Patent Application
20080069844
The present inventors isolated the flagellin flagellin proteins and subcutaneously injected mice with them (active immunization) to confirm the defensive immunities, and observed a formation of granulomatous lesions in mice skin tissues injected with flagellin proteins subcutaneously. With this result, we confirmed that the flagellin acts as a vaccine adjuvant.
The Flagella, an important factor for determining the mobility of bacteria, is composed of hooks, basal bodies and filaments in general. It is known that the flagella has various functions such as the swimming or swarming motility of bacteria, determining the taxis of bacteria, and forming the biofilm and determining the adhesiveness of bacteria (McCarter, L. L., Microbiol Mol Biol Rev. 65:445-62, 2001;
Kim, Y. K, McCarter, L. L., J Bacteriol. 182:3693-704,2000; McCarter, L. L., J Bacteriol. 177:1595-609, 1995; Boles, B. R., McCarter, L. L. J Bacteriol. 182:1035-45, 2000; Prouty, M. G., Correa, N. E., Kiose, K. E. Mol Microbiol. 2001 March; 39(6):1595-609, 2001). V. vulnificus has a polar flagellum (McCarter, L. L., Microbiol Mol Biol Rev. 65:445-62, 2001). The structural component of flagella is named the flagellin, and this flagellin forms filaments assembled regularly. According to the result of recent study, it is known that mammalian TLR-5 (Toll-like receptor-5) recognizes flagellins of both Gram-negative and Gram-positive bacteria and subsequently activates the NF-κB pathway of host cells (Hayashi, F., Smith, K D., Ozinsky, A., Hwan, T. R., Yi, E. C., Goodlett, D. R., Eng, J. K., Akira, S., Underhill, D. M., Aderem, A., Nature 410:1099-1103, 2001). The TLR, a receptor recognizing molecular patterns associated with pathogens, acts as a major component of the first line innate immune system against various infectious pathogens, and is a celluar receptor associated with the stimulation of effective adaptive immune responses (Akira, S., Hemmi, H., Immunol. Lett. 85:85-95, 2003). Therefore, TLR agonists can be the target for developments of various vaccine adjuvants.
According to the research of our inventors, genes composing the flagellum of V. vulnificus are composed of the flaA expressed DNA sequence number 1 or amino acid sequence number 2, the flaB expressed DNA sequence number 3 or amino acid sequence number 4, the flaF expressed DNA sequence number 5 or amino acid sequence number 6, the flaC expressed DNA sequence number 7 or amino acid sequence number 8, the flaD expressed DNA sequence number 9 or amino acid sequence number 10, and the flaE expressed DNA sequence number 11 or amino acid sequence number 12; the constitution of each genes is similar to that of Vibrio parahemotyticus, and their homologies are also high.
Processes proving the effect of the vaccine adjuvant comprising the flagellin, the structural component of bacterial flagella, according to this invention are as follows:
Therefore, the present invention relates to vaccine adjuvants containing flagellins, structural components of bacterial flagella, as an active component.
In addition, the present invention relates to a method of manufacturing recombinant mmunogens having the adjuvanticity induced by flagellin, which comprises substituting the genes encoding various immunogen epitopes for a part of the genes present between the N-terminal and the C-terminal of the structural genes of bacterial flagellins binding to TLR-5.
More specifically, the recombinant immunogens having the adjuvancity induced by the flagellin can be prepared by substituting the genes encoding protein antigen epitopes
The protein antigen epitopes in the present invention are the tetanus toxoid, the immunogenic epitopes of influenza virus, the specific antigens to PspA (pneumococcal surface protein A) and sperm of Pneumococcus, and so on.
The vaccine adjuvant in the present invention can be formulated into an oral form such as solution, suspension or emulsion form in an aqeous or oil solvent, or dried powder type that is aseptic state before use, and dissolved in pyrogen free water at use, or can be formulated into a non-oral administration (for example, subcutaneous injection, intravenous injection or intramuscular injection).
In the oral formulation, it could be manufactured in various formulation by common methods using carrier or forming agent, for example, tablets, troches, aqueous or oily emulsions, powder or particles that can be sprayed, emulsions, soft or hard capsules, syrup or ellixir; which can be selected according to the unit dosage or form.
The non-oral formulation could be injected by forming of sterilized injectable solution or emulsions that suspended with non-toxic available diluents or solvents like 1, 2 butadiol. Examples of diluents or solvents that can be used are water, Ringer solution and isoosmotic physiologic salines, and common solvents like ethanol, polyethileneglycol and polyprophyleneglycol can also be used. Sterilized volatile oils can be used both as solvent or emulsion solvent. In a suppository form, the medications are administered by the intra-rectal route after formulation by mixing the medications with non-irritable excipients, for example cocoa butter or polyethyleneglycol, that are solid at normal temperature but liquid at rectal temperature.
The examples of the vaccine adjuvants in the present invention are anti-toxin vaccines against tetanus etc.; live attenuated or killed vaccines against cholera, typhoid fever and so on; anti-viral vaccines against influenza, SARS, etc.; anti-cancer vaccines against uterine cervix cancer and so on; anti-sperm contraceptive vaccines; and adjuvants for recombinant vaccines, however it is not limited to these examples.
Further, the present invention is not limited to flagellin of V. vulnificus, it may be applied to other flagellated bacteria that have similar flagellin protein encoded flagellin genes to that of V. vulnificus.
Well explain the present invention more in detail below, but it is not limited to examples.
Characteristics of strains and plasmids used in the present invention are described in Table 1. Each detailed characteristic and manufacturing method is described in corresponding examples and experimental examples.
V. vulnificus
Listeria
monocytogens
Salmonella
typhimurium
Escherichia
coli
<Culture and Storage of Each Strain>
The LB (Luria Bertani) media (Difco Co.) were used for strains of E.coli, Salmonella and Listeria, and the HI(heart infusion) media (Difco Co.) were used for culturing of V. vuinificus in the following examples and experiments. After cultivation of these strains, glycerol was added to become 50% solution and they were stored at −80° C. in a deep freezer.
V. vulnificus MO6-24/O type strains (obtained from J. Glenn Morris, Division of Hospital Epidemiology, University of Maryland School of Medicine, USA) and mini-Tn5 lacZl containing E. coli SM10λpir strains (obtained from Kenneth N. Timmis, GBF National Research Center for Biotechnology, Braunschweig, Germany) were cultured overnight at 37° C., 210 rpm in a stirring incubator, each were inoculated with single colony at 10 ml of 2.5 HI(2.5% NaCl heart infusion) broth media and 20 ml of LB (containing 100 □/ml of Ampicillin and 100 □/ml of Kanamicin) broth media
The following day these were centrifuged, and washed with antibiotic-free LB broth media and centrifuged two times, then suspended at 100 □ of new LB broth media. Each bacterial suspension of E. coli and V. vulnificus were mixted together and dropped on LB agar plate. After culturing it overnight at 37° C., 800 □ of new 2.5 HI broth media was added to the grown colonies on LB agar plate and the grown colonies was scraped carefully after using sterilized glass rods. This bacterial suspension was moved to a 1.5 ml plastic test tube and suspended until becoming homogenous state. The suspension was diluted to 1/10 and 1/100, then undiluted and the dilutes dropped on TCBS (thiosulfate citrate bile sucrose) agar plate containing 200 □/ml of Kanamycin, spread until sufficiently penetrated, and cultured overnight at 37° C.
The following day only Vibrio colonies, grown on TCBS agar plate, were taken and inoculated on TCBS agar plate containing 300 □/ml of Kanamycin using toothpicks, and overnight cultured at 37° C. The following day grown Vibrio colonies were inoculated on 96-wells culture plates, containing 100 □ of 2.5 HI with 200 □l/ml of Kanamycin, and cultured overnight at 37° C. without stirring. The following day 80 □ of 50% glycerol was added to each well, containing grown bacteria, and stored at −80° C. in a deep freezer. When used for the experiments, these were inoculated to 2.5 HI broth media and cultured as needed.
Each clone, prepared in Example 1, of the V. vulnificus MO6-24/O transposon libraries was cultured overnight at 37° C., then inoculated to 0.3% agar containing semi-solid state HI (heart infusion) agar plates using sterilized toothpicks and cultured at 37° C. for 6 hours. Degrees of the motility of the bacteria were then determined by measuring the range of movement after growing the bacteria
3 transposon mutant clones that nearly completely lost motility were selected by screening procedures, and the experiment that would identify the mutant genes which insert transposons was progressed.
The cloning of genes nearby the transposon inserted region was carried out by screening the cosmid gene libraries, using DNA fragment as primer for amplification by arbitrary PCR methods. The amplification of DNA fragments nearby the transposon inserted site was used a two-step PCR amplification method. In the first PCR, the arbitrary primer 1 (5-GGCCACGCGTCGACTAGTCANNNNNNNNNNACGCCC-3) of sequence number 13, and the mini-Tn5 lacZl specific primer 1 (5-TTCTTCACGAGGCAGACCTCAGCGC-3) of sequence number 14, were used. The first PCR was set as follows; denaturing them for 30 seconds (sec) at 94° C., annealing for 30 sec at 30° C., and elongating for 1 minute (min) 30 sec at 72° C., with 5 cycles; afterwords a further 30 cycle PCR reaction was performed by denaturing for 30 seconds (sec) at 94° C., and annealing for 30 sec at 45° C., and elongating for 2 min at 72° C., with 30 cycles. The second PCR reaction was peformed using the products of the first PCR as templates. In the second PCR, the arbitrary primer 2 (5-GGCCAAGAGTCGACTAGTCA-3) of sequence number 15, and the mini-Tn5 lacZl specific primer 2 (5-CCGCACTTGTGTATAAGAGTCAG-3) of sequence number 16, were used. Reaction conditions were; denaturing for 30 sec at 94° C., annealing for 30 sec at 72° C., and elongating for 1 min 30 sec at 72° C., 30 cycles. These PCR products were electroporesed in agarose gel and the amplified DNA fragments was separated from the gel and their base sequences were determined. As a result of the described PCR reaction, 3 types of specific DNA fragments were anplified.
The result of determining the amplified DNA fragments and BLAST analyzing them with genes recorded at GeneBank databases of U.S. National Center for Biological Information showed identities with bcr, cheR and flgG genes of Vibrio parahemolyticus. According to the result of the complete decoding of the genome sequence analysis of V. vulnificus by the inventors, the described genes were located at polar flagellar flagellin operon, as shown in
DNA fragments containing the ORF of flaB gene of V. vulnificus, fliC gene of Salmonella (sequence number 18) and flaA gene of Listeria (sequence number 17) were ligated into pTYB12 vector (New England Biolabs Inc.), intein fusion expression vector, to yield each plasmid pCMM250, pCMM251, pCMM252. Each plasmids was transformed into E. coli ER2566 by electroporation, and induced the expression by adding 0.5 mM 5-bormo-indol-3-chloroisopropyl-β-D-galactopyranoside(ITPG). According to the manufacturers' (New England Biolabs Inc.) instructions, Flab, FliC and FlaA proteins were purified from Intein fusion proteins by using Chitin bead columns and 1,4-dithiothreiol. The endotoxin contained in the separated FlaB, FliC and FlaA proteins was removed by using the AffinityPak™ Detoxigel™ Endotoxin Removing Gel (Pirece Inc. Rockgord, Ill.).
Using the above described method, ORFs of genes of flaA, flaB, flaF, flaC, flaD and flaE of V. vulnificus were ligated into pGEX4T-1 vector (pCMM244-flaB, pCMM245-flaA, pCMM247-flaD, pCMM248-flaE, pCMM249-flaF). According to the manufacturers' (Amersham Pharmacia) instruction, the glutathion-S-transferase fusion protein was purified.
Seven-week-old female Balb/c mice were intranasally immunized three times with 20 □ of PBS (phosphate buffered saline), 3 □ of tetanus toxoid alone, or with combinations of 3 □ of tetanus toxoid and 1 □, 5 □ and 15 □ of FlaB of V. vulnificus, at 7-day intervals. Seven days after the last immunization, saliva, vaginal wash and serum samples were collected from the immunized mice to assess TT-specific systemic immune responses and mucosal immune responses. These responses were measured by ELISA (Enzyme linked immuno sorbant assay) methods, and the mice that were vaccinated 3 times before were observed for 7 days after systemic administration of minimally 200 folds of lethal doses of tetanus toxoid. The results are shown in
As shown in
As shown in
To confirm which of these vaccine adjuvanticities were general for flagellins of other non-V. vulnificus-flagella, the same experiments were performed with purified FlaA recombinant protein, that is flagellin structural component of Listeria monocytogenes, the Gram(+) bacterium, and with FliC recombinant protein, that is flagellin structural component of Salmonella typhimurium, the Gram(−) bacterium. The results are shown in Table 2 and
The data of Table 2 are results of administration of tetanus toxoid after intranasal immunization with the combination of FlaA of Listeria, FlaB of V. vulnificus and FliC of Salmonella, and tetanus toxoid. It is shown that these flagellin structural component proteins completely protect the host from lethal doses of tetanus toxoid.
In the results of Table 2 and
From the results of
Caco-2 cells were seeded at 2.0×105/well in 24-well plates and maintained overnight in the DMEM supplemented with 10% fetal calf serum (FCS). The following day they were washed with fetal calf serum free DMEM twice and treated with different concentrations of recombinant Vv-FlaB for 3 hours without FCS supplementation, and the level of IL-8 released to supernatants was measured using ELISA kit (R&D systems Co.). IL-8 expression in the Caco-2 cells treated with Vv-FlaB was analyzed by the real-time RT-PCR analysis. Total RNA was isolated from the Vv-FlaB treated cells. The results are shown in
Caco-2 cells seeded at 2.0×105/well in 24-well plates were transfected with a propriate amounts of expression plasmids, the reporter pIL-8-Luc or pNF-κB-Luc (obtained from professor Kim, Jeong Mok, Hanyang University Medical School) and p3Xflag-hTLR5 that encodes TLR-5 genes (obtained form Steven B. Mizel, Departments of Microbiology and Immunology, Wake Forest University School of Medicine, U.S.A). The levels of luciferase activity were normalized to the lacZsd expression using the control expression plasmid pCMV-β-ga (9BD Biosciences Clontech, Palo Alto, Calif.). Total amounts of expression vectors were kept constant by adding appropriate amounts of blank vector. 24 hours after transfection, the culture was replaced with fresh medium, containing each of recombinant FlaB of V. vuinificus, recombinant FliC of Salmonella and recombinant FlaA of Listeria, and FlaA, FlaB, FlaF, FlaC, FlaD and FlaE of V. vulnificus purified by IMPACT-CNTM system and co-adinistration of glutathion-S-transferase fusion protein. Some hours after being administered, the luciferase activity was assayed by a luminometer (MicroLumatPlus LB 96V, Berthold, Wilbad, Germany) to measure expression of IL-8, and the results are shown in
The recombinant FlaB activated the expression of IL-8 and pNF-κB in dose dependent manners. It was also shown by other flagellar structural components of V. vulnificus, FlaA, FlaF, FlaC, FlaD and FlaE with somehow difference in degree.
The peripheral blood mononuclear cells (PBMC) were separated by centrifugation using Ficoll Paque PLUS (Amersham Inc.) from human peripheral blood. The magnetic beads identifying the CD14 that selectively expresses at myeloid cells in PBMC were reacted at 6-12° C. for 20 min. The CD14 positive cells were separated by the magnetic cell sorter. CD14 positive cells were added to RPMI media containing 10% of FCS and co administration of 50 ng/ml of GM-CSF and 50 ng/ml of IL-4, and cultured for 6 days to differentiate into immature dendritic cells. After differentiation, they were treated with recombinant FlaB prepared in Example 4, and flagellin FliC of Salmonella, in doses of 6 nM concentration and cultured for 24 hours. The influence of FlaB and FliC to differentiation of human dendritic cells was observed. The reason for administration of FliC is to determine whether the present invention can be wide in use.
Monoclonal antibody that recognizes CD80, CD83 and CD86, selectively expressed on the surface of dendritic cells, and which binds to FITC (fluorescein isothicyanate) or phycoerytbrin was treated. The expression level of the cell groups that show positive signals was measured using flow cytometry.
When human dendritic cells were treated with recombinant FlaB of V. vulnificus, the percent levels of CD80, CD83 and CD86 positivity, meaning the maturity of dendritic cells, were increased by 67.3%, 23.57% and 143.29%. These levels were more increased rates than those of the control group, which showed 15.29%, 0.82% and 1.5%. However CD14, selective expressed don myeloid cells, levels were more decreased in maturation. The flagellin FliC of Salmonella showed similar traces to FlaB.
As is shown in the above results, the FlaA, FlaB, FlaF, FlaC, FlaD and FlaE, which are the structural component of flagellin of V. vulnificus, and FliC, which is the structural component of flagellin of Salmonella, and FlaA, which is the structural component of flagellin of Listeria, stimulates release of IL-8 from epithelial cells and maturation of dendritic cells. They also increase an antigen specific immune response of the host to immunostimulants uses as a vaccine.
When mice were immunized by tetanus toxoid and the mentioned flagellins via the intranasal route, they showed a remarkable increase of IgA level against antigens compared to the control group that were not administered flagellin as an adjuvant. Further, the host was protected completely from tetanus toxoid. Especially in vaginal washing, the IgA levels increased tremendously increased, so it could be available for use as an adjuvant of contraceptal vaccine that selective to sperms.
The recombinant flagellin proteins of the present invention are also available as an effective adjuvant of vaccine against other infectious diseases and anticancer therapies.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2004-0001974 | Jan 2004 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR05/00103 | 1/12/2005 | WO | 00 | 5/3/2007 |
