Classifications

• • 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
  • A61K39/02—Bacterial antigens
  • A61K39/095—Neisseria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • 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
  • A61K39/385—Haptens or antigens, bound to carriers
• • 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
• • 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
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • 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
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6068—Other bacterial proteins, e.g. OMP
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

• the present invention is related to the branch of medicine, particularly with the development of pharmaceutical compositions, of preventive or therapeutic application, which allow an increase in the quality of the immune response against vaccine antigens characteristic of diseases of diverse origin. .
• Neisser ⁇ a meningitidis a Gram negative diplococcus whose only host is man, is the causative agent of meningococcal disease. Usually this bacterium is found in the nasopharynx of people who are asymptomatic carriers, this being the most common route for their microbiological isolation. In the world, children under 2 years of age are the population most susceptible to contracting meningococcal meningitis, however, young adolescents and the population of older adults can also be affected. Meningococcal disease without treatment is fatal in the majority of affected individuals, and vaccination could prevent this situation by avoiding even bacterial colonization.
• Capsular polysaccharides have been studied and evaluated as vaccine candidates.
• a tetravalent, polysaccharide-based vaccine that confers protection against serogroups A, C, Y, and W-135 has been licensed in the United States.
• the antibodies that are generated after vaccination are serogroup-specific (Rosenstein N. ef al. (2001). Meningococcal disease. N. Engl. J. Med 344: 1378-1388).
• Serogroup B unlike the rest, continues to be an important cause of endemic and epidemic meningococcal disease, largely due to the absence of effective vaccines against it. It has been seen that serogroup B polysaccharide has a low immunogenicity, in addition to the potential risk that vaccines based on this compound can develop immunotolerance and / or induce autoimmunity given its structural homology with oligosaccharide chains present in human fetal structures (Finne J . et al. (1987). An IgG monoclonal antibody to group B meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissue. J. Immunol 138: 4402-4407).
• Vaccines composed of outer membrane vesicles were significantly more immunogenic parenterally than PME aggregates, and this immunogenicity was initially explained by greater adsorption to the aluminum hydroxide adjuvant (Wang LY and Frasch CE. (1984). Neisseria meningitidis group B serotype 2b protein vaccine and evaluation in mouse model Infeci Immun. 46 (2): 408-14136).
• the vaccine produced by the Finlay Institute in Cuba (commercially called VA-MENGOC-BC ® ) is produced from strain B: 4,7: P1,19,15 and is composed of a VME preparation of said strain and polysaccharide capsule isolated from serogroup C, adsorbed to aluminum hydroxide (Sierra GV et al. 1991. Vaccine against group B Neisseria meningitidis: protection trial and mass vaccination results in Cuba. NIPH Ann Dis. 14 (2): 195-210). This vaccine contributed to a rapid decline in the epidemic in Cuba (Rodr ⁇ guez AP, et al. (1999). The epidemiological impact of antimeningococcal B vaccination in Cuba. Mem Inst Oswaldo Cruz.
• VME vaccines appear to be effective in the presentation of PME, arranged in their natural conformation, to allow the generation of bactericidal antibodies, at least in adolescents and adults.
• the antibody responses generated increased the opsonophagocytosis of the meningococcus.
• the precise formulation of the vaccines (for example: PME content, LPS content and the presence or absence of the adjuvant) has a significant impact on the immunogenicity, there being great differences from one producer to another according to the strain and / or the methodology used (Lehmann AK, et al. (1991). Immunization against serogroup B meningococci. Opsonin response in vaccinees as measured by chemiluminescence. APMIS.
• VME vaccines have been more used than any other serogroup B vaccine and are useful in the context of outbreaks of the disease caused by a single type of strain.
• the immunogens responsible for cross-reactivity induced by this type of preparations have not been fully characterized, and many antigens present in these preparations remain to be identified.
• VME vaccine in the Netherlands was developed that contained P1 of six different pathogenic isolates (van der Ley P and Poolman JT. (1992). Construction of a multivalent meningococcal vaccine strain based on the class 1 outer membrane protein Infectious Immun. 60 (8): 3156-61; Claassen I, et al. (1996). Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer membrane protein containing vesicle vaccine. Vaccine 14 (10): 1001-8). In this case, the vesicles were extracted from two variants of the strain H44 / 76, genetically manipulated to express three independent P1 proteins. The search for a universal antigen
• TbpA and B proteins regulated by iron
• FbpA and FetA proteins regulated by iron
• NspA proteins regulated by class 5 proteins
• Opc class 5 proteins
• TbpB is part of the transferrin binding complex, together with TbpA.
• TbpA has a more important function in iron binding (Painter M, et al. (1998). Analysis of TbpA and TbpB functionality in defective mutants of Neisser ⁇ a meningitidis. J Med Microbio! 47 (9): 757-60) and is a more effective immunogen than TbpB.
• NspA protein a minor, highly conserved PME
• PME PME
• J Exp Med 185 (7): 1173-83 B cells from immunized mice were used, and the mAbs were examined to detect cross-reactivity against multiple strains of meningococcus.
• a monoclonal antibody with cross-reactivity was found that recognized a 22 kDa PME and was designated as NspA.
• mice with NspA induced bactericidal antibody response against strains of groups A to C. This protein also protects against lethal meningococcal infection (Martin D, et al. (1997). Highly conserveed Neisser ⁇ a meningitidis Surface Protein Confers Protection against Experimental Infection. J Exp Med 185 (7): 1173-83).
• the comparison of genetically divergent NspA sequences showed that the protein is highly conserved (97% homology) (Cadieux N, et al. (1999). Bactericidal and Cross-Protective Activities of a Monoclonal Antibody Directed against Neisser ⁇ a meningitidis NspA Outer Membrane Protein Infect Immun 67 (9): 4955-9).
• NspA The presence of NspA was detected by ELISA in 99.2% of the evaluated strains belonging to serogroups from A to Ia C, using monoclonal antibodies (Martin D, et al. (1997). Highly conserveed Neisser ⁇ a meningitidis Surface Protein Confers Protection against Experimental Infection. J Exp Med 185 (7): 1173-83). It has been shown that these monoclonal antibodies have bactericidal activity against numerous strains of meningococcus and are capable of reducing the bacteraemia caused by this microorganism in a murine model (Cadieux N, et al. (1999).
• the components of a vaccine should be selected based on the contribution of the antigens in the pathogenesis of N. meningitidis.
• the antigens alone can be effective vaccine candidates, or alternatively, attenuated mutants can be considered members of a vaccine.
• An important problem of the prevention and / or the therapy of meningococcal disease is that none of the vaccines available to date confers universal protection, due to the great heterogeneity of the meningococcal antigens that have been used as a vaccine.
• This invention contributes to solving the aforementioned problem by providing pharmaceutical compositions containing a protein whose sequence is highly conserved, even between different genera of pathogenic microorganisms.
• the technical objective pursued with this invention is precisely the development of compositions capable of raising and / or extending the host's systemic and mucosal immune response against several pathogens, or against a broad spectrum of varieties thereof.
• the use of the NMA0939 protein in pharmaceutical compositions of a therapeutic or preventive nature is reported for the first time.
• the invention relates to pharmaceutical compositions, containing said protein, to prevent or treat any infection caused by a bacterium of the Neisseria genus.
• pharmaceutical compositions containing said antigen are useful for the prevention or treatment of diseases caused by N. meningitidis and N. gonorrhoeae, respectively.
• compositions containing the NMA0939 protein may also contain one or more antigens. of a synthetic, recombinant or natural nature.
• the combined pharmaceutical compositions contain polysaccharide antigens, including bacterial polysaccharides. More particularly, the invention relates to the capsular polysaccharides of N. meningitidis.
• the pharmaceutical composition of the present invention may contain protein-polysaccharide conjugates, whose polysaccharide component corresponds to a bacterial polysaccharide.
• compositions containing NMA0939 contain antigens of a peptide nature, in order to broaden their protection spectrum.
• compositions of the present invention are administered parenterally, or mucosally, including oral administration.
• the NMA0939 protein can be used as immunopotentiator or carrier of peptides, polysaccharides or other antigens of less immunogenicity, in order to enhance the immune response against them.
• Example 11 illustrates that the aforementioned protein is capable of significantly raising antibody levels against a peptide derived from a viral antigen, once both molecules have been conjugated.
• the protective determinants for a given protein antigen be inserted into the sequence of the NMA0939 protein, in order to induce an increased immune response against them, giving rise to hybrid proteins that are part of A pharmaceutical composition
• the pharmaceutical compositions of the present invention may contain fragments of the NMA0939 protein, which are capable of generating in the host a protective response against meningococcus or another bacterium of the Neisseria genus.
• the pharmaceutical compositions contain mimotopes or mimetic peptides of the NMA0939 protein, in synthetic form or obtained by recombinant DNA technology.
• micromotope means any peptide that is capable of generating antibodies that combine with the NMA0939 protein, and in that way are capable of producing a protective response against Neisseria. Also part of the present invention is the detection of the meningococcal disease through the use of pharmaceutical components containing the NMA0939 protein, or the gene encoding it, either alone or in combination with other components.
• Figure 1 Vector pM238 used in cloning and expression of the NMA0939 protein.
• Figure 4 Analysis of the purity of the different fractions of the purification process of the recombinant protein NMA0939, from the rupture precipitate, by SDS-PAGE: Lane 1, precipitate of solubilization with 2M urea in carbonate-bicarbonate buffer; lane 2, solubilization supernatant; lane 3, fraction not adsorbed to the purification matrix; lane 4, pollutant of lower molecular weight eluting at another chromatographic peak; lane 5, purified protein. PM: molecular weight standard.
• Figure 5 Analysis of the purity of the different fractions of the purification process of the recombinant protein NMA0939, from the rupture precipitate, by SDS-PAGE: Lane 1, precipitate of solubilization with 2M urea in carbonate-bicarbonate buffer; lane 2, solubilization supernatant; lane 3, fraction not adsorbed to the purification matrix; lane 4, pollutant of lower molecular weight eluting at another chromatographic peak; lane 5, purified
• Antibody levels (IgG) against the recombinant protein NMA0939 obtained by immunizing mice with the same antigen adjuvant with Freund's Adjuvant (Freund), Aluminum hydroxide (Alum) or polysaccharide C (PsC) of N. meningitidis, by intraperitoneal route.
• the results obtained in an ELISA type test are represented, which were expressed as the inverse of the title, calculated as the dilution of the serum sample where the optical density of the preimmune serum sample is doubled.
• Figure 6 Recognition of antigenic determinants present in the PME of N. meningitidis, strain CU385, using sera from mice immunized with the recombinant protein NMA0939 adjuvant with Freund's Adjuvant (Freund), Aluminum hydroxide (Alum) or polysaccharide C (PsC) intraperitoneally. The results were expressed as the inverse of the title, calculated as the dilution of the serum where the optical density of the preimmune serum is doubled.
• Figure 7 Results of the search for similarity between the gene coding for the NMA0939 protein (“query”) and the annotated sequences of the genomes of different serogroups of N. meningitidis (“Sbjct”) using the BLAST program.
• NMA0939 mixed with polysaccharide C of N. meningitidis intraperitoneally.
• Figure 9 Passive protection experiments against meningococcal infection in the infant rat model, using sera obtained by immunizing with the recombinant protein NMA0939 with Freund's Adjuvant (Freund), with Aluminum hydroxide (Alum) or mixed with polysaccharide C (PsC ) of N. meningitidis.
• A Infection with strain CU385, and B: Infection with strain 233 (C: 2a: P1.5).
• C- mixture of sera from untreated animals
• C + mouse serum immunized with outer membrane vesicles of N. meningitidis of strain CU385 or 233, as appropriate with the experiment.
• the symbol * represents a statistically significant difference with respect to the negative control group (C-), in terms of bacteremia levels expressed as colony forming units per milliliter (cfu / ml).
• Figure 10 Recognition of the recombinant protein NMA0939 and a panel of unrelated antigens, by the mAbs generated (mAbs H 10/67, 3H3 / 24 and 7D6 / 18).
• Antigens P1, class 1 protein of N. meningitidis strain B: 4: P1.15; P64k, N. meningitidis] TT protein, tetanus toxoid; HBsAg, surface antigen of the Hepatitis B virus.
• the results are represented as the absorbance (492nm) in an ELISA type assay.
• Figure 11 Recognition of the NMA0939 recombinant protein by sera from convalescent patients of meningococcal disease. As a negative control, sera from healthy donors were used. The results are represented as the absorbance (492nm) in an ELISA type test.
• Figure 12 Titles of anti-JY1 peptide antibodies corresponding to the sera of animals immunized with the free peptide (JY1), the recombinant protein (NMA0939) and the conjugate JY1-NMA0939.
• Example 1 Detection of the NMA0939 protein in preparations of outer membrane vesicles of Neisse ⁇ a meningitidis, serogroup B.
• Peptides generated during digestion were extracted from the solution using microcolumns (ZipTips, Millipore, MA, E. U.) Prior to mass spectrometry analysis the peptides were eluted from the microcolumns with 60% acetonitrile solution and 1% formic acid and immediately the mixture was loaded into nano-needles (Protana, Denmark). Measurements were performed on a spectrometer hybrid mass with quadrupole and flight time (QTof-2 TM, Manchester, United Kingdom), equipped with an ionization source (nanoESI). Mass spectra were acquired in a range of m / z from 400 to 2000 in 0.98 seconds and using 0.02 seconds between each of the scans. The acquisition and processing of the data was carried out through the MassLynx program (version 3.5, Micromass).
• Protein identification based on the ESI-MS spectra was performed using the ProFound program (Zhang VV and Chait BT. (2000).
• ProFound an expert system for protein identification using mass spectrometric peptide mapping information.
• Search parameters included cysteine modification as well as possible oxidations and deamidations. From the analysis of the data obtained from the identification of the proteins present in external membrane vesicle preparations, a group of peptides was selected. From the sequencing by mass spectrometry of one of them, the existence in serogroup B of a homologue of the NMA0939 protein was verified, which was not identified or predicted from the genomes of serogroup B. This homologous candidate to NMA0939, It is called NMA0939 throughout the entire document.
• Example 3 Cloning and expression of the nmaO939 gene, coding for the NMA0939 protein of Neisser ⁇ a meningitidis, in Escher ⁇ chia coli.
• the vector pM238 was used to perform the cloning and expression of the nmaO939 gene. Said vector allows cloning using different restriction enzymes, and obtaining high levels of heterologous protein expression in the form of inclusion bodies in the cytoplasm of E. coli.
• the vector pM238 ( Figure 1) has the following main elements: tryptophan promoter, sequence corresponding to the N-terminal stabilizing segment of the P64k antigen of N. meningitidis strain CU385 (coding for 47 aa), sequence encoding a histidine C tail -terminal, sequence corresponding to the terminator of transcription of bacteriophage T4 and sequence corresponding to the gene that confers ampicillin resistance as a selection marker.
• this vector allows the selection of recombinants by means of the blue or white coloration of the colonies, product of the presence of the lac ⁇ subunit alpha gene.
• a pair of oligonucleotides 704467U and 704467L were designed to amplify the segment of said gene without the sequence encoding the signal peptide, using the genomic DNA of strain CU385 (B: 4: P1.19,15).
• Xba-I BgI-II 704467U 5 ' GTGGTATCTAGATCTGCCAGCCAGAAACTC ' 3 (SEQ ID No. 1)
• BamH-l (SEQ ID No. 2)
• the methods described in the SignalP World Wide Web server http://www.cbs.dtu.dk/services/SiqnalP-2.0) were used to predict the signal peptide.
• PCR polymerase chain reaction
• the plasmid obtained was named pM NMA0939 for later use.
• the E. coli GC 366 strain was transformed by the chemical method with the plasmid pM NMA0939 ( Figure 2).
• the expression experiment was performed in minimal M9 saline medium (Miller JH. (1972). Experiments in Molecular Genetics, CoId Spring Harbor Laboratory Press, New York, EU) supplemented with 1% glucose, 1% casein hydrolyzate, extract of 0.1% yeast, 0.1 mM CaCI 2 , 1mM MgSO 4 and 50 ⁇ g / mL ampicillin.
• the cultures were incubated for 12 h at 37 ° C at 250 rpm.
• the NMA0939 protein was obtained in the Rupture precipitate, representing 20% of the total proteins present in this fraction ( Figure 3).
• Samples of the cell precipitate were solubilized with carbonate-bicarbonate buffer (0.1 M sodium carbonate, 0.1 M sodium hydrogen carbonate) containing urea dissolved at different molarities (2M, 4M, 6M and 8M).
• carbonate-bicarbonate buffer 0.1 M sodium carbonate, 0.1 M sodium hydrogen carbonate
• urea urea dissolved at different molarities
• Example 4 Evaluation of the immune response induced by the NMA0939 protein intraperitoneally.
• an immunization scheme was designed in mice, in which the protein adjuvant with aluminum hydroxide, Freund's adjuvant or polysaccharide C of N. meningitidis was administered.
• female Balb / c mice from 8 to 10 weeks of age, were immunized, which were divided into 3 groups of 8 mice each. Three immunizations were performed intraperitoneally, separated by an interval of 7 days.
• the antibody titers (IgG) against the recombinant protein and against antigens present in the bacterium were determined by an ELISA type test, in sera obtained after the booster dose.
• Figure 5 shows the antibody titers of each of the animals against the recombinant protein. From the second inoculation, antibodies capable of recognizing the administered antigen (data not shown) are detected, although they were higher after the last inoculation. The immunological identification was also performed by Western blotting, detecting the recognition of the band corresponding to the protein (data not shown).
• the sera obtained after immunizing with the recombinant protein recognized antigenic determinants present in a PME preparation of strain CU385. These results are represented in Figure 6.
• the antibody titres obtained were transformed to achieve normal distribution of the values and / or homogeneity of variance. In general, the statistical significance of the differences between the groups was analyzed by simple classification variance analysis, followed by the Newman Keuls multiple range test.
• Example 5 Characterization of the sequence of the gene coding for the NMA0939 protein in different strains of Neisser ⁇ a meningitidis and in Neisser ⁇ a gonorrhoeae.
• Said sequences have 99% identity in serogroup B, 100% identity in serogroup A and 96% in the case of N. gonorrhoeae, with the sequence of the gene coding for the NMA0939 protein obtained (SEQ ID No. 3). Additionally, the nucleotide sequence of the gene in question was determined for 3 Cuban isolates (SEQ ID No. 5-7) belonging to serogroup B (B: 4: P1.19.15) and sequence alignment was performed using the ClustalX program (http://www.ebi.ac.uk/clustalw/). The results of the alignment show that there is a great conservation in the nucleotide sequence of the nmaO939 gene between the different strains analyzed and generally in the Neisseria genus.
• NMA0939 protein as a vaccine candidate, taking into account the high degree of similarity between the aforementioned sequences, would allow generating an effective immune response and a broad spectrum of protection against meningococcal disease, product of the wide reactivity.
• Example 6 Characterization of the immune response of a broad spectrum of action induced by the immunization of Balb / c mice with the NMA0939 protein.
• an ELISA type test was carried out in which the polystyrene plates were coated with complete cells of 5 Neisseria strains belonging to different serogroups, serotypes and serosubtypes. The plates were incubated with the mixture of the sera obtained against the NMA0939 protein mixed with different adjuvants, as described in Example 4.
• Figure 8 shows the recognition of antigens present in strains of serogroups A, B and C of N. meningitidis, by sera obtained after immunization with the recombinant protein NMA0939 mixed with polysaccharide C of N. meningitidis.
• the sera generated after the inoculation of the protein with the other adjuvants had a similar behavior.
• Example 7 Protection induced by murine sera generated against protein NMA0939 in the infant rat model
• a passive protection test was performed in the meningococcal infection model in infant rats. In this test, 24 rats of 5 to 6 days old were used, divided into groups of 6 animals each. It was determined if the sera that were administered by the intraperitoneal route protected the rats from the meningococcal infection caused by the strain CU385, inoculated by the same route one hour later. Sera from each group of immunized mice were mixed before being inoculated in infant rats and diluted 1: 10 in sterile PBS. Four hours after the challenge, the animals were sacrificed and a viable bacteria count was made in their blood.
• Example 8 Generation of monoclonal antibodies against the NMA0939 protein, capable of mediating bactericidal activity against Neisser ⁇ a meningitidis
• the immunization scheme was performed in Balb / c mice (H-2 d , female sex, 5-6 weeks) and had a total of 4 doses distributed as follows: on days 0, 15 and 30 of the scheme were inoculated 10 ⁇ g of the NMA0939 antigen per mouse (total volume 100 ⁇ l), administered subcutaneously, emulsified the first dose with Freund's complete adjuvant, and the remaining doses with Freund's Incomplete Adjuvant; Day 50, 10 .mu.g of antigen were administered per mouse in phosphate buffer (NaCl 140 mM, KCI 270 mM, KH 2 PO 4 1.5 mM, Na 2 HPO 4 x 2H 2 O 6.5 mM, pH 7.2) intraperitoneally. Extractions were performed on days 0 and 45 of the scheme.
• Figure 10 shows the results obtained in this experiment, in total 3 positive clones were obtained (mAbs H 10/67, 3H3 / 24 and 7D6 / 18) that specifically recognize the NMA0939 protein, and not the amino acid sequence corresponding to segment N -term of Ia P64k, also to the rest of the panel of unrelated antigens tested.
• bactericidal assay was performed.
• the bactericidal antibody titer was expressed as the reciprocal of the highest antibody dilution evaluated, capable of killing 50% or more of the bacteria;
• Two of the generated mAbs (3H3 / 24 and 7D6 / 18) had bactericidal titres greater than 1: 128 against the homologous strain B: 4: P1.19,15 and one (H10 / 67) greater than 1: 80.
• a SPOTScan type assay was performed. A series of overlapping peptides that cover the protein sequence were synthesized on a cellulose support and the membrane was incubated with a mixture of sera diluted 1: 100. The antigen-antibody reaction was detected by incubation with an alkaline murine-alkaline phosphatase conjugate, followed by the addition of a solution containing the Bromo-Chloro-Indolyl-Phosphate substrate. Several common antigenic regions present in the protein were observed, regardless of the preparation that was used in the immunization (data not shown). However, it was noted that in the groups immunized with protein adjuvant with Freund's Adjuvant a much broader recognition pattern was obtained.
• Example 12 Evaluation of the immune response induced by the NMA0939 protein, mucosally.
• mice To evaluate the immunogenicity of the NMA0939 protein by mucosal route, an immunization scheme was designed in mice, in which the protein encapsulated in liposomes or mixed with polysaccharide C of N. meningitidis was adiministrated. Liposomes were obtained by the dehydration-rehydration method as previously described (Carménate T, et al. (2001).
• mice Female Balb / c mice were immunized, 8 to 10 weeks of age, which received 3 doses of 50 ⁇ g of the protein intranasally, separated by a 15-day interval.
• Figure 13 shows the levels of IgA antibodies detected in the two groups analyzed.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• Immunology (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Genetics & Genomics (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Analytical Chemistry (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Pathology (AREA)
• Physics & Mathematics (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (2)

Family

ID=38218331

Family Applications (1)

Country Status (13)

Families Citing this family (1)

Family Cites Families (4)

• 2005
  • 2005-12-29 CU CU20050279A patent/CU23549A1/es active IP Right Grant
• 2006
  • 2006-12-26 AR ARP060105777A patent/AR058735A1/es unknown
  • 2006-12-28 WO PCT/CU2006/000019 patent/WO2007073705A2/es not_active Ceased
  • 2006-12-28 AU AU2006331224A patent/AU2006331224A1/en not_active Abandoned
  • 2006-12-28 US US12/097,895 patent/US20090208521A1/en not_active Abandoned
  • 2006-12-28 CA CA002633424A patent/CA2633424A1/en not_active Abandoned
  • 2006-12-28 RU RU2008131066/13A patent/RU2008131066A/ru not_active Application Discontinuation
  • 2006-12-28 BR BRPI0620867-3A patent/BRPI0620867A2/pt not_active IP Right Cessation
  • 2006-12-28 CN CNA200680049521XA patent/CN101443037A/zh active Pending
  • 2006-12-28 KR KR1020087018520A patent/KR20080081198A/ko not_active Withdrawn
  • 2006-12-28 EP EP06846929A patent/EP1977761A2/en not_active Withdrawn
• 2008
  • 2008-06-19 ZA ZA200805355A patent/ZA200805355B/xx unknown
  • 2008-07-28 NO NO20083318A patent/NO20083318L/no not_active Application Discontinuation

Non-Patent Citations (38)

Also Published As

Similar Documents

Legal Events