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WO2012070974A1 - Vaccin à immunogénéité élevée et procédés de leur fabrication - Google Patents

Vaccin à immunogénéité élevée et procédés de leur fabrication Download PDF

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Publication number
WO2012070974A1
WO2012070974A1 PCT/RU2010/000700 RU2010000700W WO2012070974A1 WO 2012070974 A1 WO2012070974 A1 WO 2012070974A1 RU 2010000700 W RU2010000700 W RU 2010000700W WO 2012070974 A1 WO2012070974 A1 WO 2012070974A1
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Prior art keywords
vaccines
increased immunogenicity
vaccine antigen
immunogenicity according
microbial
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English (en)
Russian (ru)
Inventor
Борис Славинович ФАРБЕР
Софья Борисовна ФАРБЕР
Артур Викторович МАРТЫНОВ
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Priority to PCT/RU2010/000700 priority Critical patent/WO2012070974A1/fr
Priority to EA201300488A priority patent/EA025417B1/ru
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

  • the invention relates to veterinary medicine and, in particular, to vaccinology and pharmacy and is intended for the prevention and treatment of infectious and other diseases of humans and animals where vaccination is used.
  • vaccination is one of the main methods of preventing epidemics.
  • the first group of infections includes conservative microorganisms and viruses whose antigenic composition is unchanged and the vaccine induces high levels of protective antibodies in the blood. These are infections such as diphtheria, pertussis, measles, rubella, etc.
  • influenza virus is a polymorphic virus (a virus particle does not have a clear structure and shape) with a fragmented variable genome.
  • Influenza virus is very variable and capable of persistence (lifetime in the human body) [ 5 ]. In humans and animals (including birds [ 6 ]), this virus multiplies in several stages - in the acute productive phase, an infected cell releases virus particles that can infect neighboring cells [ 7 ]. In the persistence phase (latent phase), this virus “waits” inside the cell, while losing part of the fragmented genome or capturing pieces
  • a change in the approach to vaccine design should be accompanied by the inclusion of such antigens in the vaccines that have not yet appeared as a result of virus mutations [ 10 ].
  • the so-called predictive inclusion of antigens is possible in two ways: the classical one using the methods of epidemiological prediction of antigenic drift and by partially modifying the antigens to obtain an unlimited number of antigen combinations in one antigen ampoule [ p ].
  • the first direction justified itself only partially: in no case did the prognosis of antigenic drift coincide with real mutational changes in neuraminidase and influenza hemagglutinin [ 12 , 13 ].
  • the antibodies induced by this protein will block all possible combinations of attachment sites. Accordingly, the number of induced monoclones will be an order of magnitude higher, although the protein will remain the same. Moreover, any “future” epitope of the structure of neuraminidase will be blocked by already synthesized antibodies.
  • Non-parenteral vaccination methods are based on the ability of antigens to penetrate the mucous membranes the shell is mainly the intestinal tract.
  • oral vaccines ensures the continuity of the antigenic stimulus, which is a prerequisite for maintaining a high level of collective immunity against infections controlled by specific prophylaxis.
  • this method of immunization is the simplest, physiologically adequate and psychologically attractive.
  • a known method [ 15 ] of increasing immunogenicity by expressing a cholera toxin antigen in a plant chimeric protein A known method of increasing the immunogenicity of hepatitis B virus nucleoproteins by covalent binding of the amino groups of this protein to microbial haptens. Such conjugation made it possible to increase the immunogenicity of the vaccine and induce the titer of antibodies to a level of 1: 128 (when vaccinating animals with a native protein, the titer of antibodies did not exceed 1: 32).
  • This technology cannot be used to increase the immunogenicity of other vaccines (microbial and viral), because it is designed exclusively for the protein that is induced in the cell nucleus by hepatitis B.
  • the oligan peptide was removed from the cell wall of microorganisms of the genus Nocardia, and 30-50% by weight of the peptidoglycan was acylated.
  • the use of this method made it possible to increase the immunogenicity of the microbial antigen by 25-30% against a standard injection vaccine.
  • Specific antibody titers grew to 1: 256-1: 512 two weeks after vaccination.
  • Obtaining pure peptidoglycan is an important procedure, since it is associated with multi-stage purification.
  • the objective of the invention is to develop vaccines with increased immunogenicity and effectiveness, including when administered orally, and a method for their preparation.
  • the problem is solved by developing vaccines with increased immunogenicity and a method for their preparation, characterized in that the vaccine antigen cut into oligomeric fragments, a polysaccharide, a protein, a lipopolysaccharide or a whole antigen, is used as a bacterium, a virus, a mixture of bacteria or virus proteins, and the resulting mixture (ensemble) of oligomeric fragments is modified by reversing the charge by acylation with succinic anhydride or by alkylation with monochlorax tartaric acid; Also, as a specific immunogenic component, a vaccine antigen with a partially reversed molecular charge is used, the charge change of which is carried out by acylation or alkylation to form a mixture (ensemble) of vaccine antigens with different molecular charges.
  • Supramolecular ensemble or ensemble is a term from supramolecular chemistry.
  • the objects of supramolecular chemistry are supramolecular ensembles built spontaneously from complementary, that is, having geometrical and chemical correspondence of fragments, similar to spontaneous assembly of complex spatial structures in a living cell [,]
  • This technology can be used to create other vaccines: for the prevention of infections such as influenza, hepatitis, herpes viruses, measles, rubella, HIV / AIDS, animal viral infections: Newasle disease, infectious bursal disease of the bird, classical swine fever, African swine fever and any other diseases. Due to the partial modification of the structure during the modification reaction, a huge number of various vaccine antigen derivatives with different immunogenicity and structure are formed and, accordingly, the immune system induces the synthesis of a larger number of monoclones in response to these new antigenic determinants. In addition, such a variety of new epitopes (hundreds of thousands or even millions) allows us to predictively protect the body from future nonexistent strains of the flu and mutant HIV / AIDS viruses. Brief Description of the Drawings
  • FIG. 1 - The relationship between the titer of specific antibodies induced and the degree of acylation of the corpuscular Pseudomonas antigen
  • Figure 2 The relationship between the titer of inducible specific antibodies and the degree of acylation of soluble high molecular weight Pseudomonas antigen The best embodiment of the invention
  • Example 1 Obtaining Pseudomonas vaccine based on a composition of vaccine antigens with a modified charge of molecules
  • Pseudomonas aeruginosa was cultured on solid nutrient medium (BCH with the addition of 1% glucose). After three days, the surface of the nutrient medium was completely covered with a pseudomonas aeruginosa. Washings were made from the surface of the medium in a Petri dish with a 0.9% sodium chloride solution, and the resulting suspension was washed three times and centrifuged. After re-suspension, the suspension of microorganisms was heated in a thermostat for 140 minutes at 80 0 C, and then again sown on PMA in order to control inactivation.
  • the resulting suspension corresponded to 10 billion cells / ml; then 0.1 ml of the suspension was diluted 100 times with 0.9% sodium chloride solution and the concentration of surface proteins was established using the Biuret method and in the complexation reaction with the bromophenol blue Flores method [3].
  • the acylation reaction of succinic anhydride was carried out as shown in example 1 [6].
  • Exceeding 15% completely deprives immunogenicity of succinylated proteins; therefore, derivatives with degrees of modification greater than 15% were not considered advisable to receive and use in the future.
  • Corpuscular antigen with varying degrees of acylation was further used to establish its immunogenicity.
  • the other part of the antigen was centrifuged for 40 minutes at 3 thousand rpm. The precipitate was discarded, and the supernatant was passed through a Sephadex G-75 column. The first, heaviest fraction was collected and used further to establish the protein concentration and degree of chemical modification.
  • the resulting antigen was a homogeneous fraction (one polymer substance) and had a molecular weight of 1.5 mDa and a charge of 186000. Received a soluble glycoprotein antigen with such degrees of acylation: 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%.
  • the molecular mass M of the experimental proteid was determined by comparing the volume of Ve elution with a similar parameter of marker proteins.
  • Anti-Pseudomonas serum for diagnostic purposes with a titer of specific anti-Pseudomonas antibodies (1: 1000) was obtained according to the standard scheme for immunizing mice, according to the scheme [1] of a thermally inactivated corpuscular Pseudomonas vaccine with a particle concentration of 10 billion / ml, which was administered at 3; 5 and 7 days at a dose of 0.2 ml intramuscularly.
  • 20 mice were used.
  • acylated samples of both corpuscular antigen (10 animals per group per sample, 8 groups) and acylated soluble antigen (8 samples, 10 animals per sample) were tested.
  • the first group of animals was injected with 8 samples of antigen (10 animals per oral sample of 0.2 ml) according to the Pasteur scheme (on days 1, 3 and 7), the second group - according to the scheme of Professor Babich E.M. (every other day, 0.2 ml orally for 15 days) [1].
  • the level of antibodies was established by two methods: a hemagglutination reaction and a method of fluorescent antibodies. Three animals from each group were left alive for up to 15 days, killed with ether and serum was obtained, where the level of specific antibodies was also established by the above methods.
  • a test system was prepared for the direct hemagglutination reaction, which was carried out in 96-well round-bottom immunological plates, to which 0.02 ml of a 0.1% suspension of thermostated ram red blood cells and 0.02 ml of a suspension of thermally inactivated Pseudomonas aeruginosa cells were added at a concentration 10 billion cells / ml.
  • Antibody levels were determined by serial ten-fold (but twofold) dilutions of the blood sera of mice, which were added in an amount of 0.02 ml to the wells of the plates. The presence of agglutinates testified to the formation of immune complexes.
  • mice we used normal human immunoglobulin (the titer of anti-pseudomonas antibodies ranged from 0 to (1: 10) according to the AED) and the blood serum of unvaccinated mice (titer from 0 to 1: 10).
  • the first sample was a corpuscular vaccine model based on inactivated pasteurization of Pseudomonas aeruginosa. Only surface antigens were acylated. The degree of acylation ranged from 1% to 15% in increments of 2%. A total of 8 samples of the modified soluble antigen and 8 corpuscular antigens were examined. The results of the immunogenicity study of the obtained samples are illustrated in FIG. one.
  • the average antibody titer in vaccinated animals was (1: 640).
  • oral use of the native corpuscular antigen did not induce the synthesis of specific antibodies.
  • a chemically modified antigen with a degree of modification of 1% of the protein concentration in it induced the synthesis of the same level of antibodies as the native antigen (1: 640).
  • Modification of surface antigens by 3% led to the induction of antibodies at the level of (1: 320) for the oral variant and at the level of (1: 2560) for the injection variant.
  • no induction of the synthesis of specific anti-Pseudomonas antibodies was observed.
  • the injection option was effective even to the derivative with a 15% degree of acylation.
  • a derivative with an acylation degree of 5% induced the synthesis of antibodies at the level of (1: 1280), a derivative with a 7% degree of acylation of the antigen at a level of (1: 320), a derivative of 9% at a level of (1: 40), other variants (with 11% and 15% degree of modification) - at the level of (1: 20).
  • the derivative with an acylation degree of 3% turned out to be the most effective, which induced the synthesis of specific antibodies both when using the classical L. Pasteur vaccination regimen for injectable use and when using the E. Babich vaccination regimen for oral administration of the vaccine.
  • FIG. Figure 2 shows the relationship between the level of specific antibodies induced and the degree of acylation of soluble modified antigen (first fraction).
  • This variant of the candidate antigen is a chemical monocomponent vaccine based on a glycoprotein modified high molecular weight antigen with a mass of 1.5 mDa and a molecular charge of 186,000. It was the largest molecular weight or the first fraction that comes out of the column when the fractions are divided that turned out to be the most immunogenic.
  • a correlation between the change in charge and mass of the antigen was confirmed, which confirms the state of completion of the chemical reaction of the modification of the structure of the antigen. Attention should be paid to an interesting fact discovered during injection vaccination of mice: the introduction of an unmodified vaccine (on the seventh day - the third time) caused an allergic reaction in some animals in the form of tremor, decreased motor activity and refusal to eat during the day.
  • acylated variant of soluble antigen with an acylation degree of 1% (Fig. 14), like the unacylated antigen, induced the synthesis of the same amount of antibodies in the titer (1: 1280). Oral administration of soluble antigen and antigen with an acylation degree of 1% did not lead to a significant induction of the synthesis of specific anti-Pseudomonas antibodies.
  • a soluble antigen derivative with an acylation degree of 3% activated the synthesis of antibodies at the level of (1: 5120) in the injection version and (1: 1280) when taken orally.
  • a 5% acylated derivative induced the synthesis of specific antibodies at the level (1: 640) for the oral form of administration and at the level (1: 2560) for the injectable form.
  • the derivative with an acylation degree of 7% induced the synthesis of specific antibodies at the level of (1: 640) for the oral form and (1: 1280) for the injectable.
  • the corresponding level of specific antibodies induced was (1: 320) for the oral form of the vaccine and (1: 640) for the injectable form.
  • the levels of antibody synthesis were equalized in vaccinated animals when administered orally and injected only when using a vaccine preparation with an acylation level of 13% and equal 1: 40, and when the degree of acylation was 15%, only the injection form of the candidate vaccine was immunogenic, it induced antibody synthesis at the level (1: 20).
  • the characteristic difference between the soluble high molecular weight antigen was the induction of antibody synthesis, not only derivatives with acylation degree of 3%, but derivatives with acylation degrees of 5%, 7%, 1 1%, 13% for oral administration according to the above scheme of Babich E.M.
  • the antibody titer was four times greater when injected with the most effective derivative with an acylation degree of 3% than with oral administration.
  • a 3% acylated derivative of the soluble antigen was twice as effective in immunogenicity when injected and four times as effective in oral administration.
  • a soluble glycoprotein antigen succinylated at 3% by weight of the protein was selected, which, when administered orally for 15 days, induced the synthesis of specific antibodies in the titer (1: 1280) and titer (1: 5120), when it was injected application.
  • Example 2. Obtaining an anti-diphtheria vaccine based on a composition of vaccine antigens with a modified charge of molecules
  • the microbial mass is obtained from the production strain PW - 8 Weissensee variant by cultivating C. diphtheriae bacteria in Linguud broth with the addition of 0.3% glucose or maltose. The cultivation is carried out at a temperature of 37 ° C for 36 hours, after which the microbial mass is separated from the toxin by centrifugation (6000 rpm for 30 minutes). The resulting precipitate ( ⁇ -gram wet weight) is poured with ethanol (concentration 96 °) in a volume of (2-4) p ml, kept in a refrigerator at 4 ° C for 24 hours and centrifuged in the marked mode.
  • Microbial sediment is poured (2-10) p ml of physical. solution, adjust the pH to 7.2-7.4, cool to 4-6 ° C, leave for 3-4 hours, then centrifuged (BOOOob / min. for 30 minutes).
  • a 0.8% solution of ethylenediaminetetraacetate-disodium salt (EDTA-Ka 2 ) is gradually added to the resulting precipitate, and triturated in a porcelain mortar until a white, viscous mass is obtained. It is kept in the refrigerator at a temperature of 4 ° -6 ° C for 18 hours.
  • the extract is centrifuged at bOOOob / min.
  • the selected antigenic complexes include proteins (75.3 ⁇ 3.4)%, lipids (23.3 ⁇ 4.1)% and carbohydrates (1.4 ⁇ 0.4)%.
  • aqueous suspension of somatic antigens is prepared, focusing on obtaining the necessary concentration for oral vaccination (5.0 mg / l), adjust the pH to 8.5-9.0 with a 1.0% sodium hydroxide solution or 1.0% acetic acid acid, set on a spectrophotometer (wavelength 230nm) the exact contents of the protein substance.
  • the somatic complex is modified with succinic anhydride in relation to the protein content.
  • the immunogenic properties of the obtained complexes are determined on chinchilla rabbits weighing 3.0-3.5 kg.
  • the creation of grossimmunity in animals is carried out according to the scheme: twice the introduction of antigen in a dose of 5.0 mg one hour before feeding with a daily interval for five days. Serological examination is performed 7, 14 and 21 days after the last vaccination using a standard diphtheria erythrocyte diagnosticum with a titer of 1: 3200 (see table 1).
  • modified antigens have different ability to influence humoral immunity.
  • the use of a modifier in an amount of 1.0% or less, as well as 5.0% or more with respect to the weight of the protein component did not lead to the induction of antibody titers in animals, while acylation of 2.0-4.0% by weight of the protein led to the induction of protective titers in the blood of orally vaccinated rabbits to a titer of 1: 2560 with preservation of it up to 21 days.
  • the invention relates to medicine and veterinary medicine, and in particular to vaccinology, and can be used to create means of specific prevention
  • the method allows for the modification of existing vaccine antigens directly at a pharmaceutical enterprise, using affordable equipment, significantly reducing the cost of vaccines, reduce their side effects, create oral forms of vaccines. Unique equipment for carrying out the invention is not required.

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Abstract

L'invention peut s'utiliser en médecine et dans le domaine vétérinaire pour créer des vaccins peroraux, parentéraux et transdermaux efficaces dans la prévention des maladies humaines ou animales. Les vaccins à immunogénéité élevée et les procédés de leur fabrication se distinguent en ce qu'en tant que composant spécifique immunogène on utilise un antigène de vaccin entier ou découpé en fragments oligomériques, et le mélange (ensemble) obtenu de fragments oligomériques ou l'antigène entier sont modifiés par la voie de modification de la charge des molécules en une charge opposée. L'utilisation de ces vaccins en raison de leur capacité de s'adapter à l'organisme permet de protéger l'organisme même contre les agents infectieux mutants qui n'existent pas encore dans la nature. Le moyen possède un spectre d'action large, est faiblement toxique et se prête bien à la production industrielle; il a une haute immunogénéité et n'est pas allergène, se métabolise rapidement et ne comprend pas de composants toxiques.
PCT/RU2010/000700 2010-11-22 2010-11-22 Vaccin à immunogénéité élevée et procédés de leur fabrication Ceased WO2012070974A1 (fr)

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PCT/RU2010/000700 WO2012070974A1 (fr) 2010-11-22 2010-11-22 Vaccin à immunogénéité élevée et procédés de leur fabrication
EA201300488A EA025417B1 (ru) 2010-11-22 2010-11-22 Вакцины с повышенной иммуногенностью и способы их получения

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Publication number Priority date Publication date Assignee Title
EA202090768A1 (ru) * 2018-05-04 2020-06-11 Борис Славинович ФАРБЕР Вакцины с повышенной иммуногенностью, низкой аллергенностью и реактогенностью

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1557712A (en) * 1975-08-29 1979-12-12 Anvar Immunological agents
WO1997027311A1 (fr) * 1996-01-23 1997-07-31 St. Jude Children's Research Hospital Melange de vecteurs de recombinaison du virus de la vaccine utilises comme vaccins polyenv contre le vih
WO2004000351A1 (fr) * 2002-06-20 2003-12-31 Cytos Biotechnology Ag Particules pseudo-virales enveloppees destinees a etre utilisees en tant qu'adjuvants: procede de preparation et utilisation de celles-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1557712A (en) * 1975-08-29 1979-12-12 Anvar Immunological agents
WO1997027311A1 (fr) * 1996-01-23 1997-07-31 St. Jude Children's Research Hospital Melange de vecteurs de recombinaison du virus de la vaccine utilises comme vaccins polyenv contre le vih
WO2004000351A1 (fr) * 2002-06-20 2003-12-31 Cytos Biotechnology Ag Particules pseudo-virales enveloppees destinees a etre utilisees en tant qu'adjuvants: procede de preparation et utilisation de celles-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARTYNOV A.V. ET AL.: "New approach to design and synthesis of therapeutic and preventive drugs, taking into account interspecies polymorphism of receptors (method of precision partial modification)", ANNALS OF MECHNIKOV INSTITUTE, vol. 4, 2007, pages 5 - 15 *

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EA025417B1 (ru) 2016-12-30

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