Цитирование (постоянный адрес по стандартам ВАКа и ГОСТа)
По стандарту ВАК Республики Беларусь: Rem PETROV, Rakhim KHAITOV, REALITY OF THE VACCINATION [Электронный ресурс]: электрон. данные. - Минск: Белорусская цифровая библиотека LIBRARY.BY, 18 ноября 2021. - Режим доступа: https://library.by/portalus/modules/medecine/readme.php?subaction=showfull&id=1637242755&archive=&start_from=&ucat=& (свободный доступ). – Дата доступа: 23.05.2022.
Rem PETROV, Rakhim KHAITOV, REALITY OF THE VACCINATION / Science in Russia, №3, 2014, C.18-25.
(Phenotypie conrrection of the genetically controlled immune response)
by Academician Rem PETROV, RAS adviser, Academician Rakhim KHAITOV, Director of the State Research Center "Institute of Immunology" of Federal Medical Biological Agency of Russian Federation
The paper comprises the long-term study and use of conjugated immunogens and next generation vaccines.
The Nobel Prize winner Karl Landsteiner showed that immunogenic characteristics of a protein are determined not by the whole molecule but rather by its small chemical groups: the antigenic determinants (Fig. 1, left). Joining of a small chemical group (e.g. phenyl) to a protein changes its immunogenicity though the chemical group itself is not antigenic. Such small chemical structures have been called "haptens". Hapten and protein conjugation results in the formation of a new very weak antigen. To potentiate this weak immunogenicity, the additional boosters such as Freund's adjuvant are needed. In other words, a protein itself as a hapten carrier is not strong enough to stimulate an immune response to the hapten.
Michael Sela introduced as an antigen "a model protein molecule"---a branched, multichain synthetic polypeptide (T,G)-A-L, consisting of four repeated amino acid residues (Fig. 1, right). This artificial peptide exhibited immunogenic properties but also very weak, so Freund's adjuvant was needed to enhance the response. This is typical for practically all highly purified and genetically engineered proteins and peptides.
In 1977 (Fig. 1, lower half) we published for the first time the results of our research on joining hapten groups, not to a protein carrier, but to non-antigenic synthetic linear polyelectrolytes: polyanion or polycation molecules. As a result, we got completely synthetic, non-natural, antigens, which caused strong
Fig. 2. Dependence of immune-stimulating activity on the degree of polycation and polyanion polymerization (n). The coefficients of AFC (antibody-forming cell) production were determined following simultaneous injection of antigen (5 mln sheep red blood cells, SRBC) with polymer fractions of polyconidines (1) and polyacrilic acid (2).
immune response without protein, lipoprotein or other common antigen carriers.
We would like to emphasize once more that the poly-electrolytes we used had no antigenic activity themselves. But hapten specificity of polyelectrolyte-based nanostructures revealed strong immune response without the need for Freund's adjuvant. The only necessary feature for a polyelectrolyte carrier was the length of the polycationic or polyanionic chain: usually not less than 500-1,000 links with molecular mass 50-100 kDa (Fig. 2). The low-molecular-mass analogues of these polymers had no effect on immune reactions.
The polyelectrolyte carrier itself had an immunostimulatory effect and the haptens or weak protein antigens joined to it provided a strong epitope-specific immune response without any additional adjuvants.
So we succeeded in constructing nanostructures not known before: strong immunogens with antigenic determinants that are suitable for creating vaccines with desired specificity.
Our investigations were based on the peculiarities of genetic control of immune response and were aimed at phenotypic correction of immunogenesis. The aim was successfully achieved due to chemical conjugation of an antigen with the above-mentioned polyelectrolyte molecules which bypassed genetic control of the immune response, i.e. a phenotypic correction of a weak immunity stipulated by low immune response genes.
Expanding their studies on mechanisms of the immune response, Rem Petrov et al. introduced a genetic approach into experimental work, including the quantitative analysis of interstrain variations in antibody production at both the intact organism and cell levels. This approach was very novel, as the previous history of immunology had provided numerous examples which were suggestive of the adaptive immunity to be independent of hereditary factors. Indeed, the acquired immunity against smallpox, plague, and other infectious diseases is not transmitted to the progeny.
In 1963 the experiments with Leptospira spirochetes demonstrated that animals of different genotypes produce different amounts of antibodies.
The table shows that 12 genetically different homozygous (inbred) strains and substrains of mice immunized with Leptospira spirochetes develop immune response of different strength. Maximal antibody titers were found in C57BL/10SnSn mice, and minimal in C3H/HeDiSn mice. The difference between high and low
Titer after immunization (M±m)
On the 7th day
On the 14th day
Agglutinin titer (log2) in mice of different inbred strains after immunization with Leptospira canicola.
Fig. 3. Hybrid analysis of inheritance of immune response strength in mice immunized with Leptospira spirochetes. X-axis: Antibody titers (log2). Y-axis: Percentage of animals, %.
responders on the 14th day after immunization was 20-fold.
The analysis of hybrids between oppositely responding inbred mouse strains demonstrated the dominant inheritance of high immune response phenotype (Fig. 3), with Mendelian segregation in the second generation. The inheritance of the "intensity of the immune response" was controlled by more than one gene pair and neither sex- nor color-linked.
Similar studies were carried out with sheep red blood cells (SRBC). A single immunization of several mouse strains revealed the oppositely responding genotypes. C57BL, a high responder to Leptospira, was a lowest responder to SRBC, but CBA was a high responder to SRBC; their antibody titers differed 10-fold. Hybrid analysis of inheritance of immune response strength in mice immunized with SRBC revealed the same tendency as with Leptospira: high immune response phenotype was inherited as dominant, neither sex- nor color-linked, and controlled by more than one gene pair.
In the context of these data, an important task was to determine whether genetic control is realized at the organismal level or at the level of cell populations that contain immunocytes. To this end, the researchers employed the technique of adoptive cell transfer into lethally irradiated recipients. Animals with adoptively transferred splenocytes from high responding or low responding mice being challenged with an antigen produce respectively high or low amount of antibodies. Fig. 4 shows the data on anti-Leptospira antibody production by 25 mln splenocytes from C57BL or C3H mice. The higher responding population accumulates greater number of antibody-forming cells (AFC). The results showed that the immunogenetic patterns of
Fig. 5. Interstrain differences of immune response level against sheep red blood cells in mice at different ages. X-axis: Mouse age (days) at the time of immunization. Y-axis: Antibody titers (log2).
Fig. 4. Production of antibody against Leptospira by adoptively transferred splenocytes (2.5x107) in mice of different strains. X-axis: Days after cell transplantation. Y-axis: Antibody titers (log2).
antibody production at the level of cell populations and at the organismal level were the same. Hence, the conclusion was drawn that genetic control of immunological reactivity is phenotypically manifested at the level of lymphoid cell populations.
Genetically determined differences in immune responses in mice against SRBC are manifested during the life course. Fig. 5 shows that 6-10-fold differences in immune response levels against SRBC in CBA and C57BL mice are established in the first 2 weeks of life, i.e. during immunological reactivity maturation period, and are kept up to old age (the last group of mice was immunized at the age of 547 days).
When other antigens were used (2,4-dinitrophenyl-conjugated bovine γ-globulin, cc-anatoxins of Cl. perfringes and Cl. oedematiens), animals of each strain produced different amounts of antibodies depending on the antigen, the very same strain could respond effectively to one antigen and weakly to another, and the differences between strains persisted throughout the animal's life and did not disappear following suppression of antibody production by irradiation (although the intensity of their immune responses was much lower than in non-irradiated mice). The variability of immune responses to different antigens was also investigated in humans and the obtained data provided evidence for the existence of genes that control the immune response to various antigens.
So the immune response is always specific, and its manifestation depends on genetically-determined properties of the responding organism and specific properties of the antigen.
Studies by Nobel laureate Baruj Benacerraf and by Hugh McDevitt and Michael Sela on peptide antigens identified specific immune response genes, called Ir genes. Ir genes are located in Major Histocompatibility Complex (MHC). MHC had been first described by Nobel laureates George Snell in mice (H-2) and by Jean Dausset in humans (HLA). The MHC is a family of tightly linked genes located on chromosome 17 in mouse and on chromosome 6 in human (Fig. 6).
Fig. 7 shows the structural formula of several synthetic polyelectrolyte carriers, polycations and polyanions: polyacrilic acid (PAC), poly-4-vinylpiridine (PVP), poly-2-methyl-5-vinylpiridine, poly-4-vinylpiridine quaternized salt, polyconidine quaternized salts.
Polyacrylic acid is a polyanion and polyvinylpiridine and its co-polymers (conidine derivatives) are polyca-
Fig. 6. Major histocompatibility complex genes. Left: H-2 on mouse chromosome 17. Right: HLA on human chromosome 6. Fig. 7. Synthetic polyelectrolytes--immunomodulators.
tions. Despite the essential distinctions in chemical nature and even in elementary unit structure of these polymeric macromolecules, their levels of immunostimulatory activity are fairly similar. Conjugation of any of them with any weak antigen increases the immune response by about 10-fold. It is important to mention that monomer analogs of these linked polymers don't manifest any immunomodulatory activity. Our data and the results of physicochemical studies carried out in collaboration with Academician Victor Kabanov prove that the mechanism of action of poly-electrolyte carriers is stipulated by their macromolecular nature and may be explained by their capacity to multipoint cooperative interactions with other complementary macromolecules or cooperative multipoint absorption onto chemically complementary surfaces.
While investigating the immunological mechanisms governing the function of synthetic polyelectrolytes, we analyzed the early changes in immune cell membranes (cyclase enzymes activity, ion transport system, lipid matrix microviscosity) and immunogenesis main stages, which finally define strength of the immune response. As a result the mechanisms of polyelectrolytes activity (T and B cell migration, cell cooperation, compensation of T helper functions, membrane proteins clusterization, followed by increase of cell membrane permeability for Ca2+, Na+, K+ ions and Ca2+- and Na+/K+-ATPases production) have been discovered.
Based on the results regarding the main properties of polyelectrolytes and the mechanisms of cellular responses to them, we propose that the chemical joining of antigen and polyelectrolyte molecules acts to focus the activity of the polymer on the cells interacting with antigen. That will allow to generate nanostructures with heightened immunogenicity. Besides
Fig. 8. Dynamics of AFC in the spleen of mice immunized with BSA. On the figure: BSA (1), unstable (2) and stable (3) BSA-electrolyte complexes and BSA-polyelectrolyte conjugate (4). BSA--bovine serum albumin.
the above described synthetic antigen hapten-polyelectrolyte, there is another example: immunogens obtained by joining the various antigen molecules to polyelectrolyte carriers.
Conjugation of polyelectrolyte and antigen makes it possible to focus the immunostimulatory activity of the polyelectrolyte on antigen-recognizing cells. Covalent conjugates of antigen-polyelectrolyte introduced into animal or human organisms induce antigen-specific immune responses much more strongly than antigen by itself or the non-covalent antigen+polyelectrolyte complexes.
Our studies show that conjugated irnmunogens can be made using protein and polysaccharide antigens, as well as synthetic peptides that imitate natural antigenic determinants. The conjugates with model and natural microbial antigens were studied. By covalent binding to electrolytes highly immunogenic conjugates containing salmonella O and H antigens, hemagglutinin, neuraminidase, NP and M proteins of influenza viruses A and B, brucella glycoprotein antigen, tuberculin purified protein component (PPD) and many other were created. In all cases, antigen and electrolyte binding significantly (by an order of magnitude) increased the intensity of immune reaction against the antigen (Fig. 8), even in genetically low-reacting individuals.
The peculiarities of immune reactions developing against conjugated immunogens were examined in detail. We identified increases in production of IgM, IgGl, Ig2a, Ig2b, IgG3, IgA, but not IgE antibody isotypes. After only a single injection of the conjugate, immunological memory was established. The immune response following the second injection of the conjugate was 10- to 100-fold higher than the primary response. In the lymph nodes proximal to the injection site, antigen-specific T helpers and cytolytic T cells capable of killing the target cells infected with appropriate virus were activated.
It is important to emphasize that the relative T-(T cell) and IR - (Ir genes) independence is one of the most significant peculiarities of immune responses against conjugated immunogens. Under the polyelectrolyte influence, the production of antibodies against T-dependent antigens was achieved even in T cell-deficient animals. A full-strength immune response against the antigen-polyelectrolyte conjugate developed even in mice with Ir genes encoding for only a weak immune response against the antigen (Fig. 9). Due to their T- and IR-independence the conjugated immunogens are extremely valuable in practice because they can induce an effective immune response even when there is T-cell deficiency or genetically determined weak reactivity to the particular antigen. Conventional vaccines are not effective in such cases. Phenotypic correction of immune response was proven in experiments using different strains of mice undergoing combined immunization or vaccination using conjugates of various antigens with polyelectrolytes.
In the experiments involving infections with various pathogens, the conjugated immunogens proved capable of protecting against lethal doses of the infectious agents. Conjugated immunogens, containing influenza virus hemagglutinin and neuraminidase, induced high-level protection in mice challenged with a lethal dose (10 LD50) of pathogenic influenza virus (Fig. 10).
Fig. 9. Phenotypic correction of immune response in mice immunized with [(T,G)-A-L]-polyelectrolyte conjugate.
Fig. 10. Immunogenicity and protective properties of polyelectrolyte conjugates with influenza virus surface antigens. Fourteen days prior to infection with a lethal dose of influenza virus, mice were injected with normal saline (control), hemagglutinin, hemagglutinin-polyelectrolyte conjugate, hemagglutinin-neuraminidase-polyelectrolyte conjugate.
Previously it had been thought that immune reactions specific to M protein play no significant role in defense against influenza infection. In an experimental model, we found that conjugates containing influenza virus M protein had protected mice against pathogenic viral infection.
The study of protective properties of conjugated immunogens based on Salmonella typhimurium O-specific polysaccharide and H-flagellar antigens also showed them to be highly effective vaccines. In a mouse model of typhoid fewer---Salmonella typhimurium infection---after challenge with a lethal dose (20 LD50) the protective efficacy of O-polysaccharide antigen conjugated with polyelectrolyte was 20- to 40-fold greater than of pure antigen. The use of salmonella polysaccharide O antigen as well as composition of O+H antigens conjugated with polyelectrolyte also protected mice against challenge with a significantly higher lethal dose (100 LD50) of virulent Salmonella typhimurium strain.
A special project directed at creating a synthetic polymeric immunostimulant that would meet the requirements for pharmacological preparations was carried out. Such pharmacological preparations must be safe and the organism should be able to metabolize and excrete them. We therefore needed to retain the polymer's capacity to promote multi-point cooperative interactions which provide polyelectrolyte stimulating activity. In 1990, Arkadiy Nekrasov et al. synthesized a biodegradable polymer that met all the above-mentioned requirements. It was a copolymer of N-oxide-1.4-ethylene piperazine and N-carboxiethyl-1.4-ethylene piperazinium bromide, recently named "Polyoxidonium" (PO). PO is the first innovative pharmaceutical product composed of a new class of heterochain aliphatic amines. See its chemical formula and molecular mass on Fig. 11.
PO was patented in 1990. Its degree of polymerization corresponds to a polymer length necessary for a
Fig. 11. Polyoxidonium chemical formula.
key event: protein clustering in immune cell membranes. The variety of repeated dipole NO groups provides enhanced ability to undergo multi-point cooperative interactions. Carboxyethyl groups introduce the additional absorbing properties and, most importantly, serve as universal sites for chemical binding to any antigen.
PO is degraded in the organism by biological systems utilizing N-oxides of tertiary polyamines. It is well known that oxidation of tertiary polyamines via N-oxide formation is the main way of metabolizing nitrogenous compounds in nature. The mechanisms responsible for PO destruction were studied and the kinetic and thermodynamic constants of the process were determined. It was found that PO was destroyed as a result of N-C bond break that is typical for aliphatic N-oxides. The pharmacokinetics of 3H and 14C labeled PO were studied in experimental animals in detail. PO was shown to be effectively eliminated from the organism; half-distribution and half-elimination periods, retention time, clearance and other important pharmacological indices were determined.
The immunostimulatory properties of PO were compared to those of previously studied synthetic polyelectrolytes. Phagocyte functions were significantly activated by PO and antibody production against foreign antigens was increased.
Covalent binding of PO with protein and polysaccharide antigens enhanced the immunogenicity of the conjugated antigens. Using PO as a carrier increased the immunogenicity of antigens isolated from the pathogens of influenza, brucellosis, typhoid fever, hepatitis A, tuberculosis and other bacterial and viral infections.
The development of conjugated vaccines to the above-listed pathogens has progressed gradually. The trivalent polymer-subunit conjugated vaccine against influenza has been developed and introduced into clinical practice; clinical trials of a brucellosis vaccine have been completed; preclinical trials of a typhoid fever vaccine have been completed; preclinical trials of a tuberculosis vaccine are in progress.
The successful creation of an influenza polymersubunit vaccine and its introduction into clinical practice provided a good foundation for the development of other vaccines and is worth describing in detail. We created a vaccine containing the purified proteins hemagglutinin and neuraminidase isolated from three influenza viruses type A (H1N1 and H3N2) and B, conjugated with PO. Today, the vaccine is well known as Grippol. Grippol has been manufactured since 1997 by Russian top-rated industrial enterprises producing high-quality immunobiological preparations and pharmaceuticals (Immunogen, Petrovax).
It has been proven that the immunostimulatory and membrane-active effects of polyelectrolytes critically depend on the degree of polymerization and on repeating ionogenic and dipole groups, i.e. are determined by "polymerity" itself. Thus, synthetic molecules biological activities of which are based on their effective physical and chemical interactions with cells have been introduced into medical practice for the very first time in the world. Before, only chemically neutral polymers such as polyvinylpyrrolidone were used in medicine as construction materials or passive carriers of low-molecular-mass pharmaceuticals.
Comparison of our results to the results of the other researchers allows us to claim that the presented work has no analogies and may be referred to as an important novel direction among new principles and technologies for vaccine creation.