be studied in “large-scale experiments on children.” More often this is called “large-scale observations”, but the task is one - experiments on our children!

IT would seem senseless and immoral to prove the danger of mercury salts for infants, when the consequences of their effects on the body of an adult are widely known.

Let us remember that mercury salts are more dangerous than mercury itself. However, the domestic DTP vaccine, containing 100 µg/ml of merthiolate (organomercury salt) and 500 µg/ml of formalin (the strongest mutagen and allergen), has been used for about 40 years. The allergenic properties of formalin include: Quincke's edema, urticaria, rhinopathy (chronic runny nose), asthmatic bronchitis, bronchial asthma, allergic gastritis, cholecystitis, colitis, erythema and skin cracks, etc. All this has been noted by pediatricians for more than 40 years, but the statistics are hidden behind iron doors from the general public. Thousands of children have been suffering for decades, but medical officials don’t care.

There is no data on the effect of mertiodyat and formalin; NO ONE HAS NEVER STUDYED THIS CONGLOMERATE on young animals in terms of immediate reactions and long-term consequences; let's say for teenagers. Firms WARN, therefore, do not bear any responsibility for the actions of our vaccinators and controllers! Thus, in our country, many years of “large-scale trials” continue on our children with the development of various pathological syndromes. Every day, more and more innocent babies (those who escaped abortion) are thrown into this hellish meat grinder, joining the ranks of disabled children and their unfortunate parents, unaware of the true cause of their children’s suffering. A carefully prepared and carried out “campaign to intimidate the population” with epidemics of diphtheria, tuberculosis, and influenza on the one hand and prohibitive measures against kindergartens and schools leave no chance for parents.

WE CANNOT ALLOW ONLY FIRMS AND LOW-COMPETENT VACCINators TO CORPORATELY DECIDE THE FATE OF OUR CHILDREN.

Since BCG vaccination for newborns is not carried out anywhere else in the world, the activities carried out in Russia and Ukraine are an experiment, because “they are assessing the effectiveness of combined immunization of newborns against hepatitis B and against tuberculosis against the backdrop of mass immunization.” Unacceptable stress on the body of newborns! This experiment, “large-scale vaccination for the detection of pathological syndromes,” is being carried out on a state scale, which provided an unlimited number of its own children for such observations... without informing the parents about it! In addition, “pathological syndromes” can appear a year later, or five years, or much later... There is evidence that this vaccine can cause cirrhosis of the liver after 15-20 years.

What components are included in ENGERIX (vaccine against hepatitis B)?

1. The basis of the drug is “modified” baker’s yeast, “widely used in the production of bread and beer.” The word “genetically modified” is clearly missing here, apparently due to the fact that this combination has already fairly frightened the population with the example of soybeans, potatoes, and corn imported from abroad. A genetically modified product combines the properties of its constituent ingredients, which, when used, lead to unpredictable consequences. What did genetic engineers hide in a yeast cell besides the hepatitis B virus? You can add the gene of the AIDS virus or the gene of any cancer disease there.

2. Aluminum hydroxide. It should be emphasized here that for many decades it has not been recommended (!) to use this adjuvant for vaccinating children.

3. Thiomerosal is a merthiolate (organomercury salt), the harmful effects of which on the central nervous system have been known for a long time, and is classified as a pesticide.

4. Polysorbent (not deciphered).

Vaccination can be characterized in different ways: genocide, extermination of a population, a large-scale experiment on living children, manipulation of mass consciousness. In any case, a healthy look through the looking glass shows that health and vaccines are incompatible things.

RGIV – new products in the prevention of infectious diseases. An example of such a vaccine is the hepatitis B vaccine. Armed with genetic engineering methods, medical biologists have direct access to the genome. It is now possible to insert genes, delete them, or duplicate them.

For example, a gene from one organism can be inserted into the genome of another. Such transfer of genetic information is possible even across “the evolutionary distance separating humans and bacteria.” The DNA molecule can be cut into individual fragments using specific enzymes and these fragments can be introduced into other cells.

It has become possible to incorporate genes from other organisms into bacterial cells, including genes responsible for protein synthesis. In this way, in modern conditions, a significant amount of interferon, insulin and other biological products is obtained. A vaccine against hepatitis B was obtained in a similar way - the gene of the hepatitis virus is built into the yeast cell.

Like everything new, especially a genetically engineered drug intended for parenteral administration (again, in large quantities and three hours after the birth of a child!), this vaccine requires long-term observations - that is, we are talking about the same “ large-scale trials... on children."

From numerous publications it follows: “Observations become more accurate and valuable if they are carried out during mass immunization campaigns. In such campaigns, large numbers of children are vaccinated within a short period of time. The appearance of a group of certain pathological syndromes during this period indicates, as a rule, their causal connection with vaccination.” The concept of a certain pathological syndrome can include short-term fever and cough, as well as complete or partial paralysis or mental retardation.

In addition to the Engerix vaccine against hepatitis B, the South Korean anti-hepatitis vaccine, which is being actively imposed on our country, is declared to be “just as safe and effective.” Genetically engineered vaccines are a “preventive” treatment with many unknowns. Our country is not able to verify the safety of these products due to the lack of appropriate experimental facilities. We can neither qualitatively control the purchased vaccines nor create conditions for the preparation of safe our own vaccines. Testing recombinant drugs is a high-tech experiment that requires enormous costs. Alas, in this regard we are very far from the level of advanced laboratories in the world and are practically completely unfocused on the control of such products. In this regard, in Russia (and Ukraine) everything is registered that has not passed clinical trials with foreign manufacturers of these vaccines, or has passed tests, but in an insufficient volume... Hence the avalanche-like number of vaccines from various well-wishers, “trying to help Russia” and bringing us not tomorrow’s or today’s technologies, but those of the day before yesterday – “essentially, waste from their modern production, or those vaccines that need to be studied in “large-scale experiments on children.” More often this is called “large-scale observations”, but the task is one – experiments on our children!

IT would seem senseless and immoral to prove the danger of mercury salts for infants, when the consequences of their effects on the body of an adult are widely known.

WE CANNOT ALLOW ONLY FIRMS AND LOW-COMPETENT VACCINators TO CORPORATELY DECIDE THE FATE OF OUR CHILDREN.

What components are included in ENGERIX (vaccine against hepatitis B)?

2. Aluminum hydroxide. It should be emphasized here that for many decades it has not been recommended (!) to use this adjuvant for vaccinating children.

3. Thiomerosal is a merthiolate (organomercury salt), the harmful effects of which on the central nervous system have long been known and belongs to the category of pesticides.

4. Polysorbent (not deciphered).

Genetically engineered vaccines contain pathogen antigens obtained using genetic engineering methods and include only highly immunogenic components that contribute to the formation of protective immunity.

There are several options for creating genetically engineered vaccines:

Introduction of virulence genes into avirulent or weakly virulent microorganisms.
Introduction of virulence genes into unrelated microorganisms with subsequent isolation of Ag and its use as an immunogen.
Artificial removal of virulence genes and the use of modified organisms in the form of corpuscular vaccines.

Immunobiotechnology is based on the antigen (AG)-antibody (AT) reaction. IN

An example of an immunobiotechnological gene process is the production of polio virus from a tissue culture of a living person

to receive the vaccine. Bioproducts (vaccines) must undergo rigorous testing for safety and effectiveness. This stage of vaccine testing typically takes up about two-thirds (2/3) of the cost of the vaccine.

Let's take a closer look at vaccines.

Vaccines are preparations made from killed or weakened pathogens or their toxins. As is known, vaccines

used for the purpose of prevention or treatment. The introduction of vaccines causes an immune reaction, which is followed by the acquisition of resistance of the human or animal body to pathogenic microorganisms.

If we consider the composition of the vaccine, they include:

The active ingredient, representing specific antigens,

A preservative that extends the shelf life of the vaccine

Stabilizer, which determines the stability of the vaccine during storage,

A polymer carrier that increases the immunogenicity of an antigen (AG).

Under immunogenicity understand the property of an antigen to cause an immune response

In the role antigen can be used:

1. live weakened microorganisms

2. non-living, killed microbial cells or viral particles

3. antigenic structures extracted from a microorganism

4. waste products of microorganisms, which use toxins as secondary metabolites.

Classification of vaccines according to the nature of the specific antigen:

Non-living

Combined.

Let's take a closer look at each of them.

Live vaccines are received

a) from natural strains of microorganisms with weakened virulence for humans, but containing a full set of antigens (an example is the smallpox virus).

b) from artificial weakened strains.

c) some vaccines are obtained by genetic engineering. To obtain such vaccines, a strain carrying a gene for a foreign antigen is used, for example, a smallpox virus with an integrated hepatitis B antigen.

2. Non-live vaccines are:

a) molecular and chemical vaccines. In this case, molecular vaccines are constructed on the basis of a specific antigen, which is in molecular form. These vaccines can also be obtained by chemical synthesis or biosynthesis. Examples of molecular vaccines are toxoids. Anatoxins are bacterial exotoxins that have lost their toxicity as a result of prolonged exposure to formalin, but retain their antigenic properties. This diphtheria toxin, tetanus toxin, butulinic toxin.

b) corpuscular vaccines, which are obtained from a whole microbial cell that is inactivated by temperature, ultraviolet irradiation or chemical methods, for example, alcohol.

3. Combined vaccines. They are combined from individual vaccines,

turning into polyvaccines that are capable of immunizing

from several infections at once. An example is the DTP polyvaccine containing diphtheria and tetanus toxoids and pertussis corpuscular antigens. This vaccine is known to be widely used in pediatric practice.

Let's take a closer look toxins from the point of view of them as products of the vital activity of microorganisms.

1 group of toxins is exotoxins:

Exotoxins are protein substances released by bacterial cells into the external environment. They largely determine the pathogenicity of microorganisms. Exotoxins have two centers in their structure. One of

They fix a toxin molecule on the corresponding cellular receptor, the second - a toxic fragment - penetrates into the cell, where it blocks vital metabolic reactions. Exotoxins can be heat labile or heat stable. It is known that under the influence of formaldehyde they lose their toxicity, but retain their immunogenic properties - such toxins are called toxoids.

Group 2 toxins are endotoxins.

Endotoxins are structural components of bacteria, representing lipopolysaccharides of the cell wall of gram-negative bacteria. Endotoxins are less toxic and are destroyed when heated to 60-80 0 C for 20 minutes. Endotoxins are released from the bacterial cell during its decomposition. When introduced into the body, endotoxins trigger an immune response. The serum is obtained by immunizing animals with pure endotoxin. However, endotoxins are a relatively weak immunogen and serum may not have high antitoxic activity.

Getting vaccines

1. live vaccines

1.1.live bacterial vaccines. This type of vaccine is the easiest to obtain. Pure weakened cultures are grown in the fermenter.

There are 4 main stages in obtaining live bacterial vaccines:

Growing

Stabilization

Standardization

Freeze drying.

In these cases, producer strains are grown on a liquid nutrient medium in a fermenter with a capacity of up to 1-2 m3.

1.2. live viral vaccines. In this case, vaccines are obtained by cultivating the strain in a chicken embryo or in animal cell cultures.

2. molecular vaccines. To have an idea about this type of vaccine, you need to know that in this case, a specific antigen or exotoxins are isolated from the microbial mass. They are purified and concentrated. The toxins are then neutralized and toxoids. It is very important that a specific antigen can also be obtained by chemical or biochemical synthesis.

3. corpuscular vaccines. They can be obtained from microbial cells that are pre-cultured in a fermenter. The microbial cells are then inactivated by temperature, or ultraviolet irradiation (UV), or chemicals (phenols or alcohol).

Serums

Application of serums

1. Serums are widely used in cases of prevention and treatment

infectious diseases.

2. Serums are also used for poisoning by microbial or animal poisons - for tetanus, botulism, diphtheria (to inactivate exotoxins), serums are also used for the venom of cobra, viper, etc.

3. Sera can also be used for diagnostic purposes, to create various diagnostic kits (for example, in pregnancy tests). In this case, antibodies are used in reactions that form complexes with antigens (antigen (AG) - antibody (AT), when the presence of the corresponding antigens is confirmed, which can be used in various reactions.

The preventive or therapeutic effect of serums is based on the antibodies (AT) contained in the serum.

For mass production of serum, donkeys and horses are vaccinated. Introduction

such serum gives the formation of passive immunity, that is, the body

receives ready-made antibodies. Sera that are obtained by immunizing animals must be monitored according to such indicators as antibody titer in animals to take blood from them during the period of maximum antibody content. Blood plasma is isolated from the blood of animals, then fibrin is removed from the plasma and serum is obtained. This is one way to obtain whey.

Another way to obtain serum is from cultured animal cells.

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 is a recombinant vaccine against viral hepatitis B, a vaccine against rotavirus infection. Finally, there are positive results from using the so-called. vector vaccines, when the carrier - a live recombinant vaccinia virus (vector) - is coated with the surface proteins of two viruses: glycoprotein D of the herpes simplex virus and hemagglutinin of the influenza A virus. Unlimited replication of the vector occurs and an adequate immune response develops against viral infection of both types.

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 dangerous diseases such as meningitis or pneumonia (hemophilus influenza, pneumococci) have antigens that are difficult to recognize by the immature immune system of 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. The cowpox virus is used to create recombinant vector vaccines, in particular against HIV infection. Similar studies are carried out with weakened bacteria, in particular salmonella, as carriers of hepatitis B virus particles.

Currently, vector vaccines have not been widely used.