Combined bacterial meningitis vaccines to be introduced through mucous membrane
SUBSTANCE: invention refers to medicine, namely to manufacturing of vaccines for intranasal introductions. There is offered composition to be delivered through mucous membrane, containing two or more following agents: a capsular oligosaccharide antigen which induces immune response on Haemophilus influenzae; and (b) a capsular oligosaccharide antigen which induces immune response on Neisseria meningitidis; with specified antigens conjugated with a carrier protein.
EFFECT: combination allows for single introduction to immunisation against two separate agents of the systemic disease, namely bacterial meningitis.
20 cl, 4 dwg
All documents cited herein are entirely incorporated in it as a reference.
The SCOPE of the INVENTION
This application is related to the meningitis vaccine for administration through mucosa, especially vaccines for intranasal.
PREREQUISITES TO the CREATION of INVENTIONS
Meningitis is an inflammation of the tissues that cover the brain and spinal cord. He may have a bacterial or viral; bacterial meningitis is usually more severe.
The main causative agent of bacterial meningitis is Neisseria meningitidis (the meningococcus), however, other existing pathogens of meningitis include Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae (Hib) and Streptococcus agalactiae (GBS). N.meningitidis causes meningococcal septicaemia, which is a main feature of infection, a life-threatening.
Vaccine for protection against infection Hib exist for many years. The vaccine, which protects against meningococcus serogroup C ("MISP"), was introduced in several European countries in 1999-2000-pneumococcal vaccine, it has become customary in America in 2000
Vaccines against the three pathogens based on antigenic capsular polysaccharides are conjugated to protein carriers to enhance the immunogenicity of polysaccharides. These vaccines is lead by injection, although there have been described studies in mice on delivery via the mucous membrane, for example, reference 1 describes intranasal introduction of conjugate Hib vaccines, and reference 2 describes intranasal introduction of conjugate vaccines MISP (see also reference 3). Delivery of vaccines through the mucosa is an attractive approach to overcome the problem of the large number of injections that produce small children. In addition, since most pathogens first infect the surface of the mucous membranes, the acquisition of immunity to the mucosa at the site of infection may contribute to optimal protective immunity.
Also described intranasal and oropharyngeal delivery of vaccines against meningococcus serogroup B ("MAV") [e.g., reference 4], as well as intranasal delivery of bacteria .pertussis that Express transferrin-binding protein In N.meningitidis . In publication 6 described intranasal delivery of pneumococcal conjugated vaccines [see references 7 and 8].
The aim of the present invention to provide an improved delivery of vaccines against meningitis through the mucous membrane.
Description of the INVENTION
The present invention relates to compositions for delivery via the mucous membrane, containing two or more of the following agents: (a) the antigen is which induces an immune response against Haemophilus influenzae; (b) an antigen which induces an immune response against Neisseria meningitidis; and (C) an antigen which induces an immune response against Streptococcus pneumoniae.
The combination of different antigens reduces the number of doses that must be entered for the purpose of immunization against multiple pathogens. This is usually represented as an advantage for vaccines administered by injection, when the number of painful injections decreases, but this is less important for vaccines delivered through the mucous membrane (for example, intranasal vaccine), due to less pronounced discomfort associated with delivery. However, the combined antigenic compositions are advantageous even for delivery via the mucous membrane, as is improving the patient's compliance regime and schemes of vaccination, and facilitated the transportation and storage of medicines.
Although the Association of antigens in a single dose is attractive [for example, links 9-12], it presents difficulties because of the interactions between the various components after the merger, especially in liquid compositions . Emerging issues include the interaction of antigens, competition antigens [14, 15], the degradation of antigens, inhibition of epitopes and compatibility with adjuvants. Quality control mixtures is also more t is odnim. In addition, existing knowledge in the field of combining antigens are vaccines, which are administered by injection, and not through the mucous membrane.
Despite these difficulties, the applicants have unexpectedly found that the antigens of Haemophilus influenzae, Neisseria meningitidis and/or Streptococcus pneumoniae can be combined for delivery via the mucous membrane without negative consequences that could be expected. Combining antigens of the three microorganisms is also advantageous because it allows a single dose to deal with three separate causes of common diseases, namely, bacterial meningitis. Messages about combined meningitis vaccines of this type had previously , but about delivery via the mucous membranes have been reported.
Shipping through the mucous membrane
The composition according to the invention is intended for delivery via the mucous membrane.
From all the available paths for delivery via the mucous membrane of the intranasal route is the most practical, since it is connected with easy access using relatively simple devices that are already being produced on an industrial scale. In addition, intranasal immunization is more effective compared with alternative ways. Thus, the preferred route for delivery through slit the out shell is intranasal path, and composition according to the invention is preferably adapted for intranasal, such as the introduction through a nasal spray, nasal drops, gel or powder [for example, references 17 and 18].
Alternative routes for vaccine delivery via the mucous membranes are oral, intragastric, lung, putting, rectal, ocular and vaginal path.
(A.) Antigen Haemophilus influenzae
Antigen H.influenzae in the composition will usually be a capsular charigny antigen. Sacharine antigens from H.influenzae b is well known.
Advantageously, if the Hib saccharide covalently anywhereman with protein carrier to enhance its immunogenicity, especially for children. Production of polysaccharide conjugates in General and capsular polysaccharide Hib, in particular, is well documented [e.g., links 19-27, etc.]. In the present invention can use any suitable conjugate Hib.
Sahariana part of the conjugate may be a polysaccharide (e.g., polyribosylribitol (PRP) full length), but it is preferable to carry out the hydrolysis of polysaccharides (e.g., acid hydrolysis) to obtain oligosaccharides (for example, with molecular mass from ~1 to ~5 kDa). If you are hydrolysis, the hydrolysate can be sorted by size to remove too short oligos Aridi, which are not full immunogenum. Divided according to the size of the oligosaccharides are preferred sharedname antigens.
Preferred protein-carriers are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The latter are widely used in conjugated vaccines. Diphtheria toxoid CRM197 is especially preferred . Other suitable carrier proteins include protein N.meningitidis outer shell , synthetic peptides [30, 31], heat shock proteins [32, 33], pertussis proteins [34, 35], protein D from H.influenzae , cytokines , lymphokines , hormones , growth factors , toxin a or b from .difficile , proteins, exciting iron , and the like, it is Possible to use mixtures of proteins vehicles.
Sharenow part directly or through a linker to konjugierte with protein carrier. A direct connection can be made by oxidation of the polysaccharide followed by reductive amination using protein, as described, for example, in references 40 and 41. Communication through the linker group can be accomplished using any known procedure, for example, procedures described in the links 42 and 43. Suitable linkers include carbonyl, adipic acid, In propionamido , nitrophenylamino , halogenoalkane, glycosidic bonds , 6-aminocaproic acid , ADH , the part from4to C12, etc.
Before conjugation saccharide will usually be activated or functionalized. Activation may include, for example, Cinelerra reagents, such as CDAP (for example, tetrafluoroborate 1-cyan-4-dimethylaminopyridine [51, 52]. Other suitable methods are used carbodiimide, hydrazides, active esters, norburn, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction to the reference 53). The preferred technique is reductive amination.
The preferred conjugate comprises a Hib saccharide covalently linked to CRM197 via amber diapir adipic acid [54, 55].
Compositions according to the invention can include more than one antigen Hib.
(b) Antigen of Neisseria meningitidis
Antigen N.meningitidis in the composition will usually be a capsular charigny antigen (for example, from serogroups a, C, W135 or Y). Sacharine antigens from N.meningitidis well known. In the case of antigen from serogroup b, however, it is preferable that the antigen was a protein antigen, because the native capsular polysaccharide Mepv contains autoantigens. If you want to use charigny antigen from serogroup B, it is preferable to use a modified sugar is command antigen [for example, links 56, 57, 58], for example, an antigen, a modified N-Propionibacterium. Possible chemical modification of saccharides from other serogroups.
The saccharide is preferably an oligosaccharide, i.e. a fragment of the capsular polysaccharide. Polysaccharides can be processed with getting shorter oligosaccharides, and the latter can be obtained by cleaning and/or sorting by size of native polysaccharides (for example, by hydrolysis in a weak acid, by heating, by chromatography sorted by size and the like). Preferred oligosaccharides MISP described in the links 59 and 60.
The saccharide is preferably kongugiruut with protein carrier, as described above.
Compositions according to the invention may include more than one meningococcal antigen. Preferred may be the inclusion of capsular sharidny antigens of at least two (i.e. 2, 3 or 4) of serogroups a, C, W135 and Y of N.meningitidis .
When the mixture includes capsular saccharides from both serogroups a and C, it is preferable that the ratio (wt./mass.) saccharide Mopa: saccharide MISP was greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1 or above). Unexpectedly observed improved the immunogenicity of the component Mopa, when he was present in excess (weight/dose) in relation to a component of the MISP .
When the mixture consisted of capsular Saha is the ides of serogroup W135 and at least one of the serogroups A, C and Y, it has been unexpectedly found that the immunogenicity of the saccharide MenW135 above in the introduction, in combination with a saccharide (sugar) from the other serogroups (serogroups)than when introduced separately (in the same dose, etc.) . Thus, the ability of antigen MenW135 to cause a more intense immune response than the immune response induced by an equivalent amount of the same antigen when delivered out of touch with antigens from other serogroups. Such enhanced immunogenicity can be determined by injection of the antigen MenW135 the control animals, but a mixture of experimental animals and comparing antibody titers against these two immunogens using standard surveys such as the titles of bacteria, radioimmunoassay analysis, ELISA and other Vaccines containing synergistic combinations of saccharides from serogroups W135 and other serogroups have immunological advantages, because they reinforce anti-W135-answers and/or lower dose W135.
When used protein antigen from serogroup B, it is preferable to use one of the proteins described in the links from 62 to 71. Preferred protein, antigens include protein "287" or its derivatives (for example, ΔG287).
It is also possible to use the antigen from outer membrane vesicles (OMV) for serogroup In [for example, 72, 73].
Compositions according to the invention may include SEB is more than one meningococcal antigen.
(C) Antigen of Streptococcus pneumoniae
Antigen S.pneumoniae in the composition will usually be a capsular charigny antigen, which is preferably anywhereman with protein carrier, as described above [e.g., 74, 75, 76].
It is preferable to include saccharides from more than one serotype S.pneumoniae. For example, widely used mixture of polysaccharides from 23 different serotypes, as well as conjugated vaccine polysaccharides from 5-11 different serotypes . For example, PrevNar™ contains antigens from seven serotypes (4, 6B, 9V, 14, 18C, 19F and 23F), each saccharide separately anywhereman with CRM197 by reductive amination.
Compositions according to the invention can thus contain more than one pneumococcal antigen.
Additional components - adjuvants
Compositions according to the invention will typically include adjuvants for mucosal. Adjuvants for mucosal contain, without limitation, (A) thermo-labile enterotoxin of E. coli ("LT") or its detoxificaton mutants, such as mutants C or R72 include [e.g., Chapter 5 references 78]; (C) cholera toxin ("CT") or its detoxificaton mutants [e.g., Chapter 5 references 78]; or (C) microparticles (i.e. a particle diameter from ~500 nm to ~10 μm)formed from materials that are biodegradable and non-toxic (for example, the and(α-hydroxycitrate, polyhydroxyalkane acid, polychaetes, polyanhydride, polycaprolactone etc); (D) polyoxyethylene ether or polyoxyethylene ester ; (E) surface-active agent is a polyoxyethylene ether and sorbitan in combination with octoxynol  or surface-active agent polyoxyethyleneglycol ether or ester in combination with at least one additional nonionic surface-active agent such as an octoxynol ; (F) chitosan [for example, 82]; (G) an immunostimulating oligonucleotide (such as a CpG oligonucleotide), (H) Dunaeva RNA; (I) saponin ; (J) mimetics of monophosphorylated And such as derivatives aminoalkylindole, for example, RC-529 ; or (K) of polyphosphazene (RSRR). Available are also other adjuvants for mucosal [for example, see Chapter 7 references 85].
Preferred adjuvants for mucosal are bacterial ADP-reboilers toxins or their mutants. For example, cholera toxin (CT) or thermo-labile toxin of E. coli (LT) are efficient adjuvants for mucosal and their detoxificaton twins . ST and LT are homologous and are usually interchangeable.
Detoxification ST or LT can carry chemical or, preferably, genetic means. Suitable examples include LT with the OS is atok lysine at amino acid position 63 ["LT-K63" - link 87], and LT with the arginine residue at amino acid position 72 ["LT-R72 include" link 88]. Other suitable mutants include LT with tyrosine at position 63 ["Y63" link 89] and various other mutants described in the link 90, namely, D53, K, K 104 and S106, and their combinations (for example, LT with mutations D53 and C).
The composition may include bioadhesive [91, 92], such as microspheres esterified hyaluronic acid , or in preferred embodiments, implementation, mucoadhesive selected from the group consisting of crosslinked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose.
Compositions according to the invention may include more than one adjuvant for mucosal.
Additional components - antigens
The combination of antigens from H.influenzae, N.meningitidis and S.pneumoniae is advantageous because all of these micro-organisms causing bacterial meningitis. Antigens that induce immune responses against other organisms, can also be incorporated into compositions according to the invention, for example,
antigens from Helicobacter pylori such as Garden [94-97], VacA [98, 99], NAP [100, 101, 102], Nor [for example, 103], HopY [for example, 103] and/or urease.
- an antigen from hepatitis a virus, such as inactivated virus [e.g., 104, 105];
- an antigen from a virus hepatitis is In, such as surface and/or core antigens [e.g., 105, 106];
- an antigen from hepatitis C virus [e.g., 107];
- an antigen from Bordetella pertussis, such as pertussis, holotoxin (PT) and filamentous hemagglutinin (FHA) of .pertussis, optionally in combination with pertactin and/or agglutinogens 2 and 3 [for example, the links 108 and 109];
- diphtheria antigen, such as diphtheria toxoid [e.g. Chapter 3 references 117], for example, the mutant CRM197[for example, 83];
- tetanus antigen, such as tetanus toxoid [e.g. Chapter 4 references 114];
- an antigen from Chlamydia pneumoniae [e.g., 110, 111, 112, 113, 114, 115, 116]; p>
- an antigen from Chlamydia trachomatis [for example, 117];
- an antigen from Porphyromonas gingivalis [e.g., 118];
- polio antigen (antigens) [for example, 119, 120], such as IPV or OPV;
- antigen (antigens) rabies [e.g., 121], such as freeze-dried inactivated virus [e.g., 122, RabAvert™];
- antigen measles, mumps and/or rubella [e.g., chapters 9, 10 and 11 references 123];
- antigen (antigens) influenza [e.g., Chapter 19 references 123], such as hemagglutinin and/or neuraminidase surface proteins;
- antigen (antigens) from paramyxovirus, such as respiratory syncytial virus (RSV [124, 125]) and/or parainfluenza virus (PIV3 );
- an antigen from Moraxella catarrhalis [for example, 127];
- an antigen from Streptococcus agalactiae (Streptococcus group who) [for example, 128, 129];
- an antigen from Streptococcus pyogenes (group a Streptococcus) [e.g., 129, 130, 131]; p>
- an antigen from Staphylococcus aureus [e.g., 132].
The composition may include one or more of these additional antigens.
In the case of a conjugate composition may also include free a carrier protein .
Preferably, the composition did not contain the bacteria as a whole (no matter intact or lysed).
Compositions according to the invention may include proteins that mimic sacharine antigens, for example, mimotope  or antiidiotypic antibodies. They can replace individual sacharine components or to Supplement them. As an example, the vaccine may contain a peptide mimetic of capsular polysaccharide MISP  or Mopa  instead of the saccharide.
Compositions according to the invention can include nucleic acid "genetic immunization" [e.g., 137]. Nucleic acid will encode a protein component of the composition and can replace individual protein components (including those mentioned in the previous paragraph) or to Supplement them. As an example, the vaccine may contain DNA, which encodes the tetanus toxin.
Additional components - composition
The composition according to the invention preferably includes pharmaceutical preparations which is automatic acceptable carrier.
"Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose , lipid aggregates (such as oil droplets or liposomes), and inactivated viral particles. These carriers are well known in the art. Vaccines may also contain diluents, such as water, saline, glycerol, etc. in Addition, there may be auxiliary substances, such as moisturizing or emulsifying agents, buffering agents and the like, the Carrier will be compatible with the introduction through the mucous membrane. A detailed discussion of pharmaceutically acceptable excipients can be found in Remington''s Pharmaceutical Sciences.
The composition according to the invention is preferably sterile.
The composition according to the invention preferably contains a buffer.
The composition according to the invention is preferably pyrogen-free.
The composition according to the invention can be Packed with components (a), (b) and/or (C) in the mixture, or data component may exist separately from each other until they will not need to enter the patient, and at this stage they will be United. When individual components are separate from each other, they can be in lyophilized form or in solution/suspension. When the components are in a mixture, they will all be in lyophilized form or in solution/suspension. Lyophilized components will be resuspendable (e.g., buffer) before the introduction of the patient. Components such as adjuvants may be present in the buffer or in dried material.
The composition according to the invention preferably is an immunogenic composition (e.g., vaccine). Preparation of vaccines based on saccharides or saccharide conjugates protein is well known to specialists.
Immunogenic compositions include immunologically effective amount of antigens, as well as any other listed components, if necessary. By "immunologically effective amount" is meant that the introduction of a specified number of the individual, as a single dose, or as part of a series, is effective for treatment or prevention. This number varies depending on the health and physical status of the individual being treated, age, the taxonomic GRU the dust of the individual, being treated (e.g., Primate, non-human, Primate, and the like), the ability of the immune system of an individual to synthesize antibodies, the desired degree of protection, vaccine composition, evaluation by the attending physician clinical situation and other factors. It is expected that this amount will fall in a relatively broad range that can be determined by the usual tests.
Methods of treatment
After manufacture of the composition according to the invention can be administered to the patient, usually a person. The human is preferably a child or teenager. Another preferred class of patients are adult women and especially women of childbearing age or pregnant women. Compositions according to the invention are particularly suitable for passive immunization of children through the mother's body.
The antigens in the compositions induce immune responses against certain bacteria. These immune responses are preferably protective, i.e. they protect the patient from further infection by bacteria. Thus, compositions according to the invention is preferably used for the prevention (i.e. to prevent infection), although they can also be used for therapeutic purposes (i.e. to treat disease after infection). Immune response preferably is clucalc in itself the production of bactericidal antibodies in a patient.
The present invention relates to a method for producing an immune response in a patient, comprising the administration to the patient of the vaccine through the mucous membrane (e.g., intranasal). The immune response is preferably protective against bacterial meningitis and/or bacteremia caused by Haemophilus influenzae, Neisseria meningitidis and/or Streptococcus pneumoniae. Individual antigenic components of the compositions is preferably introduced simultaneously and in combination. In other embodiments, implementation of the present invention, however, they can be administered separately, simultaneously or sequentially. In the case of the introduction of the smaller components are preferably delivered to the same surface of the mucous membrane.
The present invention relates also to compositions according to the invention for use as a medicine.
The present invention relates also to the use of (a) an antigen which induces an immune response against Haemophilus influenzae, (b) an antigen which induces an immune response against Neisseria meningitidis, and (C) an antigen which induces an immune response against Streptococcus pneumoniae, for the manufacture of a medicine for immunization of the patient.
These methods and application according to the invention may include initial/booster mode. Methods and applications according to izobreteny may represent the initial dose, followed by a booster dose, with a booster dose may be injected through the mucous membrane or parenteral route. Similarly, methods and applications according to the invention can cause the booster response in a patient who has already been immunized initial dose, and the initial dose can be entered through the mucous membrane or parenteral route. Booster doses may contain fewer antigens than in the original doses, for example, they can be used only antigen.
Treatment for primary exposure to antigen and/or revaccination may be a single dose or multiple doses. Compositions according to the invention can be presented in the form of dosage units.
The manufacturing methods
The present invention relates to a method of manufacturing a composition according to the invention, comprising a stage of mixing two or more of the following agents: (a) an antigen which induces an immune response against Haemophilus influenzae, (b) an antigen which induces an immune response against Neisseria meningitidis, and (C) an antigen which induces an immune response against Streptococcus pneumoniae, and manufacture of the mixture composition for delivery via the mucous membrane.
BRIEF DESCRIPTION of DRAWINGS
Figure 1 shows the geometric mean Titus is s of IgG antibodies against the MISP in the serum. Mark on the X-axis refers to adjuvant that was used. Not shaded column on the left in each pair corresponds to the data obtained after the introduction of Zaharenko antigen separately, while the shaded column on the right corresponds to the data obtained after administration of the United sharidny antigens. On figa shown anti-MISP-answers, and figv shown anti-ib responses. The variance of the errors above represents the standard deviation within one standard error.
Figure 2 shows the titers of bactericidal antibodies against the MISP. As in figure 1, the shaded data were obtained using the combined sharidny antigens.
Figure 3 shows the titers of IgA against the MISP in flushing out the nasal cavity. As before, the shaded data were obtained using the combined sharidny antigens.
WAYS of IMPLEMENTATION of the PRESENT INVENTION
The combined composition Hib/MenC
Capsular oligosaccharide With serogroup of Neisseria meningitidis was obtained by selective activation of the reducing end groups sorted by size of the oligosaccharide. The same method was used for Haemophilus influenzae type B. Sugars conjugatively with protein carrier CRM197 via a hydrocarbon spacer  (Chiron Siena, Italy). The conjugates were diluted with saline phosphate buffer (PBS) and the volume of inali (i) mutant thermolabile enterotoxin E. coli LTK63 or LTR72, (ii) with aluminum hydroxide (Superfos Biosector a/s) or (iii) by cholera toxin (CT) from Sigma. For the combined introduction of the above composition was mixed before use.
The introduction of the composition through the mucous membrane
Simultaneously conducted two identical studies on the introduction. Groups of 10 female BALB/C mice aged 6-10 weeks were immunized intranasally 10 µg MISP or no Hib separately, in combination with CT (1 μg) or with mutant LT (1 µg and 10 µg). For comparison, an additional group of mice were immunized/m 10 ág MISP or Hib adsorbed on Alum. Compositions were made on the day of immunization, the mice were immunized at 0, 21 and 35 day. 50 μl of the compositions were injected with hip or buried into different the nostrils of mice under anesthesia. Blood samples were taken on day 49, together with samples of swabs from the end portion of the nasal cavity (NW).
In order to assess, is not violated if the immunogenicity of the conjugates by mixing two vaccines, conducted at the same time the third study, in which two vaccines were administered simultaneously to the same group of mice, in the same doses and according to the same scheme as described above.
Immunological responses to composition
Or antibody-based test answers against the conjugate MISP was determined by ELISA using a modified method as described previously . KRA is to, tablets for ELISA were covered with a saccharide MISP, derivational adipinate dihydrazide, over night at 4°C. antibodies were detected using HRP-conjugate goat artemisinin IgG and horseradish peroxidase. Titers of IgG antibodies against MISP test samples and internal control was expressed as the reciprocal serum dilution giving OP=1,0. Each serum sample was analyzed in duplicate, and used the average value to calculate the geometric mean and standard deviation within the standard error. Or antibody-based test answers against Hib PRP was determined similar to ELISA for the MISP way, except that the tablets were covered with PRP, conjugated with BSA (PRP-BSA). Titers were expressed as OD450 nmfor serum, diluted 1:50.
Conducted analysis of swabs from the nasal cavity to IgA against the MISP using bioluminescent analysis (BIA) . Briefly, we used the same reagents and procedure coverage for the measurement of IgG against the MISP in the serum. Then as the first antibody was added specific biotinylated goat antimurine IgA. Titers represent logarithmic values of breeding, extrapolated from data log RLU shut-off value, calculated by at least two standard deviations above the average background.
Mediated by the complement system is a bactericidal activity against bacteria MISP was measured in pooled serum samples, as was previously described . Titers were determined by calculation of the serum dilution required to achieve a 50% reduction in the number of CFU after incubation for 1 hour.
On figa shows the geometric average titers of serum IgG antibodies against the MISP alone (not shaded bars)and in combination with Hib antigen (shaded bars). Serum or antibody-based test answers, caused both mutants LT were significantly higher responses to antigen alone. LTR72 demonstrated more pronounced adjuvant properties than LTK63, in lower doses. Most notably, or antibody-based test responses induced by intranasal immunization with both LT mutants were compared with the responses obtained when using ARTICLE wild-type or responses induced by intramuscular immunization with a vaccine using the adjuvant alum. It is important that adding a second conjugated Zaharenko antigen does not adversely impact on or antibody-based test answers against each of the antigens.
On FIGU shows the geometric average titers of serum IgG antibodies against saccharide Hib PRP. As in the case of the MISP, or antibody-based test answers, caused by both LT mutants were higher than responses to the antigen alone. And again, LTR72 demonstrated more pronounced adjuvant properties. Against the appropriate titers were induced in mice immunized intranasally mutants LT, and vaccine using adjuvant alum intramuscular immunization. In addition, no data on competition after combined intranasal immunization two sharedname conjugated vaccines;
the responses induced against Hib in combination with the MISP, were comparable to responses induced by immunization with Hib separately.
Levels of bactericidal antibodies induced by intranasal immunization with LT mutants, was strongly correlated with serum IgG responses by ELISA results, and again were comparable to responses induced ARTICLE, or intramuscular immunization vaccine, adsorbed on alum (figure 2).
Samples obtained from swabs of the nasal cavity after intranasal immunization MISP with both the LT mutants showed higher titers of IgA than samples obtained intranasal immunization in the absence of adjuvants (figure 3). As expected, intramuscular immunization caused a very low titers of IgA.
A powerful serum or antibody-based test answers against N.meningitidis and H.influenzae can be induced by intranasal immunization with conjugated vaccines in combination with adjuvants for insertion through the mucous membrane. In addition, in the case of antigen MISP, antibodies, induced by intranasal immunization, have m is Snoy bactericidal activity, which is known to correlate with protective immunity . In addition, the IgA responses in the nasal cavity were induced only in animals immunized by intranasal. The induction of secretory immunity is important, because the upper parts of the respiratory system are the input gates for several pathogens, including N.meningitidis and .influenzae.
Based on the antibody titers obtained with the use of conjugated vaccines administered separately or in combination, and on the basis of bactericidal activity, measured against the MISP, it can be concluded that the combination of the two vaccines administered in conjunction with adjuvant for mucosal, does not adversely impact on or antibody-based test answers against the MISP or Hib. Thus, the results suggest that intranasal immunization is an effective way of immunization for conjugated vaccines, polysaccharide-protein in combination with adjuvants for mucosal, such as mutants of LT.
For reliable increase the immunogenicity of both conjugated vaccines administered at the same time, it was quite the same dose of LT mutants. This is particularly important as this should reduce the number of required adjuvant and the risk associated with potential toxicity. It is important that the existing immunity against Muta is the LTK63 does not affect the ability of the mutant to act as an adjuvant for the second antigen . In addition, the effectiveness of the vaccines delivered through the mucous membrane, can be further improved by making vaccines in bioadhesive delivery systems .
In conclusion we can say that the combination of conjugated vaccines, polysaccharide-protein mutants LT for intranasal immunization is an effective approach to immunization through the mucous membrane for pediatric purposes.
1. Composition for administration through mucosa containing adjuvant for administration through mucosa, as well as (a) antigen capsular oligosaccharide, which induces an immune response against Haemophilus influenzae; and (b) the antigen capsular oligosaccharide, which induces an immune response against Neisseria meningitidis; moreover, these antigens conjugated to a protein carrier.
2. The composition according to claim 1, adapted for intranasal administration.
3. The composition according to claim 2 in the form of a nasal spray, nasal drops, gel or powder.
4. The composition according to claim 1, in which the antigen H.influenzae is a capsular charigny antigen conjugated to a protein carrier.
5. The composition according to claim 4, in which charigny antigen is an oligosaccharide.
6. The composition according to claim 1 in which the antigen N.meningitidis is particularly the capsular charigny antigen from serogroups A, C, W135 or Y conjugated to a protein carrier.
7. The composition according to claim 6, in which charigny antigen is an oligosaccharide.
8. The composition according to claim 1, containing antigens N.meningitidis at least two of serogroups a, C, W135 and Y.
9. Composition according to any one of claims 4 to 8, in which the carrier protein is a diphtheria or tetanus toxoid.
10. The composition according to claim 9, in which the carrier protein is a CRM197.
11. The composition according to claim 1, in which each of the antigens: antigen H.influenzae and antigen N.meningitidis is an oligosaccharide fragment of the capsular polysaccharide conjugated to protein carrier.
12. The composition according to claim 11, in which the antigen H.influenzae anywhereman with the first protein-carrier antigen N.meningitidis anywhereman with a second protein carrier.
13. The composition according to claim 11, in which the antigen H.influenzae and antigen N.meningitidis conjugated with the same protein carrier.
14. The composition according to item 12, in which the first and second carrier proteins are individually CRM197.
15. The composition according to claim 1, in which adjuvant for administration through mucosa is a detoxified enduring mutant of a bacterial ADP-reboilers toxin.
16. The composition according to item 15, in which adjuvant for administration through mucosa is a LT-K63 or LT-R72 include.
17. The composition according to claim 1 for use the education as a medicine.
18. The method of obtaining immune response in a patient, comprising the administration to a patient of a composition according to any one of claims 1 to 16.
19. The application of (a) antigen capsular oligosaccharide, which induces an immune response against Haemophilus influenzae, (b) antigen capsular oligosaccharide, which induces an immune response against Neisseria meningitidis, and (C) adjuvant for administration through mucosa for the manufacture of a medicine for immunization of the patient, and these antigens are conjugated to a protein carrier.
20. A method of manufacturing a composition according to claim 1, comprising the following stages: (i) mixing (a) antigen capsular oligosaccharide, which induces an immune response against Haemophilus influenzae, (b) antigen capsular oligosaccharide, which induces an immune response against Neisseria meningitidis, and (C) adjuvant for administration through mucosa, and (ii) the manufacture of the mixture composition for delivery via the mucous membrane, and these antigens are conjugated to a protein carrier.
SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.
EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.
35 cl, 13 dwg, 36 tbl, 14 ex
SUBSTANCE: invention relates to medicine, in particular to gynecology and concerns treatment of patients with sterility induced by anovulation at the background of chronic and acute inflammation of small pelvis organs. For this purpose mixture of the following composition is introduced paracervically: amoxicillin 1000 mg, clavulanic acid 200 mg, longidase 1500 U and Novocain in form of 0.5% solution - 8 ml. Preparation is introduced 1 time a day during 10 days. Additionally serotonin in dose 1.0 ml 2 times a day during 5 days is introduced.
EFFECT: method ensures normalisation of ovulation and menstrual cycle, increase of libido, elimination of premenstrual syndrome, algodismenoria, acne in short terms with possibility of treatment in the outpatient setting.
SUBSTANCE: invention relates to medications and concerns means for activation of stem cells, containing active beginning of chemical origin and solution of sodium chloride for injections, differs by the fact that, as active beginning it contains formaldehyde with the following components ratio, wt %: formaldehyde 0.00003-0.004, solution of sodium chloride for injections of 0.85-0.95% concentration - the remaining part.
EFFECT: invention is aimed at creation of non-toxic and not having side effects means for activation of organism's own stem cells.
3 tbl, 7 ex
SUBSTANCE: kit includes material object, containing preparation which after interaction with human organism provides chemical impact on its tissues. Additionally it contains at least one device including element made with possibility of its introduction into mechanical contact with human in area of location on the latter of acupuncture points, which are supposed to be subjected to impact. Said device includes made with possibility of electric connection with human, made from conductor or semiconductor electrically monopolar physical body, whose capacity satisfies the condition: C > 0.1 pF, where C is electrical capacity of said body as isolated conductor.
EFFECT: application of claimed kit will allow to influence acupuncture points in highly efficient way, simultaneously reducing doses of applied preparations due to complex medication acupuncture influence on certain organ.
14 cl, 6 dwg
SUBSTANCE: invention is related to compound represented by formula (1) , in formula A represents nitrogen-containing saturated ring; m represents integer number, equal to 0, 1 or 2; n represents integer number, equal to 1, 2, 3 or 4; G1 represents atom of hydrogen, hydroxyl group or alkoxygroup; G2 represents atom of halogen, hydroxyl group, cyanogroup, alkyl group, alkenyl group, alkinyl group, which may be substituted with hydroxyalkyl group, alkoxygroup, alkyl thiogroup, aminogroup or aryl group; G3 represents atom of hydrogen; G4 represents hydroxyl group or -N(R1)(R2) (R1 and R2 may be identical or different, and independently represent atom of hydrogen, alkyl group, aralkyl group, alkenyl group or saturated heterocyclic group); and G5 represents substituent at carbonic atom, which constitutes nitrogen-containing saturated ring, represented with A, and represents atom of hydrogen, to medicinal agent on the basis of this compound for treatment and prophylaxis of glaucoma, to inhibitor of phosphorylation of regulatory light chain of myosin and inhibitor of kinase Rho/Rho path, and also to method of therapeutical and/or prophylactic treatment of glaucoma.
EFFECT: new compounds have been produced and described, which efficiently inhibit phosphorylation of regulatory light chain of myosin.
32 cl, 42 ex, 4 tbl
SUBSTANCE: invention is related to the area of pharmacy, pharmacognosy, to preparation of medicinal mixtures. Mixture contains horse gowan bloom, meadow clover grass, licorice roots, hop cones, bottle brush grass and sweetflag rhizome.
EFFECT: infusion and thick extract of mixture displays complex estrogen-like, antimicrobial and anti-inflammatory effect.
SUBSTANCE: there are presented compounds of formula I wherein W, R, R1, R2, R3, R4, R5, R6 and R7 have values specified in cl. 1 of the patent claim, and to method for making these compounds, a based medicinal agent used for treating conditions affected by inhibition, regulation and/or modulation of mitotic motor protein Eg5, to a mixture and application of said compounds for making the medicinal agent.
EFFECT: there are produced and described new compounds which can find application in treating tumours.
40 cl, 654 ex, 3 tbl, 25 dwg
SUBSTANCE: invention refers to chemical-pharmaceutical industry, namely to development of an antiaggregatory and stress-limiting agent as a reactant containing ecdysteroids-containing substance in the form of mixed 20-hydrohyecdysone in amount 75% and more, and 25S-inokosteron in amount 10% and more. The reactant is recovered from herb of Serratula sort of Asteraceae family, preferentially leaves collected during vegetation, mass budding or beginning of blossoming.
EFFECT: extended range of antiaggregatory and stress-limiting agents without manifesting the by-actions on a human body.
SUBSTANCE: invention refers to medicine and can be used for prevention of inflammatory processes in animals. That is ensured by putting in a dark glass bottle at the relation g (wt %) of mixed dry powdered elecampane rhizomes and roots - 20.0 (5,9) plaster clover leaves, blossom, fruits, thin bodies - 14.5 (4.4), oak barks - 15.0 (4.4), touch-and-heal herb - 20.0 (5.9), calendula blossom basket - 20.0 (5,9), burnet bloodwort rhizomes and roots - 20.0 (5.9), lavender blossom clusters - 20.0 (5.9), peppermint leaves, bodies and blossom - 20.0 (5.9), costmary blossom baskets - 15.0 (4.4), common plantain leaves - 20.0 (5.9), horse gowan blossom - 20.0 (5.9), common licorice rhizomes, soboles - 20.0 (5.9), nosebleed herb - 20.0 (5.9), thyme herb - 20.0 (5.9), full-maturity hips - 20.0 (5.9), eucalyptus leaves - 20.0 (5.9), killwort leaves, bodies, blossom, fruits - 15.0 (4.4), and nonfrozen stoned sea-buckthorn fruits- 20.0 (5.9). Then agitated mixture is filled with 1500.0 g of corn or sunflower oils either plain or refined, or olive oil either plain or salad. The prepared mixture is kept at room temperature with constant mixing for 25-30 days with following separation of vegetable sediment and preparing of oil-vegetable agent. The prepared vegetable sediment is filled with 1500.0 g of 0.9% sterile solution of table salt at temperature no more than 50 C, and kept in agitation for 25-30 days to produce thereby an aqueous-vegetable agent. For each 100.0 g of prepared oil-vegetable or aqueous-vegetable agent, there are added in agitation natural may honey 6.0 g (6.0%) and formalin 0.6 g (0.6%); then the prepared agent is packed. The agent is applied externally, once or manifold on affected body part, or introduced internally.
EFFECT: invention allows ensuring higher clinical effectiveness with respect to inflammatory processes ensured by various actions of the components of the mixture.
SUBSTANCE: invention refers to medicine, namely to dermatology, and can be used for treatment or rejuvenation of human skin. It is ensured by introduction of autogenous fibroblasts suspended in physiologic saline and hyaluronic acid in treatment or rejuvenation region. The skin is prepared with growth fibroblast medium with biologically active substances (BAS) - growth medium factors including fibroblast growth in the form of gel.
EFFECT: invention provides smoothing of wrinkles, higher skin elasticity.
2 ex, 2 tbl, 52 cl
SUBSTANCE: group of inventions relates to medicine and aims at treatment of cancer in an individual. It involves introduction to the specified individual of amount of purified heat-shock protein (HSP) preparation. The purified HSP preparation contains purified HSP-peptide complexes containing HSP covalently or non-covalently attached to peptide and where HSP-peptide complexes express antigenicity of said cancer. In addition, an immunogenic reagent is introduced to an individual, where the immunogenic reagent specifically reacts with antigen chosen from the group consisting of VEGF, EGF-R, HER2/NEU, CD25 and CD20, or is being anti-CTLA-4 or anti 41BB antibody.
EFFECT: combined introduction of HSP-peptide complexes and immunogenic reagent allows intensifying immune response in antibody therapy of cancer.
36 cl, 4 ex, 2 dwg
SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.
EFFECT: improved pharmacokinetic properties of erythropoietin.
23 cl, 14 ex, 6 tbl, 11 dwg
SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.
EFFECT: enlarging the application range of medicine.
SUBSTANCE: invention concerns area of medicine and concerns compositions and medicinal forms on the basis of Gastrinum, application and reception methods. The essence of the invention includes the bond of Gastrinum representing conjugates of fragments of amino-acid sequence of Gastrinum, possessing functional ability to contact Gastrinum/SSK receptor, with various carriers, including application amino-acid spacers, and application of bifunctional sewing agents, and also a method of treatment by the compositions including bond of Gastrinum, sick of diabetes.
EFFECT: advantage of the invention consists in action prolongation.
7 cl, 8 ex, 3 tbl, 2 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention concerns vaccines, particularly vaccines for meningococcal infections and diseases. Invention claims immunogenic composition for transport via mucosa, including capsular saccharides of at least two of the following serological groups: A, C and W135 and Y N. Meningitidis, as well as trialkylated chitosan.
EFFECT: enhanced production of immune response to meningococci in mucosa, possible balance shift of Th1/Th2 type responses.
16 cl, 24 dwg
SUBSTANCE: invention pertains to modified polysaccharide in particular to modified polysaccharide Neisseria meningitidis of serogroup A, which preserves immunogenicity, but has improved stability. The modified polysaccharide is obtained from reaction of capsular polysaccharide, or its fragment - oligosaccharide, with CDI type bifunctional reagent, accompanied by reaction with an amino-compound, such as dimethylamine. Description is also given of modified polysaccharide conjugates and vaccines, which are obtained from such conjugates.
EFFECT: obtaining modified saccharide.
70 cl, 17 dwg
FIELD: medicine; veterinary science.
SUBSTANCE: method of higher meat production of broilers provides single injection for day birds of liposomal forms of preparation containing chimeric protein with water insoluble enzyme-inactive chloramphenicol acetyltransferase without 10 S-terminal aminoacids, aminoacid spacer (Sp)n, where n=1, 2, 4, 8 and somatostatin-14 with aminoacid sequence AGCFWKTFTSC, with median size of liposomes 250±50 nm. And preparation is introduced in combination with Marek's disease factor vaccine.
EFFECT: invention allows for higher effective meat production of broilers using single injection of preparation during the whole fattening period.
2 cl, 1 tbl
SUBSTANCE: invention concerns aldehyde derivatives and conjugates of di-, oligo- or polysaccharide, of the general formula (I), methods of obtaining them, and pharmaceutical composition based on them and capable of staying in blood flow for prolonged time. , where R is -CH(CHO)CH2OH, -CH2CHO, -CH(CH2NHR1)CH2OH, -CH(CH2NHNHR1)CH2OH, -CH(CH=NNHR1)CH2OH, -CH2CH2NHR1, -CH2CH=N-NHR1, -CH2CH2NHNHR1; R1 is polypeptide or albumen; GlyO is a sialic acid bond; R3 is H; R4 is OH; n is 2 or more.
EFFECT: obtaining pharmaceutical composition based on aldehyde derivatives of sialic acid capable of staying in blood flow for prolonged time.
20 cl, 7 tbl, 22 dwg, 10 ex
FIELD: medicine; pharmacology.
SUBSTANCE: invention group refers to compositions containing hapten-carrier conjugate within arranged and repeating matrix, and method of related composition production. Offered hapten-carrier conjugate used for induction of agent-specified immune reaction in case of addiction or abuse, contains cortex particle including at least one first apposition site, where specified cortex particle is virus-like particle of RNA-phage, and at least one nicotine hapten with at least one second apposition site, where specified second apposition site is associated by at least one covalent non-peptide bond with specified first apposition site, thus forming arranged and repeating hapten-carrier conjugate. Offered conjugates and compositions under this invention can include virus-like particles connected to various haptens including hormones, toxins and agent, especially agents causing addiction, as nicotine and can be applied for induction of hapten immune reaction for therapeutic, preventive and diagnostic purposes.
EFFECT: vaccines can induce stable immune reactions for nicotine and fast reduce nicotine availability for brain absorbing.
31 cl, 6 dwg
FIELD: medicine, virology, immunology, molecular biology.
SUBSTANCE: invention involves a composition comprising a regulated and repeated matrix of antigens or antigen determinants and, in particular, matrix comprising RANKL protein, RANKL fragment or RANKL-VLP peptide. Invention relates to a composition comprising viral-like particle and at least one RANKL protein, RANKL fragment or RANKL peptide bound with its, and to a method for preparing conjugates and regulated and repeated matrices, respectively. Proposed compositions can be used for preparing vaccines used in treatment of bone diseases and as a pharmaceutical vaccine used for prophylaxis or treatment of bone diseases, and for effective induction of immune responses, in particular, humoral responses. The advantage of invention involves enhancing induction of immune responses to RANKL protein.
EFFECT: valuable biological and medicinal properties of matrices.
28 cl, 7 dwg, 20 ex
SUBSTANCE: invention concerns medicine, namely to creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria. The immunogene compositions containing a transferrin-binding fiber and Hsf, and a way of their reception are offered. It is shown, that the combination of these two antigens synergically influences on production of antibodies with high activity in the analysis of bactericidal Serum. The composition can be used in vaccines against Gram-negative bacteria, including Neisseria meningitides, Neisseria gonorrhoeae.
EFFECT: creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria.
56 cl, 10 ex, 1 dwg