KR20110124060A - Vaccine composition comprising wta as effective component - Google Patents

Abstract

PURPOSE: A vaccine composition containing WTA is provided to effectively prevent Staphylococcus infection such as acute respiratory disease. CONSTITUTION: A vaccine composition contains WTA(wall teichoic acid) as an active ingredient. WTA is obtained from Staphylococcus aureus. The composition is used for preventing Staphylococcus infection. The composition is inoculated to an individual without plasma MBL(mannose binding lectin) or with plasma MBL of low concentration. The composition is inoculated to an individual without anti-WTA antibody or with anti-WTA antibody of low concentration. The individual is infant.

Description

Vaccine composition containing VTA as active ingredient {VACCINE COMPOSITION COMPRISING WTA AS EFFECTIVE COMPONENT}

The present invention relates to a vaccine composition containing WTA (wall teichoic acid) as an active ingredient, more specifically Staphylococcus aureus) Staphylococcus aureus is related to a vaccine composition for the prevention of infection containing, as an active ingredient from the tablets WTA.

Innate immunity is an evolutionarily conserved primary host defense system that detects pathogens through "pattern-recognition" that recognizes conserved molecular patterns of pathogens. In particular, the complement system is an innate immune defense system mediated by soluble pattern recognition molecules such as mannose-binding lectin (MBL).

MBL is a collagen lectin in the collectin family consisting of collagen, neck and carbohydrate recognition domains and is present in the serum of mammals, including humans. MBL binds to mannose and N-acetylglucosamine (GluNAc) in glycopolymers of pathogens. The binding activates the lectin complement system via MBL-associated serine protease-1, 2 and 3 (MASP-1, 2 and 3). MASP-2 cleaves C4 and then cleaves C2 to form the C3 convertase, C4bC2a, and MASP-1 cleaves factor D through other pathways.

The deficiency of serum MBL was first identified in humans with respect to deficiencies in opsonization in children. MBL deficiency has also been shown to increase infection susceptibility, including acute respiratory infections in children. Recent studies have confirmed that MBL- or MBL / MASP complex mediated opsonophagocytosis is required to inhibit the growth of S. aureus .

S. aureus is a human pathogen known to cause wound infections and sepsis. The major cell wall related glycopolymers of S. aureus include wall teichoic acid (WTA) and lipoteichoic acid (LTA). WTA and LTA contain repeating units of ribitol phosphate and glycerol phosphate and can be modified with GlcNAc substituents or D-Ala esters. While LTA is attached to the cell membrane, WTA is covalently bound to the peptidoglycan (PG) layer and is known to be involved in the attachment of S. aureus to nasal mucosal cells.

Recent studies have reported that MBL can bind to S. aureus and activate the complement system, but it is not confirmed whether the glycopolymers of S. aureus are recognized by MBL.

The problem to be solved in the present invention is to provide a vaccine composition for preventing staphylococcal infection, containing WTA obtained from S. aureus as an active ingredient.

In order to solve the above problems,

The present invention provides a vaccine composition containing WTA (wall teichoic acid) as an active ingredient.

The WTA is Staphylococcus aureus ).

The WTA is obtained from WTA-attached peptidoglycan of Staphylococcus aureus.

The composition is used for the prevention of staphylococcal infection.

The staphylococcal infection may be acute respiratory tract infections.

The composition is preferably inoculated subjects who do not have plasma MBL (mannose binding lectin) or present in low concentration.

The composition is preferably inoculated subjects who do not have an anti-WTA antibody or are present at low concentrations.

The composition is preferably to be inoculated subjects in the absence or low concentration of plasma MBL and anti-WTA antibodies.

The subject is preferably an infant.

When the vaccine composition according to the present invention is inoculated with an MBL and / or anti-WTA antibody or in low concentrations of an individual, especially a child, there is an effect of effectively preventing staphylococcal infections such as acute respiratory infections. .

1 is a schematic diagram showing that Staphylococcus WTA is obtained from Staphylococcus peptidoglycan by enzyme treatment,
Figure 2 shows the results of the purification of Staphylococcus WTA,
3 shows that MBL binding to Staphylococcus requires Staphylococcus tagO gene,
4 shows the results showing that Staphylococcus WTA is a ligand of human MBL,
5 is a result showing that the serum MBL of adults do not bind to Staphylococcus WTA,
6 is a result showing that the anti-WTA antibody inhibits the binding of MBL to WTA,
7 is a result showing the MBL binding and IgG binding in pediatric serum,
8 is a result showing that neutrophilic phagocytosis occurs by MBL bound to Staphylococcus aureus WTA,
9 is a result showing that the binding of MBL to staphylococci is mediated by the carbohydrate recognition domain of MBL,
10 shows the results showing MBL binding and C4 deposition from adult serum,
11 is a result showing that MBL binding to Staphylococcus is recovered in IgG deficient adult serum,
12 shows the results showing MBL binding and C4 deposition from pediatric serum.

Hereinafter, the present invention will be described in detail.

The present invention provides a vaccine composition containing WTA as an active ingredient. The WTA is Staphylococcus aureus ), a surface antigen present in the peptidoglycan layer of Staphylococcus aureus.

Since WTA is attached to the peptideglycan layer, it is possible to isolate WTA using known peptidoglycan degrading enzymes. The degrading enzyme is not particularly limited, and examples thereof include lysozyme or lysostaphin.

The vaccine composition of the present invention can be used for the prevention of staphylococcal infection, and preferably for the prevention of acute respiratory tract infections caused by Staphylococcus aureus.

The subject to be inoculated with the vaccine composition of the present invention is preferably a subject to be inoculated with a subject without or without plasma mannose binding lectin (MBL). In addition, the vaccine composition of the present invention is preferably to be inoculated subjects who do not have an anti-WTA antibody or present at a low concentration. Furthermore, the vaccine composition of the present invention preferably targets the inoculation subjects in which neither plasma MBL nor anti-WTA antibodies are present or at low concentrations. The low concentration means a case where the concentration in plasma is 2% or less. The subject may be a mammal, including a human, preferably an infant of 12 months or less.

Vaccine compositions of the present invention may include WTA and optionally one or more pharmaceutically acceptable carriers, diluents and / or adjuvant and the like.

Carriers used in the vaccine composition according to the invention are selected based on the method and route of administration, and standard drug compositions, and include, for example, carrier proteins (ie, bovine serum albumin (BSA), egg white albumin (OVA), human serum). Albumin (HSA) and keyhole limpet hemocyanin (KLH)), solubilizers (ie ethanol, polysorbate and Cremophor EL ^™ ), isotonic agents, preservatives, antioxidants, excipients (ie lactose, starch, crystalline cellulose, Mannitol, maltose, calcium hydrogen phosphate, light anhydrous silicic acid and calcium carbonate), binder (ie starch, polyvinylpyrrolidone, hydroxypropylcellulose, ethylcellulose, carboxymethylcellulose and gum arabic), lubricant (ie magnesium magnesium stearate) , Talc, and cured oils, and the like) and stabilizers (ie, lactose, mannitol, maltose, polysorbate, macrosol, polyoxyethylene cured castor oil). If desired, glycerin, dimethylacetamide, 70% sodium lactate, a surfactant or a basic substance (ie sodium hydroxide, ethylenediamine, ethanol amine, sodium bicarbonate, arginine, meglumine or trisaminomethane) and the like can be added. have. Specifically, the vaccine composition comprising the WTA according to the present invention may be combined with a known KLH solution (Calbiotec, dissolving 125 mg per 1 ml of 50% glycerol solution) as a carrier protein to enhance antigenicity.

Diluents used in the vaccine composition according to the present invention may be selected based on the method and route of administration and the actual standard drug composition. Examples of diluents include water, saline, phosphate buffered saline and bicarbonate solutions.

Adjuvants used in the vaccine composition according to the invention are selected based on the method and route of administration and the actual standard drug composition. Examples of adjuvant include cholera toxin, Escherichia coli's dimeric enterotoxin (LT), liposomes and immune stimulatory complex (ISCOM).

The route of administration may vary depending on the age, body weight, sex and general health of the subject with the risk of staphylococcal infection, but administration may be oral and parenteral (eg, intravenous, arterial and topical). It can be administered by any of the routes. Among them, parenteral administration is preferable.

Formulations for oral and parenteral administration and methods for their preparation are well known to those skilled in the art. Formulations for oral and parenteral administration may be prepared by conventional processes, for example by mixing the vaccine composition according to the invention with, for example, the aforementioned pharmaceutically acceptable carrier. Examples of oral dosage forms include solid or liquid dosage forms such as solvents, tablets, granules, powders or capsules. Examples of parenteral formulations include solvents, suspensions, ointments, creams, suppositories, eye drops, nasal drops and ear drops. If sustained release of the formulation is desired, biodegradable polymers (eg, poly-D, L-lactide-co-glycoside or polyglycoside) can be added to the buck substrate (e.g., US Pat. 5,417,986, 4,675,381 and 4,450,150. In the case of oral administration, flavoring agents and coloring agents may be added. Suitable pharmaceutical carriers, diluents and pharmaceutically necessary materials for their use are described in Remington's Pharmaceutical Sciences.

The dosage of the vaccine composition according to the present invention is determined based on the type of adjuvant, the method and frequency of administration, and the desired effect and may generally be from 1 μg to 100 mg of WTA once in adults. When adjuvant is added to the vaccine composition of the present invention, the dosage may generally be from 1 μg to 1 mg of WTA once per adult. The administration can be administered several times if necessary. For example, initial vaccination followed by three booster vaccinations can be performed at weekly intervals. Alternatively, adjuvant injection and second adjuvant vaccination may be performed at week 8-12 and week 16-20, respectively, from the first immunization using the same agent.

[ Example ]

1. Proteins, Serums and Bacteria

The native human MBL / MASP complex and human L-ficolin were expressed in Matsushita, M. and Fujita, T., J. Exp. Med. 176, 1497-1502 (1992) and Matsushita, M. et al., J. Biol. Chem. 271, 2448-2454 (1996).

Recombinant human MBL was expressed in CHO cell line and purified by chromatography as described in Kang, H. J. et al., Immunology 122, 335-342 (2007). MBL deficient serum was obtained from homozygous humans for the codon 54 mutation of the MBL gene, and C1q-deficient serum was obtained from Calbiochem / EMD Biosciences (San Diego, CA). Infant serum was obtained from a clinical laboratory and adult serum was obtained from healthy volunteers.

S. aureus surface component mutants are derivatives of RN4220 and are listed in Table 1. All bacteria were cultured in Luria Bertani medium supplied with antibiotics.

T002 species (RN4200 Δ oatA :: erm ) were constructed through integration of the pMutinT3 plasmid harboring the central open reading frame region of the oatA gene. p StagO is a pND50 plasmid harboring the S. aureus tagO gene. T258 and T358 were constructed by phage transduction using phage80.

2. S. aureus of WTA -Attach PG , WTA -Not attached PG  And WTA

WTA-attached insoluble PG is described in Park, JW et al., Proc. Natl. Acad. Sci. The method described in USA 104, 6602-6607 (2007) was modified from S. aureus . In summary, WTA-attached insoluble PG is S. aureus , a PG O -acetyltransferase-deficient oatA mutant that is sensitive to lysozyme. Obtained from T002 species.

WTA-deficient PG was replaced with WTA-deficient S. aureus Δ tagO. Obtained from the mutation. To purify WTA, insoluble WTA-attached PG (20 mg) was incubated with lysostaphin (0.17 mg, Sigma-Aldrich) in 1 ml of buffer A (20 mM Tris-HCl, pH 7.0) at 37 ° C. for 16 hours, and lysozyme (1 mg, Bioshop) was incubated at 3737 ° C. for 18 hours.

The cleaved material was boiled for 10 minutes, passed through a 0.45 μm membrane filter and 1/10 volume was loaded into a Hi-Trap-Q column (1 ml) homogenized with buffer A. The column was washed and eluted with 20 ml gradient of 0-1 M NaCl in buffer A. Fractions (1 ml) were collected and measured by inorganic phosphate and silver staining PAGE to detect WTA.

The combined WTA fractions (8 mg per 20 mg PG) were precipitated with acetone and dissolved in PBS (pH 7.5) and used for the experiment.

3. MBL of S. aureus Binding to

S. aureus (2.0 × 10 ⁹ cells) was fixed with 70% ethanol, washed, and then 1 μg MBL and L in 500 μl buffer B (20 mM Tris-HCl, pH 7.4 containing 150 mM NaCl, 20 mM CaCl ₂ and 0.05% Tween20). Incubated with -ficolin at 4 ° C. for 2 hours.

Cells were harvested by centrifugation, washed and treated with 500 μl of 20 mM Tris-HCl (pH 8.0) containing 10 mM EDTA. The supernatant and eluted protein were precipitated with trichloroacetic acid and analyzed by 12% SDS-PAGE.

4. S. aureus top MBL  Combined and C4  Calm Flow cytometry  analysis

IgG-binding protein A-deficient S. aureus Δ spa Mutations (M0107) and analogs thereof were used. S. aureus immobilized with ethanol (1.0 × 10 ⁹ cells) with MBL / MASP (1 μg) in 500 μl of buffer C (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM CaCl ₂ , 1% BSA and 0.05% Tween20) for 2 hours at 4 ° C. Incubated.

After washing with washing buffer (10mM Tris-HCl (pH 7.4), 140mM NaCl, 0.05% Tween20, 1mM CaCl ₂ ), C4 (800ng, Complement Tech.) was washed with S. aureus in buffer C 150μl for 1 hour at 37 ° C. During incubation.

In addition, immobilized S. aureus was incubated with 2-20% human serum in 150 μl of buffer C for 1 hour at 37 ° C.

To detect bound C4b, goat F (ab ') ₂ anti-mouse IgG Ab (Beckman coulter, conjugated with mouse anti-human C4 monoclonal antibody (mAb) (Bioporto, Denmark, diluted 1: 500) and FITC diluted 1: 200) was used. To detect bound MBL, mouse anti-human MBL mAb (Dobeel, Korea, diluted 1: 1,000) was used.

S. aureus was sonicated for 30 min to make single cells and flow cytometry analysis was performed (Beckman Coulter).

5. ELISA assay

MBL binding to purified WTA and the resulting C4 deposition were assessed using ELISA. In summary, 5 μg of each ligand in 60 μl PBS (pH 7.5) was applied to Immulon 4HBX 96-well plates (duplicate, Nunc) and adsorbed overnight at room temperature.

Each well was washed with washing buffer and blocked with buffer D (20 mM Tris-HCl, pH 7.4), 150 mM NaCl and 1% BSA for 1 hour at room temperature. After washing, each well was incubated for 2 hours on ice with 100 μl of MBL or MBL / MASP in 50 μl of buffer C.

When C4 deposition was determined, the wells were washed and incubated for 1 hour at 37 ° C. with C4 (100 ng) in 50 μl of buffer C. Immobilized ligands were also incubated with 6% human serum (3 μl) in 50 μl buffer C.

Primary antibodies against MBL and C4b were diluted 1: 1,000 and 1: 3,000 and used for flow cytometry analysis. The secondary antibody was goat anti-mouse IgG (H + L) Ab conjugated with HRP (Beckman coulter, diluted 1: 10,000).

The resulting plate was developed in the dark with substrate 3,3 ', 5,5'-tetramethylbenzidine (Zymed Laboratories) and recorded using a microplate reader (Thermo Scientific USA). To measure inhibition of MBL binding to WTA by total IgG or anti-WTA-IgG, the tested material and MBL were mixed and used for ELISA.

6. WTA Coated Nitrocellulose Membrane  Used human Ig  Purification of anti-WTA-IgG from Serum

anti-WTA-IgG is described in Park, J. W. et al., Proc. Natl. Acad. Sci. Purification as described in USA 104, 6602-6607 (2007). In summary, 1 μg of purified WTA or WTA-free PG in 5 μl of PBS was spotted onto nitrocellulose membrane (10 × 3 mm, Whatman, pore 0.45 μm) and baked at 100 ° C. for 1 hour. The membrane was washed with buffer E (20 mM Tris-HCl, pH 7.4), 150 mM NaCl, 0.05% Tween20) and blocked with buffer D for 1 hour at room temperature.

Twenty membranes were prepared for each sample and incubated for 2 hours on ice with 250 mg of commercially available human IVIG (SK Chemicals, Korea) in 25 ml buffer E. After washing with buffer E, bound IgG was eluted with 1 ml of 0.1 M glycine (pH 2.8) and immediately neutralized to pH 7.5 with 1N KOH.

7. Human neutrophil With separation of Opsonophagocytosis assay

Peripheral blood neutrophils were isolated from healthy donors using dextran precipitation and differential centrifugation through a Ficoll-Hypaque density gradient. The donor was a person who did not receive any anti-inflammatory drug until at least 3 weeks of sampling. Mixed monocytes were removed using anti-CD14 Ab-coated magnetic bead (Milteny Biotec Inc., Auburn, Calif.).

CD14 ⁺ cells in isolated neutrophils were <0.1% in flow cytometry using FITC-conjugate anti-CD14 Ab. The proportion of neutrophil was found to be greater than 98% by CD66b staining.

neutrophil (1 × 10 ⁷ / ml) was labeled with PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), and recombinant MBL- or MBL / MASP-treated S. aureus (1 × 10 ⁸ / ml) was labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich). Cells were washed three times with PBS. PKH67-labeled ^{neutrophil (1 × 10 5 / 100μl} ) of PKH26-labeled wild-type or mutant S. aureus (1 × 10 ⁶ / 100μl) in the presence or incubated for 1 hour at 5% or without 1% lutamine, 100U / ml penicillin and 37 ℃ of 100μg / ml streptomycin are from the supply RPMI1640 5% CO2 atmosphere in the absence of FBS in the absence It was.

The percentage of PKH26 stained cells in total neutrophil was measured by flow cytometry. The neutrophil was 99% viable with trypan blue, and 98-99% pure by Giemsa staining.

8. anti - WTA IgG  A positive decision

The amount of anti-WTA IgG in human serum was determined by ELISA. WTA-coated microplates were incubated with 0.1 μl of human serum in 50 μl buffer D containing 1% BSA at 4 ° C. for 2 hours, and the bound IgG was conjugated with mouse monoclonal anti-human-IgG antibody (Sigma-Aldrich) and HRP. It was identified as goat anti-mouse IgG (H + L) antibody conjugated to.

9. result

(1) human MBL Required to join with S. aureus tagO gene

S. aureus It is known that PG binds to human MBL and activates the PG dependent lectin complement pathway. In the present invention, in order to identify the extracellular membrane molecules of S. aureus that recognize human MBL, WTA-deficient Δ tagO mutant, LTA-deficient ΔltaS D-Ala modification-deficient Δ dltA of mutant, LTA and WTA mutant, lipoprotein maturation-deficient Δ lgt mutant, lysyl-phosphatidylglycerol-deficient Δ mprF mutant, glycolipids-deficient Δ ypfP mutant, IgG-binding A-deficient Δ spa mutant and S. aureus RN4220 was used to confirm the binding capacity with MBL.

As shown in FIG. 3, WTA-deficient Δ tagO was the only mutant did not bind to MBL, tagO the Gene Δ tagO Mutant recovered the MBL binding capacity.

In addition, MBL / MASP complex-mediated C4 deposition in S. aureus was confirmed to confirm whether the binding capacity of MBL is related to complement activation. As a result, Δ tagO The mutant did not show C4 deposition, and the Δt a gO mutant introduced with the tagO gene showed C4 deposition.

(2) neutrophil On by MBL - mediated opsonophagocytosis In S. aureus  Δt a gO The role of genes

To determine if tagO-dependent MBL binding causes opsonization, wild-type or WTA-deficient Δ tagO Mutant was incubated with MBL or MBL / MASP complex and neutrophil phagocytosis was confirmed (FIG. 3, FIG. 8).

As can be seen in Figure 4, in the case of wild type phagocytosis occurs, Δ tagO In the mutant, phagocytosis did not occur or occurred weakly. From these results, S. aureus of MBL or MBL / MASP complex With WTA The binding increased the neutrophil phagocytosis.

(3) human MBL of As a ligand S. aureus WTA

Soluble WTA containing N- GlcNAc- N- MurNAc with tetrapeptide of L-Ala-D-isoGln-LLys-D-Ala as shown in FIG. 1 was treated with insoluble PG by the known PG degrading enzymes lysozyme and lysostaphin. Obtained. As expected, WTA obtained a single peak on ion exchange chromatography, confirmed by silver staining of phosphorus assay and PAGE (Fig. 2).

The purified WTA was immobilized, and the binding capacity and C4 deposition with the MBL / MASP complex were confirmed (FIG. 4 and FIG. 10).

(4) adult serum MBL of WTA  Combined or not

To determine whether WTA acts as a ligand of MBL in human serum, WTA-mediated C4 deposition was confirmed using MBL-sufficient or MBL-deficient adult serum. As a result, it was confirmed that C4 deposition was induced in both MBL-sufficient and MBL-deficient serum, WTA-mediated C4 deposition was not induced in C1q-deficient serum, and IgG-mediated C4 deposition was induced in C1q deficient serum. It wasn't. In addition, mannose dependent C4 deposition was induced in a MBL dependent manner, it was confirmed that induced independently of C1q (Fig. 5). These results indicate that WTA-mediated C4 deposition in adult serum is induced by C1q, not by MBL / MASP complex.

In addition, serum from six adults with different MBL serum concentrations was harvested to confirm MBL binding and C4 deposition on wild type and ΔtagO mutant (Table 2).

As a result, MBL binding did not occur in 20% of MBL-sufficient adult serum, and MBL binding and C4 deposition were observed in 2% of MBL serum (FIG. 5). From these results, it can be expected that the binding of WTA and MBL in adults is inhibited by any serofactor.

(5) Item WTA  By antibody WTA Wow MBL Inhibit binding to

From the above results, anti-WTA antibody was anticipated as a serum component that interferes with WTA and MBL binding. It was confirmed that binding of WTA and MBL was inhibited by anti-WTA antibody, and IgG deficiency from MBL-sufficient adult serum It was confirmed that the binding capacity of and MBL restored (Fig. 6, Fig. 11).

(6) Serum of Children with Immature Acquired Immune System MBL of WTA In combination with

During pregnancy, maternal IgG antibodies are delivered to the fetus via the placenta, so that the newborn has an antibody concentration comparable to that of the mother. In children aged 6-12 months, IgG is metabolized, and the concentration of antibodies gradually decreases. At the same time, children's own antibodies are produced. In particular, MBL-deficient children who do not have a fully acquired immune system are known to be susceptible to S. aureus . Children's serum MBL was expected to bind WTA and S. aureus due to low concentrations of anti-WTA antibodies.

To demonstrate this prediction, sera with different MBL concentrations from six adults and six children were prepared (Table 2). The concentration of anti-WTA IgG in adults was 3-14 times higher than that in children, and the acquired immune system was found to be immature in all children. The concentration of anti-WTA antibodies was higher in MBL-defficient adults than in MBL-sufficient adults, indicating that MBL deficiency increased the production of anti-WTA antibodies.

In children showing 20% MBL concentration, MBL was confirmed to bind to S. aureus , and when serum was deficient in MBL, this binding was not confirmed (FIG. 7). From these results, it was confirmed that the acquired immune system was not established, but children with sufficient MBL could recognize S. aureus WTA (FIG. 12).

Claims (9)

Vaccine composition containing WTA (wall teichoic acid) as an active ingredient. The method of claim 1,
The WTA is Staphylococcus aureus ). The method of claim 2,
Wherein said WTA is obtained from WTA-attached peptidoglycan of Staphylococcus aureus. The method of claim 1,
The composition is Staphylococcus aureus ) composition for preventing infection. The method of claim 4, wherein
The staphylococcal infection is acute respiratory tract infections. The method of claim 1,
The composition is a composition characterized in that the subject is inoculated subject to the absence or low concentration of plasma MBL (mannose binding lectin). The method of claim 1,
The composition is characterized in that the anti-WTA antibody (anti-WTA antibody) is present in the inoculation subject to the absence or low concentration. The method of claim 1,
The composition is a composition characterized in that the subject is inoculated subject to the absence or low concentration of plasma MBL (mannose binding lectin) and anti-WTA antibody (anti-WTA antibody). The method according to any one of claims 6 to 8,
The subject is a composition, characterized in that the infant (infant).

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