WO2009056075A1 - Composition pharmaceutique comprenant la protéine nmb0873 - Google Patents

Composition pharmaceutique comprenant la protéine nmb0873 Download PDF

Info

Publication number
WO2009056075A1
WO2009056075A1 PCT/CU2008/000008 CU2008000008W WO2009056075A1 WO 2009056075 A1 WO2009056075 A1 WO 2009056075A1 CU 2008000008 W CU2008000008 W CU 2008000008W WO 2009056075 A1 WO2009056075 A1 WO 2009056075A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
nmb0873
pharmaceutical composition
composition according
neisseria
Prior art date
Application number
PCT/CU2008/000008
Other languages
English (en)
Spanish (es)
Inventor
Maité DELGADO ESPINA
Olivia Niebla Perez
Darién GARCIA DIAZ
Sonia Gonzalez Blanco
Gretel SARDIÑAS GARCIA
Original Assignee
Centro De Ingenieria Genetica Y Biotecnologia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centro De Ingenieria Genetica Y Biotecnologia filed Critical Centro De Ingenieria Genetica Y Biotecnologia
Publication of WO2009056075A1 publication Critical patent/WO2009056075A1/fr

Links

Classifications

    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria

Definitions

  • the present invention is related to the branch of medicine, particularly with the development of a pharmaceutical composition, of preventive or therapeutic application, which allows an increase in the quality of the immune response against vaccine antigens characteristic of diseases of origin diverse.
  • Neisseria 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 vaccine based on polysaccharides, which confers protection against serogroups A, C, Y, and W-135 ha been licensed in the United States.
  • the antibodies that are generated after vaccination are serogroup-specific (Rosenstein N. et 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.
  • 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).
  • the development of vaccines against serogroup B has focused on the use of subcapsular antigens.
  • 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 a mouse model. I infected Immun. 46 (2): 408-14136).
  • VME-based vaccines Several efficacy studies have been carried out using VME-based vaccines, in different formulations. The two most widely studied vaccines were developed in the 1980s, in response to outbreaks of meningococcal disease in Cuba (Sierra GV e ⁇ al. (1991).
  • Vaccine against group B Neisseria meningi ⁇ idis protection trial and mass vaccination resuits in Cuba. NIPH Ann Dis. 14 (2): 195-210) and Norway (Bjune G, ei al. (1991). Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway. Lance ⁇ 338 (8775): 1093-6), respectively.
  • 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 meningifidis protection trial and mass vaccination resuits in Cuba. NIPH Ann Dis. 14 (2): 195-210). This vaccine contributed to a rapid decline in the epidemic in Cuba (Rodr ⁇ guez AP, e ⁇ al. (1999). The epidemiological impact of antimeningococcal B vaccination in Cuba. Mem Inst Oswaldo Cruz. 94 (4): 433-40).
  • the vaccine produced by the National Institute of Public Health of Norway (NIPH) was initially used during a hyperendemic period of the disease caused by a strain belonging to clone ET-5 (B: 15: P1.7,16).
  • This monovalent vaccine was also produced from purified VME and adsorbed to aluminum hydroxide (Bjune G, et al. (1991). Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway. Lancei. 338 (8775): 1093 -6).
  • 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. Antibody responses generated increased the opsonophagocytosis of the meningococcus.
  • the precise formulation of the vaccines (for example: content of PME 1 content of LPS 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.
  • P1 protein is an antigen with a significant level of variability, which seems to undergo continuous variation between and during outbreaks (Jelfs J, ei. (2000). Sequence Variation in the porA Gene of a Clone of Neisseria meningitidis during Epidemic Spread Clin Diagn Lab Immunol. 7 (3): 390-5).
  • the bactericidal antibodies are predominantly directed after vaccination and after the disease.
  • its variability makes it questionable that the protection product of the immunization with VME vaccines of a single strain (monovalent) is broad spectrum.
  • RIVM VME vaccine in the Netherlands
  • VME vaccines The discrete cross-reactivity induced by VME vaccines has encouraged the search for an outer membrane antigen (or a group of antigens), which induces functional antibodies and is present in all strains of meningococcus. These antigens must be the basis for a truly universal meningococcal vaccine, which will eliminate the potential problem of capsular modification in pathogenic strains, after vaccination with polysaccharide. Due to the variability of the immunodominant protein P1, its use in a universal vaccine is limited and therefore other majority PMEs were considered candidates for a vaccine and many of them are in development. Some of those that have been included are: iron-regulated proteins (TbpA and B, FbpA and FetA), NspA and class 5 proteins (Opc).
  • 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 Neisseria meningitidis. J Med Microbiol 47 (9): 757- 60) and is a more effective immunogen than TbpB.
  • the NspA protein a minor, highly conserved PME, has been discovered through a novel technique, which consists of using combinations of PME from different strains to immunize mice (Martin D, et al. (1997). Highly conserveed Neisseha meningitidis Surface Protein Confers Protection against Experimental Infection.
  • 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 1 et al. (1997). Highly conserveed Neisseria 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).
  • Genome sequencing of MC58 a strain of serogroup B meningococcus (Tettelin H, et al. (2000). Complete Genome Sequence of Neisseria meningitidis Serogroup B Strain MC58. Science 287 (5459): 1809-15172), and of Z2491 , a strain of serogroup A (Parkhill J, ei al. (2000). Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404 (6777): 502-6173), were published during the year 2000.
  • the components of a vaccine should be selected based on the contribution of the antigens in the pathogenesis of N. meningitidis.
  • the antigens alone they can be effective vaccine candidates, or alternatively, the 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.
  • compositions comprising 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.
  • pharmaceutical compositions, of a therapeutic or preventive nature, that comprise the NMB0873 protein are reported.
  • the invention relates to pharmaceutical compositions for preventing or treating any infection caused by a bacterium of the Neisser ⁇ a genus, which comprise said protein.
  • pharmaceutical compositions comprising said antigen are useful for the prevention or treatment of diseases caused by N. meningitidis and N. gonorrhoeae.
  • the object of the present invention is a pharmaceutical composition wherein the NMB0873 protein is present in a range between 0.5-100 ⁇ g / dose, in a pharmaceutically acceptable excipient.
  • Said composition optionally comprises a vaccine adjuvant, capable of enhancing the immune response against the active ingredient, the NMB0873 protein.
  • the pharmaceutical compositions comprising the NMB0873 protein 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-conjugates. polysaccharide, whose polysaccharide component corresponds to a bacterial polysaccharide.
  • composition comprising
  • NMB0873 also contains antigens of a peptide nature, in order to broaden the protection spectrum of said composition.
  • This invention reveals a pharmaceutical composition characterized by being a vaccine capable of generating in the recipient organism a protective response against infections caused by bacteria of the Neisseria genus. More particularly, the pharmaceutical composition of the present invention is a vaccine capable of generating in the recipient organism a protective response against infections caused by Neisseria meningitidis or Neisseria gonorrhoeae.
  • the compositions of the present invention are administered parenterally, or mucosally, including oral administration.
  • the NMB0873 protein can be used in combination or fused with other antigens.
  • the protein acts as immunopotentiator or carrier of peptides, polysaccharides or other antigens of lower immunogenicity, in order to enhance the immune response against them.
  • Example 12 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. It is also part of the present invention, that the protective determinants for a given protein antigen be inserted into the sequence of the NMB0873 protein, in order to induce an immune response imcremented 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 NMB0873 protein, which are capable of generating in the host a protective response against meningococcus or another bacterium of the Neisseria genus, in the presence of pharmaceutically acceptable excipients.
  • the pharmaceutical compositions contain mimotopes or mimetic peptides of the NMB0873 protein, in synthetic form or obtained by recombinant DNA technology.
  • mimotope means any peptide that is capable of generating antibodies that combine with the NMB0873 protein, and in that way are capable of producing a protective response against Neisseha.
  • a method for the diagnosis of the infection caused by bacteria of the Neisseria genus that comprises the detection of the NMB0873 protein, or its fragments, or of the gene coding for the NMB0873 protein, identified as Seq. ID. No 3, independently or in conjunction with other components, within a biological sample obtained from an individual.
  • Vector pM238 used in the cloning and expression of the NMB0873 protein.
  • Figure 2 Final construction obtained from the cloning of the nucleotide sequence corresponding to the NMB0873 gene in the vector pM238.
  • Figure 3. Analysis by SDS-PAGE of the fractions obtained in the cell rupture; lane 1: molecular weight standard (PM); lane 2: Control of expression of the NMB0873 protein (Mi); lane 3: Breaking supernatant (SR); lane 4: Rupture precipitate (PR); lane 5: Unrelated control of the expression (NR).
  • PM molecular weight standard
  • Mi Control of expression of the NMB0873 protein
  • SR Breaking supernatant
  • PR Rupture precipitate
  • NR Unrelated control of the expression
  • Figure 4 Analysis of the purity of the different fractions of the purification process of the NMB0873 recombinant protein, by SDS-PAGE: Lane 1: Solubilization Supernatant (S6M); lane 2, fraction not adsorbed (pass); lane 3: fraction eluted with a buffer containing 10 mM Imidazole (1OmM); lane 4: fraction eluted with a buffer containing 100 mM Imidazole (10OmM); lane 5: eluted fraction with buffer containing 300 mM Imidazole (30OmM); lane 6: fraction eluted with buffer containing 1 M Imidazole (1M); lane 7: Molecular weight pattern.
  • Lane 1 Solubilization Supernatant (S6M); lane 2, fraction not adsorbed (pass); lane 3: fraction eluted with a buffer containing 10 mM Imidazole (1OmM); lane 4: fraction eluted with a buffer containing 100 mM Imidazo
  • Antibody levels (IgG) against the recombinant protein NMB0873 obtained by immunizing mice with the same antigen adjuvant with Aluminum hydroxide (Alum); Freund's adjuvant (Freund), or mixed with the serogroup C polysaccharide (PoIiC) of N. meningitidis, subcutaneously.
  • 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 sample (individual sera) where the optical density of the preimmune serum sample is tripled.
  • Figure 6 Recognition of antigenic determinants present in PMEs of N.
  • Figure 9 Passive protection experiment against meningococcal infection in the infant rat model, using sera obtained by immunizing with the recombinant protein NMB0873 adjuvant with Aluminum hydroxide; Freund and mixed with polysaccharide C (PoIiC) from N. meningitidis.
  • the strain Z4181.C- was used: mixture of sera from untreated animals, C +: mouse serum immunized with outer membrane vesicles of N. meningitidis of strain Z4181.
  • 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 Passive protection experiment against meningococcal infection in the infant rat model, using sera obtained by immunizing with the recombinant protein NMB0873 adjuvant with Aluminum hydroxide; Freund and mixed with polysaccharide C (PoIiC) from N. mening
  • Antigens P1, class 1 protein of N. meningitidis strain B: 4: P1.15; P64k, P64k protein of N. meningitidis; TT, tetanus toxoid; HBsAg, surface antigen of the Hepatitis B virus.
  • the results are represented as the absorbance (492nm) in an ELISA type assay.
  • FIG 11. Recognition of the NMB0873 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 JY1 anti-peptide antibodies corresponding to the sera of animals immunized with the free peptide (JY1), the recombinant protein (NMB0873) and the conjugate JY1-NMB0873.
  • Example 1 Detection of the NMB0873 protein in preparations of outer membrane vesicles of Neisseria meningitidis, serogroup B.
  • 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 ESI-MS spectra was performed using the ProFound program (Zhang W and Chait BT. (2000).
  • ProFound an expert system for protein identification using mass spectrometric peptide mapping information.
  • Example 3 Cloning and expression in Escher ⁇ chia coli of the nmbO873 gene, coding for the NMB0873 protein of Neisseria meningitidis.
  • the vector pM238 was used. 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 a), sequence coding for a tail of C-terminal histidine, 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 gene / acZ subunit alpha.
  • oligonucleotides (0873U and 0873L) were designed to amplify the segment of said gene without the sequence encoding the signal peptide, using the genomic DNA of The strain B: 4: P1.19.15.
  • Xba ⁇ 0873U 5 ' CGTTTTCT AG ATCTCGCGC AATTACCTC ' 3
  • the NMB0873 protein is obtained fused with the N-terminal segment of the P64k.
  • Sequence sequencing of the cloned nmbO873 gene was performed using the automatic sequencer ALFexpressIl (Thermo Sequenase TM Cy TM 5 Dye Terminator Kit, Amersham Biosciences) and oligonucleotides 1573 (No. Sequence identification: 8) and 6795 (No. Sequence identification : 9), which hybridize in the sequence corresponding to the stabilizing segment of the P64k and in the transcription terminator of the bacteriophage T4, respectively.
  • the plasmid obtained was named pM238-NMB0873 for later use.
  • the strain of E. coli GC 366 was transformed by the chemical method with plasmid pM238-NMB0873 ( Figure 2).
  • the expression experiment was conducted in minimal M9 saline medium (Miller JH. 1972. Experiments in Molecular Genetics, CoId Spring Harbor Laboratory Press, NEW York, USA) supplemented with 1% glycerol, 1% casein hydrolyzate, CaCI 2 0.1 mM, 1mM MgSO 4 and 50 ug / mL ampicillin.
  • the NMB0873 protein was obtained in the rupture precipitate, representing 60% of the total proteins present in this fraction ( Figure 3).
  • the NMB0873 protein present in the rupture precipitate is solubilized.
  • the supernatant after solubilization was subjected to affinity chromatography by metal chelates to perform the purification of the protein of interest and a sample was obtained where the molecular species that migrates in the expected size corresponds to 85%.
  • Figure 4 shows the electrophoretic pattern of some samples taken in the purification process. Finally, dialysis was performed and its evaluation was carried out in laboratory animals.
  • Example 4 Evaluation of the immune response induced by the NMB0873 protein administered subcutaneously.
  • mice To evaluate the immunogenicity of the NMB0873 protein, an immunization scheme was designed in mice, in which the protein adjuvant with aluminum hydroxide, Freund's adjuvant or combined with the serogroup C polysaccharide of N. meningitidis was administered. With these preparations mice were immunized
  • mice Female Balb / c, 8 to 10 weeks old, which were divided into 3 groups of 10 mice each. Three immunizations were performed subcutaneously, separated by an interval of 7 days. Table 1 describes the composition administered to each group:
  • the antibody titers (IgG) against the recombinant protein and against antigens present in the bacterium were determined by an ELISA type assay, in sera obtained after the third 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 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 an outer membrane protein preparation (PME) of strain H44 / 76 (serogroup B) and Serogroup A. These results are shown in Figure 6.
  • PME outer membrane protein preparation
  • Example 5 Characterization of the sequence of the gene coding for the NMB0873 protein in different strains of N. meningit ⁇ dis and in N. gonorrhoeae.
  • Figure 7 shows the results of the sequence comparison for those sequences that produce a significant alignment in each of the genomes analyzed. Said sequences have 100% identity in serogroups B, 95% identity in serogroup A, and 96% in the case of N. gonorrhoeae, with the sequence of the gene coding for the NMB0873 protein obtained (No. Identification Sequence: 3). Additionally, the nucleotide sequence of the gene in question was determined for 3 Cuban isolates (No. Sequence identification: 5-7) belonging to serogroup B (B: 4: P1.19,15) and sequence alignment was performed using the program ClustalX (http://www.ebi.ac.uk/clustalw/).
  • Example 6 Characterization of the immune response of a broad spectrum of action induced by the immunization of Balb / c mice with the NMB0873 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 NMB0873 protein in the three compositions, as described in Example 4.
  • Figure 8 shows the recognition of antigens present in strains of serogroups B and C of N. meningitidis, by sera obtained after immunization with the recombinant protein NMB0873 adjuvant with Freund's Adjuvant. The sera generated after the inoculation of the protein in the other two compositions had a similar behavior.
  • Example 7 Protection induced by murine sera generated against the NMB0873 protein in the infant rat model.
  • Example 8 Immunogenicity of the NMB0873 protein in an animal model of neonatal immunization.
  • mice 7 days old is considered a model that reproduces the characteristics of the humoral immune response in the human neonatal stage, for many antigens. Therefore, 3 groups of mice (6 mice / group) were immunized, at 7, 10 and 14 days after birth.
  • the mice of the first group, in each dose received 10 ⁇ g of the NMB0873 protein adjuvant with aluminum hydroxide (400 ⁇ g / dose).
  • mice of the second group in each dose received 10 ⁇ g of the NMB0873 protein adjuvant with aluminum phosphate (400 ⁇ g / dose), while the mice of the third group only received 400 ⁇ g / dose of aluminum hydroxide without antigen.
  • mice of the third group At 21 days of age blood samples were obtained from the mice and the serum was analyzed by ELISA, to determine the levels of antibodies against Ia NMB0873 protein.
  • the anti-NMB0873 antibody titers detected in the three groups of mice are shown below.
  • Example 9 Generation of monoclonal antibodies against the NMB0873 protein, capable of mediating bactericidal activity against Neisser ⁇ a meningitidis.
  • mAbs monoclonal antibodies
  • a preparation of the NMB0873 protein was used in an immunization scheme with a 85% purity percent.
  • 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 NMB0873 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; on day 50, they were administered 10 .mu.g of antigen per mouse in Phosphate Buffered Saline (140 mM NaCl, 270 mM KCl, KH 2 PO 4 1.5 mM, Na 2 HPO 4 x 2H 2 O 6.5 mM, pH 7.2) via ntraperitoneal.
  • Phosphate Buffered Saline 140 mM NaCl, 270 mM KCl, KH 2 PO 4 1.5 mM, Na 2 HPO 4 x 2H
  • Extractions were performed on days 0 and 45 of the scheme.
  • 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 (3H2 / 64 and 7D2 / 15) had bactericidal titres greater than 1: 128 against the homologous strain B: 4: P1.19,15 and one (H8 / 92) greater than 1: 80. They also had titers greater than 1: 64 against heterologous strains whose classifications are B: 15: P1.7,16 and C: 2a: P1.5, respectively.
  • Example 10 Characterization of the target regions of the murine immune response against the NMB0873 protein.
  • 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 reaction antibody was detected by incubation with an alkaline murine phosphatase G-conjugated anti-immunoglobulin conjugate, followed by the addition of a solution containing the Bromo-Chloro-Indolyl-Phosphate substrate.
  • Example 11 Recognition of the NMB0873 protein by human sera.
  • the plates were coated with the NMB0873 protein obtained by preparative electrophoresis (5 ⁇ g / ml). After blocking the plates with 3% skimmed milk powder in PBS with Tween-20, the sera were diluted (1: 50) in the same solution and incubated on the plates.
  • the immunoassay continued as has been widely reported.
  • As a negative control sera from healthy donors were used.
  • a mixture of sera from vaccines with recombinant vaccine against Hepatitis B was also used as an unrelated control.
  • Figure 11 shows the results obtained with 5 convalescent sera in this test.
  • human sera recognized the Io protein that indicates that it is expressed during meningococcal infection and that it is immunogenic.
  • Example 12 Protein NMB0873 as a carrier of a peptide.
  • a synthetic peptide of 15 amino acid residues was conjugated thereto, derived from the V3 region of the gp120 protein of HIV-1, JY1 isolation. The conjugation was performed by the glutaraldehyde method. The free JY1 peptide, the recombinant protein NMB0873 and the conjugate JY1-NMB0873, was administered to adult mice in a 3 dose scheme, where the immunogens were emulsified with Freund's Adjuvant.
  • Example 13 Evaluation of the immune response induced by the NMB0873 protein, mucosally.
  • mice To evaluate the immunogenicity of the NMB0873 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 administered. Liposomes were obtained by the dehydration-rehydration method as previously described (Carménate T, et al. (2001). Recombinant Opc protein from Neisser ⁇ a meningitidis reconstituted into liposomes elicits opsonic antibodies following immunization. Biotechnol. Appl. Biochem. 34: 63-69).
  • mice Female Balb / c mice were immunized, from 8 to 10 weeks of age, which received 3 doses of 50 ⁇ g of the protein, intranasally, separated by an interval of 15 days.
  • IgA antibodies present in samples of lung washes from immunized mice were detected.
  • Figure 13 shows the levels of IgA antibodies detected in the two groups analyzed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne le domaine de la médecine, et notamment la mise au point d'une composition pharmaceutique comprenant la protéine NMB0873. La composition de la présente invention confère une protection contre diverses maladies causées ou non par des agents pathogènes. La protéine NMB0873 a été identifiée comme constituant des préparations de vésicules de membrane externe (VME) de Neisseria meningitidis. Elle a été obtenue au moyen de la technique de l'acide désoxyribonucléique (ADN) recombinant et son immunogénicité et son activité protectrice ont été évaluées chez des modèles animaux. Compte tenu du haut degré de conservation du gène codant pour la protéine NMB0873, la composition comprenant cette dernière présente une valeur élevée en tant qu'antigène induisant une réponse immunitaire à large réactivité. La composition de l'invention peut être utilisée en médecine humaine.
PCT/CU2008/000008 2007-10-30 2008-10-17 Composition pharmaceutique comprenant la protéine nmb0873 WO2009056075A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CU2007-0239 2007-10-30
CU20070239 2007-10-30

Publications (1)

Publication Number Publication Date
WO2009056075A1 true WO2009056075A1 (fr) 2009-05-07

Family

ID=40398329

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CU2008/000008 WO2009056075A1 (fr) 2007-10-30 2008-10-17 Composition pharmaceutique comprenant la protéine nmb0873

Country Status (1)

Country Link
WO (1) WO2009056075A1 (fr)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EPO Proteins [online] 31 May 2006 (2006-05-31), "Sequence 7089 from Patent WO2000071725.", XP002518352, retrieved from EBI accession no. EPOP:CS295645 Database accession no. CS295645 *
DATABASE Geneseq [online] 7 March 2003 (2003-03-07), "N. gonorrhoeae amino acid sequence SEQ ID 3428.", XP002518354, retrieved from EBI accession no. GSP:ABP78449 Database accession no. ABP78449 *
DATABASE UniProt [online] 1 December 2000 (2000-12-01), "RecName: Full=Outer-membrane lipoprotein lolB; Flags: Precursor;", XP002518353, retrieved from EBI accession no. UNIPROT:P57024 Database accession no. P57024 *
DATABASE UniProt [online] 15 March 2005 (2005-03-15), "SubName: Full=Putative lipoprotein carrier;", XP002518355, retrieved from EBI accession no. UNIPROT:Q5F9F7 Database accession no. Q5F9F7 *

Similar Documents

Publication Publication Date Title
ZA200604492B (en) Protein NMB0928 and use thereof in pharmaceutical formulations
WO2007112702A2 (fr) Composition pharmaceutique comprenant la protéine nmb0938
WO2007112703A2 (fr) Composition pharmaceutique comprenant la protéine nmb0606
WO2009000217A1 (fr) Composition pharmaceutique contenant la protéine nmb1796
WO2007073705A2 (fr) Compositions pharmaceutiques contenant la proteine nma0939
WO2009056075A1 (fr) Composition pharmaceutique comprenant la protéine nmb0873
ES2334138T3 (es) Proteina nmb1125 y uso de la misma en formulaciones farmaceuticas.
MXPA06006267A (en) Protein nmb0928 and use thereof in pharmaceutical formulations
MX2008008580A (en) Pharmaceutical compositions containing protein nma0939
MXPA06006266A (en) Protein nmb1125 and use thereof in pharmaceutical formulations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08843746

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08843746

Country of ref document: EP

Kind code of ref document: A1