JP2001011096A - Human antibody against neisseria gonorrhoeae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new human antibody specifically recognizing the sugar chain of lipooligosaccharide of Neisseria gonorrhoeae and bonding to Neisseria gonorrhoeae, exhibiting bactericidal activity on Neisseria gonorrhoeae and useful for various passive immunotherapy against Neisseria gonorrhoeae resistant to antibiotic substance. SOLUTION: The objective antibody specifically recognizes the sugar chain of lipooligosaccharide of Neisseria gonorrhoeae, bonds to Neisseria gonorrhoeae and exhibits bactericidal activity on Neisseria gonorrhoeae. The sugar chain is preferably expressed by formula (Gal is galactose; Glc is glucose; Hep is heptose; X and Y are each one or more monosaccharides; - is a bond). The antibody can be produced by using a lipooligosaccharide of Neisseria gonorrhoeae confirmed to bond with normal human serum by ELISA method, preferably JW31R strain, as a ligand and subjecting to affinity column chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human antibody which specifically recognizes a sugar chain of lipo-oligosaccharide of Neisseria gonorrhoeae and binds to Neisseria gonorrhoeae, and has a bactericidal activity against Neisseria gonorrhoeae.

[0002]

BACKGROUND OF THE INVENTION Gonorrhea caused by Neisseria gonorrhoeae is one of the major sexually transmitted diseases, and many researchers are working on the development of vaccines to prevent infection due to the problem of antibiotic resistance. Neisseria gonorrhoeae has lipooligosaccharide (LOS) in the outer cell membrane, and since this LOS shows immunogenicity, it has been attracting attention as a target for vaccine development.

[0003] LOS is a combination of an oligosaccharide and lipid A, and the oligosaccharide is composed of a structurally variable sugar chain and a structurally invariant core sugar chain. The structure of the core sugar chain is N-acetylglucosamine (GlcNAc) -heptose (Hep) -Hep, and this core sugar chain is lipidated via 2-keto-3-deoxy-mannoocturonic acid (KDO). Bound to A. The variable-structure sugar chain is bound to one or both of the two Heps present in the core sugar chain [Yamasa
ki, R. et al., J. Biol. Chem. 269, 30345-30351 (199
Four)]. Among them, it has been revealed that the structure of the structure-variable sugar chain biosynthesized by Neisseria gonorrhoeae on Hep near KDO is the same as that of human glycolipid [Yamasaki, R. et al., Biochemistry
30, 10566-10575 (1991)]. Neisseria gonorrhoeae, L
It has been reported that a part of the sugar chain structure of OS is altered to show bactericidal resistance by complement [Mandrell, RE et al., J. Ex
p. Med. 171, 1649-1664 (1990)].

[0004] For this reason, gonococcus is thought to be not recognized as a foreign substance by the human immune surveillance mechanism by imitating the host antigen and changing the serum sensitivity.
[Mandrell, RE et al., Immunobiolo. 187, 382-402 (1
993)] Therefore, there is a need for the development of a vaccine that can specifically recognize only the gonococcal LOS.

[0005]

[Problems to be Solved by the Invention] In the vaccine development research using LOS, attention has been paid to the monoclonal antibody MAb 2C7 [Gulati, S. et al., J. Infect. DisI.
174, 1238-48 (1996)]. This antibody does not cross human glycolipids, binds to an epitope expressed only on the LOS of gonococci, and has gonococcal bactericidal activity. Also, the anti-idiotopic antibody of this MAb produces an antibody that recognizes LOS. However, this antibody is derived not from human but from mouse, and there is no information that such antibody exists in human or is expressed in human. Also,
As described above, despite the elucidation of the basic structure of L. gonorrhoeae LOS, knowledge has been obtained on what kind of structure of LOS is recognized by anti-LOS antibodies of healthy individuals or patients after infection. Absent. Furthermore, antibodies obtained by affinity column chromatography using Escherichia coli lipopolysaccharide (LPS), which has a different structure from gonococcal LOS, have been reported to have bactericidal activity against gonococci [Apicell
a, MA et al., J. Infect. Dis. 153, 520-526 (1986)],
It has not been reported that such antibodies bind to LPS or L. gonorrhoeae LOS. Therefore, in order to proceed with vaccine development research using LOS, it is essential to understand human immune responses to immunogenic LOS, since gonorrhea is a disease unique to humans.

[0006]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted intensive studies on the human immune response, particularly on the presence of an antibody recognizing LOS of gonococci present in healthy human serum. As a result, LOS derived from a serum-sensitive strain
The present inventors have found that an antibody recognizing LOS is present in human serum by affinity chromatography to ligand, and completed the present invention. Therefore, according to the present invention, there is provided a human antibody which specifically recognizes the glycan of LOS of Neisseria gonorrhoeae and binds to Neisseria gonorrhoeae and exhibits bactericidal activity against Neisseria gonorrhoeae.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The human antibody in the present invention is a glycan of LOS of gonococcus, [In the sugar chain, Gal is galactose, Glc is glucose, He
p represents heptose, X and Y are one or more monosaccharides, and-represents a bond), binds to gonococci, and exhibits bactericidal activity against gonococci.

[0008] In this sugar chain, X and Y may be one or more monosaccharides, such as sugars such as galactose and glucose, or
It may be any of amine sugars such as N-acetylgalactosamine or N-acetylglucosamine. However, as described above, since Neisseria gonorrhoeae has Hep (hereinafter, referred to as Hep (I))-Hep (hereinafter, referred to as Hep (II))-GlcNAc as a core sugar chain, Y may be GlcNAc. preferable.

[0009] Such a human antibody is a gonococcus whose binding to healthy human serum has been confirmed by ELISA, preferably a gonococcal strain JW31R [Gibson, BW et al., Proc. Natl. Acad.
i. USA 86, 17-21 (1989), obtained from Dr. Mandrel] using LOS as a ligand and performing affinity column chromatography.

[0010] The ELISA method and the affinity column chromatography can be performed by methods known to those skilled in the art. For example, an ELISA method is used to analyze gonococcus by analyzing healthy human serum with an enzyme such as alkaline phosphatase, peroxidase, or galactosidase, preferably an anti-human antibody chemically bound to alkaline phosphatase.
Antibodies that recognize LOS can be found in human serum. In addition, affinity column chromatography
An agarose gel with a large pore size and low non-specific adsorption, such as Sepharose as a carrier.
By immobilizing LOS of gonococci found by ELISA as a ligand and then flowing healthy human serum to the column,
Human antibodies can be obtained that bind to specific L. gonorrhoeae LOS.

According to the study of the present inventors, the human antibody of the present invention is present in healthy human serum, but has low gonococcal bactericidal activity in the form of the serum itself. It shows a bactericidal activity that is about 40% higher than that of serum in a form purified according to an ordinary method using chromatography or the like.

Accordingly, the isolated and purified human antibody of the present invention can be used in any immunologically acceptable excipient in an immunologically effective amount capable of eliciting a protective response against gonococcal infection. For example, as a composition for passive immunization combined with aluminum hydroxide, muramyl dipeptide or Freund's adjuvant, it is effective not only for normal gonococci but also for antibiotic-resistant gonococci in passive immunotherapy such as vaccination. Can be used. The epitope is
It can be used to develop safe and effective vaccines using genetic engineering techniques.

[0013]

EXAMPLES The samples used in the present invention are shown below, and will be described in detail based on Examples. Sample 1: Lipooligosaccharide (LOS) The following gonococcal strain and Salmonella minnesota (Salmonella
minnesota) 's mutant Re [Sigma]
LOS was used. Each LOS was diluted in phosphate buffer containing _{10mM MgCl 2 (PBS): JW31R} ; 15253 [Yamasaki, R. et al.,
J. Biol. Chem. 269, 30345-30351 (1994)]; WG [Yama
saki, R. et al., Eleventh international pathogenic Nei
sseria conference, 298. (1988)]; PID2 [Schneider,
H. et al., J. Exp. Med. 174, 1601-1605 (1991)]; F62 [Y
amasaki, R. et al., Biochemistry 30, 10566-10575 (1991)
]; 24-1 [Gulati, S., et al., J. Infecti. Dis. 174, 1223-
1237 (1996)]; WR 302 [Schneider, H. et al., Infect. I
mmun. 50, 672-677 (1985)]; MS11mk [Kerwood, D.
E. et al., Biochemistry 31, 12760-12768 (1992)]

Sample 2: Human serum Human serum was used in healthy individuals (8) who had no history of gonorrhea infection.
From the pooled blood was used. Serum
Stored at -80 ° C and used for determination of bactericidal activity. For Western analysis, serum obtained after inactivating complement by treatment at 56 ° C. for 30 minutes and centrifugation (10,000 g, 4 ° C., 10 minutes) was used.

Sample 3: Monoclonal antibody Monoclonal antibody Mab2C7 was prepared by Dr. Peter Rice.
[Boston University, Boston]. The following secondary antibodies were purchased from Sigma: goat anti-human polyvalent immunoglobulin alkaline phosphatase conjugate; mouse monoclonal anti-human IgG alkaline phosphatase conjugate; mouse monoclonal anti-human IgM (μ chain specific)
Alkaline phosphatase complex; goat anti-mouse IgG (γ
Chain specific) alkaline phosphatase complex; goat anti-mouse IgM (μ chain specific) alkaline phosphatase complex

Example 1 : Antibody search LOS produced by a serum-sensitive strain JW31R of gonococcus (hereinafter referred to as JW31)
R LOS) was searched by ELISA for the presence of human antibodies in human healthy serum. ELISA method uses microtiter plate [Nunc plate, Maxi Soa
B] containing 10 mM MgCl ₂
(1% in PBS, Sigma), treated with pooled human healthy serum (75 μl, diluted 10-fold in PBS), and treated with anti-human antibody (goat anti-human polyvalent immunoglobulin alkaline phosphatase complex, substrate: p -Nitrophenyl phosphate). As a result, the human antibody in the pooled human serum hardly bound to the mutant Re LOS used as a negative control, but the JW31R LO
Binding to S was confirmed. For this reason, JW31R LO
To obtain a human antibody that binds to S, affinity chromatography was performed using JW31R LOS as a ligand.

Example 2 : Antibody fractionation After treating JW31R LOS with hydrazine, an affinity column was prepared by the method reported by Apicela et al.
[J. Infect. Dis. 153, 520-526 (1986)]. MAb2C7 is JW
Since this antibody binds to 31R LOS, JW31R LOS
Was confirmed to bind to Sepharose. The pooled human healthy serum (8 ml) is applied to a LOS affinity sepharose column (2 ml), and 50 mM Tris-HC in 0.1 M NaCl (pH 7.4) is applied.
After washing with l, 200 mM glycine in 0.5 M NaCl (pH 2.
8, 300 ml). The fraction eluted with glycine was 0.5
Neutralized with MTris-HCl (pH 8.0). When the antibodies present in these fractions were analyzed by ELISA in the same manner as in Example 1,
Immunoglobulin is present in the fraction adsorbed on the column and JW31
Human antibodies that bind to R LOS were found to be present in healthy human serum.

Example 3 Identification of Antibody Class The adsorbed fraction obtained above was subjected to SDS / PAGE (6% acrylamide) and Western blot analysis. For PAGE and electroblotting, use a mini gel device [Mini-Protean I
I and Mini Transfer Unit, both Biorad, Auckland] and a power supply model 100/500 [Biorad]. After PAGE, one gel was stained with silver and the other was transferred to nitrocellulose membrane and immunostained with anti-human IgG-alkaline phosphatase complex and anti-human IgM-alkaline phosphatase complex (both monoclonal antibodies) [Yamasaki, R. et al., Mol. Immunol. 28, 123
2-1242 (1991)]. As a result, it was confirmed that most of the immunoglobulin in the adsorbed fraction was IgG (150 kDa), and that the amount of IgM (970 kDa) was very small.

Example 4 Purification and Identification of IgG Antibodies These IgG and IgM were separated by a protein G column [Amersham / Pharmacia]. IgM is 20 mM Na-
In phosphate buffer (pH 7.0), IgG is 0.1M glycine-H
Each was eluted with Cl (pH 2.7). In addition, the isolated Ig
Binding of G and IgM to JW31R LOS was performed in the same manner as in Example 1 using EL.
Confirmed by ISA. After LOS (200 ng / well each) of Neisseria gonorrhoeae strains F62, JW31R and Mutant Re were adsorbed to the plate, the plate was treated with IgG purified on a protein G column and analyzed with anti-human IgG. The IgG fraction was JW31R L
It has been confirmed that it binds to the OS. On the other hand, IgM is JW31R
Did not bind to LOS. Subclass of this IgG fraction,
Analysis was performed using a monoclonal antibody IgG / IgA subcluster typing kit for human tissue culture supernatant. as a result,
It was found that most of the IgG fraction was IgG1 and contained a small amount of IgG2. From the above results, the IgM fraction obtained by affinity chromatography was derived from JW31R and other strains.
Since LOS is not recognized, the following experiment focused on the IgG fraction and clarified the specific structure of the oligosaccharide chain of JW LOS recognized by the IgG fraction.

Example 5 : Analysis of recognition site 1 By the PAGE / Blot method performed in Example 3, gonococcal strain PID2,
15253, WR302, JW31R, WG, MS11mk, F62 and 24-1
After PAGE (14%), each LOS (200 ng) was silver-stained or transferred to a nitrocellulose membrane, and immunostained with anti-human IgG (goat antibody alkaline phosphatase complex). As a result, the IgG fraction was converted to the Hep (I) of the core sugar chain Hep (I) -Hep (II).
Does not bind to LOS, which only has a structurally variable sugar chain,
It was suggested that JW31R LOS and 15253 LOS (hereinafter referred to as 15253 LOS), that is, LOS having a structurally variable sugar chain in both Hep (I) and Hep (II), were recognized.

Example 6 : Analysis of recognition site 2 Since the IgG fraction bound to JW31R LOS and 15253 LOS, it was examined whether the binding was inhibited by the respective antigens. Following the ELISA method, JW31R (200 ng / well) was adsorbed to the plate and blocked with BSA.
Human IgG and JW31R LOS (diluted 2-fold sequentially from 400 ng / well) as an inhibitor were added. The 15253 LOS was operated similarly. Thereafter, bound human IgG in the same manner as in Example 1 was detected with anti-human IgG-alkaline phosphatase. In each LOS, the binding was shown to be inhibited by the respective antigen.

Example 7 : Analysis of recognition site 3 It was examined whether the binding of IgG to JW31R LOS and 15253 LOS is inhibited by MAb2C7 that recognizes both LOS. Plate 15253 LOS and JW31R according to ELISA method
After adsorbing LOS, human IgG and MAb2C7 as inhibitor
A two-fold dilution was added. As shown in FIG.
All binding to LOS was inhibited by MAb2C7. From the above results, it was clarified that this IgG recognized LOS having a structurally variable sugar chain in both Hep (I) and Hep (II) and shared an epitope with MAb2C7.

Example 8 : Analysis of recognition site 4 On the other hand, LOS (hereinafter referred to as WG LOS) produced by Neisseria gonorrhoeae strain WG has a structure-variable sugar chain in both Hep (I) and Hep (II). Nevertheless, it does not bind to this IgG (Example 5). For this reason, the epitope of IgG obtained in Example 4 is the same as that of LOP having structurally variable sugar chains in both Hep (I) and Hep (II).
Even within S, it is clear that it is at a specific site.
Since WG LOS has a tetrasaccharide of Gal-GlcNAc-Gal-Glc as a structural variable sugar chain on Hep (I), Gal-GlcNAc was removed from the non-reducing end by galactosidase treatment and hexoaminidase treatment. . Then, according to the PAGE / Blot method, each LOS was separated by PAGE, one was stained with silver, the other was transferred to a nitrocellulose membrane, and subjected to Western blot using human IgG. As shown in FIG. 2, it was confirmed that the removal of Gal-GlcNAc from the non-reducing end of WG LOS allows the IgG to bind.

Example 9 : Analysis of Recognition Site 5 Neisseria gonorrhoeae strain 15253 was prepared by adding Hep (I) to Gal-Gl
It has a disaccharide of c. Therefore, when galactose at the non-reducing end is removed by β-galactosidase treatment,
We examined whether its binding ability was lost. Also, lgtE
The gene was deleted, and binding to 15253 LOS having only Glc in each of Hep (I) and Hep (II) was also examined. Each of these LOS
After separation by PAGE according to the PAGE / Blot method, one was stained with silver, the other was transferred to a nitrocellulose membrane, and subjected to Western blot using human IgG. The results shown in FIG. 3 were obtained, and when the galactose at the non-reducing end was removed, this IgG
Was found to lose its binding ability. Also, this IgG
Showed no binding to lgtE gene-deficient LOS having only Glc in Hep (I) and Hep (II).

Therefore, this IgG binds He to its binding.
p (I) requires a structure in which a Gal-Glc disaccharide, ie, lactose, is present. If there is a sugar chain that extends beyond this, it was found that the bond could not be established. IgG is WG LO
Although it does not bind to S, MAb2C7 recognizes WGLOS, revealing that this IgG epitope shares some but not the same as that of Mab2C7.

Example 10 : Measurement of bactericidal activity The bactericidal activity of human IgG was measured by the method reported by Schneider et al. [J. Gen. Microbiol. 128, 13-22. (1982)].
The serum was diluted 80-fold with GBSS into an ELISA plate, human IgG was added thereto, and then a suspension of Neisseria gonorrhoeae was added. After incubating for 45 minutes, TTC-GB
After adding SS and GCD agar and incubating for 20 hours, the number of surviving cells was counted. Without serum, with and without serum, serum + IgG, serum + JW31R
The number of sterilized cells was determined from the number of remaining cells in each system. as a result,
When human IgG was added to serum, its bactericidal activity was increased by 40% compared to serum alone. The increase in bactericidal activity is
Since IgG was lost when adsorbed by JW31R LOS, it was confirmed that this human IgG had bactericidal activity.

[0027]

According to the present invention, a human antibody against Neisseria gonorrhoeae is provided. This antibody is present in healthy human serum, specifically recognizes LOS produced by gonococci, and has gonococcal bactericidal activity as its immunochemical function. Therefore, this antibody can be used not only for normal gonococci but also for various passive immunotherapy against antibiotic-resistant gonococci, and its epitopes can be used for developing safe and effective vaccines against gonococcal infection. Can be.

[Brief description of the drawings]

FIG. 1 shows the inhibition of human IgG binding to LOS by MAb 2C7. In the figure, ▲ indicates 15253 LOS and ● indicates JW31R LOS.
The figure shows the inhibition rate (%) when the OD of the well to which no inhibitor was added was set to 1.

FIG. 2 shows binding of human IgG to enzyme-treated WG LOS. A is the result of silver staining, and B is the result of immunostaining. Each lane is 1: JW31R LOS, 2: WG LOS, 3: Enzyme treated WG L
OS, 4: 15253 LOS.

FIG. 3 shows the binding of human IgG to enzyme-treated 15253 LOS. A is the result of silver staining, and B is the result of immunostaining. Each lane is 1: JW31R LOS, 2: 15253 LOS, 3: Enzyme treated 15253 LOS, 4: 15253 lgtE LOS.

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Claims (4)

[Claims] 1. A human antibody which specifically recognizes a glycan of lipo-oligosaccharide of Neisseria gonorrhoeae and binds to Neisseria gonorrhoeae and has a bactericidal activity against Neisseria gonorrhoeae. 2. The sugar chain of a lipooligosaccharide of Neisseria gonorrhoeae, [In the sugar chain, Gal is galactose, Glc is glucose, He
p represents heptose, X and Y are one or more monosaccharides, and-represents a bond]. 3. The human antibody according to claim 1, which is obtained by performing affinity column chromatography using ligooligosaccharide of Neisseria gonorrhoeae whose binding to healthy human serum was confirmed by ELISA. 4. The human antibody according to claim 3, wherein the Neisseria gonorrhoeae is a JW31R strain.