JPH07503238A - Detoxified LPS-cholera toxin conjugate vaccine for cholera prevention - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Field of the invention: Detoxified LPS-cholera toxin conjugate vaccine for the prevention of cholera The invention disclosed herein generally relates to vaccines for recovery from bacterial infections. related to the manufacture of More specifically, the present invention provides detoxified livers derived from target bacterial strains. by binding the popolysaccharide to proteins further produced by the bacterial target. The production of antibacterial vaccines by

Background technology ] Lera persists as a cause of illness and death in fewer than 40 countries on three continents. Since the outbreak began in Peru in January 1991, approximately 340,000 cases have been reported [16.33]. Global sensitization Effective cholera prevention has not been achieved due to the limited availability of vaccines.

Research on new vaccines has led to consensus on the ingredients that best elicit a protective immune response. This is difficult because there is no opinion. Absence of bacterial invasion characterizing the disease, systemic disease The absence of symptoms and intestinal inflammation indicate that cholera is a toxin-mediated disease of the luminal surface of the jejunum. led to the understanding that local intestinal responses are required for protective immunity. [4.10-12.21.22.24.26.32.36.44].

Lipopolysaccharide (LPS) of Vibrio cholerae is a protective antigen. [3, 18, 22, 36, 38, 43, 51, 60], but The structures, pathogenic roles and host components involved in protective immunity against Lera are fully understood. It has not been. Vibrio cholerae 01 LPS contains lipid A and 4-amino-4-deoxy Ci-shi-arabinose, quinobosamine, D-glucose, D-fructose and and a core oligosaccharide consisting of heptose [23, 30, 47]. 3-deoxy -D-Mannooctulosonic acid (KDO) has recently been identified and is located next to lipid A. It is believed to be located in the adjacent core [5]. Inaba serotype collection The O-specific polysaccharide (0-3P) of Bacillus laminar O has an amino group of 3-deoxy-glycerol. - about 1 consisting of 1→2-linked D-berosamine acylated with tetronic acid It contains two saccharide residues [23,30,47]. Synthesize Vibrio cholerae LPS Sequence of the gene encoding the enzyme and Inaba and Ogawa serum The relationship between type (LPS type) [23,36] and serological specificity has not been clarified. stomach.

Cellular vaccines or partially purified LPS administered parenterally have no statistical impact on cholera. induces significantly significant protection in adults (approximately 60%) for approximately 6 months [3.7.18 ．． 22.38.45]. Cell-based vaccines are less effective in infants and children It is not effective in controlling cholera outbreaks [38.51]. These vaccines The protective immune component elicited by [1.3.18.22.38.40]. cellular vaccine also serum antitoxin [37] and, by analogy with similar products, secreted antibodies [5 6] Does not bring. Similar effects were also demonstrated by orally administered inactivated Vibrio cholerae. obtained [7.10-12]. For this vaccine formulation, the B subunit of CT The addition of additional defenses does not provide additional protection [12].

Although considered by many researchers to be a "marker" and not a protective component, Vibriocidal antibody concentration is a reliable method for predicting cholera resistance.

Serum vibriocidal activity was determined by administration of toxigenic bacterial strains of the main genus or alone or with CT Vibrio cholerae together with the B subunit of Correlates with resistance to Additionally, age-related vibriocidal antibodies in endemic areas The acquisition of [1.3.4.7.10.11.18.22.38.50]. these The inventor's interpretation of the data is that the cellular cholera vaccine and that it is poorly immunogenic and T-cell dependent, as observed with polysaccharides. and [37.49].

Based on the same reason, Kabir has developed a method for treating Vibrio cholerae 395 (Ogawa). NaOH-treated LPS from Inaba and Ogawa serotypes bound to proteinaceous extracts synthesized a bivalent conjugate consisting of [27]. This binding in complete Freund's adjuvant 1 mg of the combined product contains rabbit antibodies with vibricidal activity against two serotypes. Brought. The route of immunization using CFA and the dosage used are as per clinically acceptable standards. I can't stand it.

The use of another component from Vibrio choraceae is brought about by the inventor's conjugate. Although CT may obscure the nature of the protection provided by Haemophilus influenzae (H.

Inf 1 uenzae) serves as an immunogenic carrier for both type B and Vi polysaccharides. [49,54], the inventors chose it. Furthermore, specific CT of infectious bacterial strains Different serum antitoxins may be protective or synergistic protection by LPS antibodies may exhibit activity [28.43.52].

Conjugate vaccines have a number of advantages compared to cellular vaccines. That is, (1) LPS 11 degrees is low, so no serious side effects are expected; (2) Combined Sugars are predicted to be more immunogenic and T cell dependent than cell-mediated vaccines. [9, 14, 31, 48] and therefore the conjugate is more secure. and may be a more immunogenic (and thereby more effective) vaccine. (3) The conjugate is effective against diphtheria and tetanus toxoids, whooping cough ( DTP) and influenza B package [48] , thus the conjugate is the age group with the highest incidence in cholera endemic areas. can be incorporated into routine immunization of infants and children [38], and (4) the composition of the inventor's conjugate has been determined by laboratory assays to determine the potency of new lots. It can be standardized so that it can be adjusted.

Summary of the invention Side effects of LPS in cell-mediated vaccines, less immunogen in infants and children In order to solve the problem of gender and T cell dependence, the present inventor has developed several A conjugate consisting of detoxified LPS of Vibrio cholerae inaba serotype was applied to this protein. Synthesized. Synthesis and immunological properties of these vaccines using CT as a carrier The quality is described below.

The production of conjugates that elicit LPS antibodies requires (1) complete construction of LPS of two serotypes; (2) the dimensions of the inaba o-sp are relatively small (approximately It is difficult because the molecular weight (d) is 6,000 Daltons. (alone or in combination) The immunogenicity of kallides is directly related to their size) [2, 17, 54]. strain LPS from 569B (Inaba) was treated with 1% acetic acid at 100°C for 90 minutes. By controlling the amount of endotoxin that is clinically acceptable, hyperimmune serum can This yields a product of about 5,900 d that does not settle. Treatment with hydrazine is LPS with a molecular weight of approximately 13,000 and 6.0OOd. produce products and maintain their antigenicity. Therefore, hydrazine-treated Conjugates are made using LPS. Shigelladysent As demonstrated for type 1 o-sp (E. eriae) [9], multipoint coupling The conjugate (DeA-LPS-CTI I) produced by single-point bonding (DeA-LPS-CTI). proposal Injected subcutaneously in saline at one-tenth of the proposed human dose, our The conjugate elicits LPS antibodies with vibricidal activity in young outbred mice. I put it out. This immunization scheme was shown to be effective in infants co-injected with DTP. The immunogenicity of the influenza supra-tetanus toxoid conjugate was predicted [49]. L Low levels of "endotoxin" activity as measured by AL and rabbit pyrogen assays , our conjugate almost eliminates the side effects encountered with cellular cholera vaccines. or all (gives confidence that it will not occur) [25].

The most general explanation of the invention is that the ribopolysaccharide component derived from the target bacterial strain and the ribopolysaccharide component derived from the same bacterial strain are The present invention concerns an antibacterial vaccine formulation containing a conjugate with a protein. The vaccine is , this conjugate and a pharmaceutically acceptable compound known in the field of vaccine production. It is formulated using any of carriers, stabilizers, adjuvants, etc. Such a carrier may be a sterile saline solution for the manufacture of injectable vaccines. The conjugate is also small At the same time for use in infant immunization protocols, formulations, especially for children in the United States. Commonly administered diphtheria and tetanus toxoids, pertussis (DTP) Can be incorporated into vaccines.

A major advantage regarding the clinical utility of conjugate vaccines is that the LPS component of the vaccine Obtained by detoxification. Such detoxification can be achieved using hydrazine. By removing esterified fatty acids from the lipid A component of PS or LPS This can be achieved by acid hydrolysis of Therefore, one object of the present invention developed an LPS-protein conjugate vaccine using such detoxified LPS components. It is to provide.

Additionally, binding of detoxified LPS to proteins synthesized by bacterial targets In particular, it provides useful vaccines when using proteins localized on the bacterial surface. It is expected that there will be. Neutralizing antibody response to toxins secreted by bacteria The resulting vaccine is also considered to be a useful vaccine. Therefore, the first aspect of the present invention The second purpose is to develop vaccines in which detoxified LPS binds to such secreted toxins. It is to provide chin.

A preferred embodiment of the invention provides a method for treating toxin proteins produced by bacterial subjects. This is the binding of detoxified LPS.

Binding reactions can be carried out using a variety of reagents. The bond is LPS and tan The linkage can be carried out directly or with the aid of a cross-linking agent. this Such crosslinking agents can be divalent linkers. can be used in the present invention Examples of divalent linkers include, but are not limited to, adipic acid dihydrazide, dihydrazide, Aminohexane, amino-ε-caproic acid and N-hydrosuccinimide acid free There are hydrate-based heterotrifunctional linkers.

In a preferred embodiment, for LPS detoxified by hydrazine treatment. Two types of attachment methods are available: N-succinimidyl-3-(2-pyridyldithi). e) Reaction with propionate (SPDP) or adipic acid dihydrazide (A 1-ethyl-3(3-dimethylaminopropy)carbodiyl followed by reaction with DH) The reaction with EDAC is described. The latter method is based on LPS-protein binding. causes the formation of covalent aggregates (lattices) of the body. A preferred embodiment of the invention is as follows. Use one of these combination methods.

Polyclonal or monoclonal disease resulting from administration of conjugate vaccines to laboratory animals Antibodies can be used as components of diagnostic kits or therapeutic methods for infections caused by target organisms. It can be used as a component of Therefore, another object of the present invention is to Detection of organisms with combined LPS or protein moieties or both The objective is to provide a diagnostic kit that allows The final objective of the present invention is to or to treat infections caused by organisms that have protein moieties or both. toxins secreted by such organisms that can be used to treat The purpose of the present invention is to provide antibodies that neutralize.

Brief description of the drawing Figure 1 shows Vibrio cholerae inaba serotype (lane 1) and Escherichia coli (Escherich Silver staining of 2.5 mg of LPS from Ia coli) Olli (lane 2) 5 DS-PAGE gel (14%) is shown.

Figure 2 shows the characterization of LPS and LPS-CT conjugates by double immunodiffusion method. vinegar. Left, A, hyperimmune Vibrio cholerae inaba serotype serum, outer Ueno I/:1-Inaba L PS, 250m1m1.2-Inaba DeA-LPS, 250m1m1.3-Inaba Pa o-sp, 250m1m1゜Right: A, hyperimmune Vibrio cholerae inaba serotype serum, B, Hyperimmune cholera toxin antiserum, 3-Inaba DeA-LPS, 250ml Combined DeA-LPS-CT I I0 Figure 3 shows hydrazine-treated lipopolysaccharide (DeA- 13C nuclear magnetic resonance spectrum of LPS) is shown. The 10 main signals are This is the same as that reported by Kenne et al. [30]. acid treated The '3C-N, M, R, spectrum of lipopolysaccharide (0-8P) is based on this spectrum. It was almost the same as Toru.

Figure 4 shows 0.2M NaC110, 01M Tris, O, OOIM EDTA, 0 ．． Superose 12 in 25% deoxycholic acid, pH 8 High speed liquid of 100 mL sample (1.0 mg/ml) using lOx 300 mm column The body chromatography profile is shown. a, LPS Inaba serotype: b, De A-LPS inaba: C, 0-8P inaba.

Disclosure of invention Scientific papers and other references cited in this application are incorporated herein by reference. fully included.

Preferred embodiments of the invention are described in detail below by way of exemplary examples.

These examples are merely illustrative of the invention and do not limit the scope of the invention. It should not be taken as limiting.

Chemical reagents for carrying out the procedures described in the examples were supplied by the manufacturers listed below. It can be obtained from.

Anhydrous hydrazine (lot 104F-3523), adipic acid dihydrazide (A DH, Lot 77F-5016), Dithiothreitol (DTT, Loft 49) F-0138), 1-ethyl-3(3-dimethylaminopropyl)carbonimide (EDAC, lot 105F-0308), EDTA disodium (lot 119F-0275), KDO, RNase (lot 128F-0462), DNase (Loft 89F-9605) and Pronase (Loft 99F-03) 91) is Jig 7-Chemical Campa =-(Sigma Chemical C o, ), St. Louis, MO. HEPES (Lot) Lot 051790) and deoxycholic acid (Lot 264101) It can be purchased from Calbiochem, La Jolla, CA. Ta N-succinimidyl-3-(2-pyridyldithio)propio for protein determination Nate (SPDP, Lot 900707084), Alum (Lot 891 120103) and BCA reagents from Pierce Chemical Company (Pi Obtained from eRc eChemical Co., Rockford, IL. can be done. Cyanogen bromide (CNBr, Lot 014783A) Eastman Chemical, Ochester, NY It can be purchased from Sephadex for molecular weight assay G-25 (Lot P10036), Sephacryl (Sephacryl I) S -300, lOx300mm Superose 12 column and dextran - Mana (Pha rmac ia) - Purchased from LKB, Piscataway, NJ can be entered. LPS from Vibrio cholerae strain 569B (Inaba) is List Biologicals, Kambubel, C It can be purchased from A. Limulus amoeboid cell lysis The product (LAL) is produced by Associates of Cape Cotto (Associates of Cape Cotto). Can be purchased from sofCape Cod), Woods Hole, MA. Ru. p-Nitrophenyl phosphate was purchased from Fluka, Ronkonkoma, NY. It can be obtained from. The American Standard for Endotoxin was created by Donald Hofstein. (Donald Hochstein), available from Food and Drug Administration. It is possible [25]. Cholera toxin variety 1, lot 582, is Pasteur Mérieux. Serums and Vacuums (Pasteur MerieuxSerum Vaccines), Lyon, France, and Cholera toxin variant 1, lot rst, was purified from V. cholerae inaba strain 569B. [21.28] Anti-mouse IgG and IgM alkaline phosphatase binding The body is manufactured by Kirkegaard & Perry Laboratories, Inc. (Kirkegaard & Perry Laboratories, Inc. ), Gaithersburg, MD.

The bacterial strains used in the examples were the classical biotype of Vibrio cholerae, Inaba serotype. strain 569B and Vibrio cholerae, classical biotype, Ogawa serotype strain NlH41. It is used for vibricidal assay. Vibrio cholerae classical Inaba strain 2524 (Ki Katherine Green, C, D, C, At Ranta, GA) was used to raise antiserum against LPS in mice. Ru. Vibrio cholerae inaba serotype, EI Tor biotype, cholera toxin (CT) variant 2, Strain 075 is a new isolate from South America (Richard Haberberger (Richard Haberberger), Naval Research Med. Cal Institute (Naval Research Medical I nst i tute), Bethesda, MD). All of these strains are Laboratory, the laboratory of Dr. ・Of Developmental and Molecular Immunity, National ・Institute of Child Health and Human Development Belovment, National Institutes of Health (Labora) story of Developmental and Molecular I mmunity, National Institute of Child H earth and Human Development, National Institute of Health), Bethesda, MD 20892 or Same addressee, Dr. John B. Robbins. It can be obtained by contacting the laboratory. The equivalent strain is American ・Type Culture Collection/Yon (American Type Cu1t) available from ureCal Iect 1on), Rockville, MD. Ru. These strains were divided into Vibrio cholerae (Inaba), ATCC9459 and Vibrio cholerae. (Inaba, EI Tor biotype) Listed as ATCC 14033.

Example 1: Production and characterization of detoxified ribopolysaccharide LPS by two methods Detoxify. For acid hydrolysis, 10 mg/ml LPS in 1% acetic acid was Heat at 0° C. for 90 minutes [59]. The reaction mixture was heated at 60,000 x g at 10°C. Ultracentrifugation was performed for an hour, and the supernatant was filtered through a sterile 0.22 micron filter (Nalji). ge), Rochinister, NY) and lyophilized (referred to as 0-8P). ). For detoxification by base hydrolysis, 10 mg/ml LPS was Treat at 37° C. for 2 hours. Hydrazine treatment releases esterified fatty acids. has been reported to be removed from DeA- It is called LPS. Mix this material with acetone in a water bath until a precipitate forms. approximately 90% acetone) and the reaction mixture at 15,000 x g at 10°C. Centrifuge for 0 minutes. Approximately 3 mg/ml of the precipitate was added to 0.15 M NaCl, pH 7.0. Dissolve to ml. The reaction mixture was heated to 60.　000xg at 10℃ for 5 hours Centrifuge and dialyze the supernatant extensively against 820 and pass through a 0.22 micron filter. and lyophilized. Tan of 0-8P and DeA-LPS The protein and nucleic acid concentration was 1%. EI Tor biotype Ogawa serotype strain 3 LPS extracted from acetone-dried Vibrio cholerae cells of No. 083-13 was added to Vibriocidal. Used to inhibit activity.

Various preliminary characterizations of LPS were carried out using both in vitro and in vivo techniques. Let's do it. 5DS-PAGE is used for detection of LPS [56]. Inspected by LAL The determined LPSa degree is expressed in endotoxin units (EU) with respect to the American National Standards. [25]. LPS, 0-3P and deacylated LPS (DeA-LPS) The molecular dimensions are 0.2M NaCl, 1mM EDTA, 10mM Tris, 0.2 By gel filtration through superose 12 in 5% deoxycholic acid, pH 8.0. Estimated using dextran standards to calibrate the column. KDO is KDO as a standard [5]. double exemption The diffusion method is performed in 1% agarose in phosphate buffered saline (PBS). .

NMR spectrum data was obtained using a JEOL G5X-500 spectrometer. recorded. Each spectrum is a 90° 10-m5ec carbon observation pulse; 32,000 data zero-filled to 64,000 points before Fourier transformation point; 30KHz spectral window (acquisition time 0.54 seconds); pulse wide width 1) with a delay of 3.0 seconds between cycles (obtained by decoupling; Before the Elier transform, each free induction decay signal is converted into a frequency domain spectrum. The additional 4 Hz width increases exponentially. Approximately 10 mg of each sample was added to D2 Dissolve in 0.5 mL of 0 and record at ambient probe temperature (21°C).

Silver-stained SDS PAGE of 2.5 mg of LPS from Inaba shows a “ladder-like” shape in the center. ” and two dark bands near the bottom of the gel ( Figure 1). A typical “ladder” of high molecular weight o-sp is L from E. coli 0111. Formed by PS. For a 10 mg sample of o-sp or DeA-LPS, No bands were observed. According to the LAL test, the LPS of Inapa serotype is 3 to 6. x103EU/mg and DeA-LPS has 3EU/mg. shows a >1ooo-fold reduction. The immunodiffusion method uses LPS and hyperimmune LPS blood. A single precipitated band is shown in the middle (Figure 2). The band is not too dark (the more diffuse band is , observed with DeA-LPS resulting in a partial identity reaction with LPS. 0-8P Neither CT nor CT are precipitated by this hyperimmune serum. LPSlo-8P and De The molecular dimensions of A-LPS were determined by high performance liquid chromatography on Superose 12. It is estimated that (Figure 3). LPS and DeA-LPS show two peaks.

That is, the Kd value of LPS is 0.40 (16,000d) and 0.40 (16,000d). 46 (8,700d), and the Kd value of DeA-LPS is 0.38 (13,0O Od) and 0.50 (6,000d). o-sp is DeA-LPS Only one peak (Kdo, 51.5,900d) is shown, which corresponds to the second peak of did. Due to its more pronounced antigenicity and high molecular weight, DeA-LPS is used as a conjugate support. Preferably, it is used as a tucharide. The present inventor has developed a method for thiobarbic acid assay. Therefore, KDO in 0-8P or DeA-LPS cannot be detected [ 5,57] o DeA-LPS and o-sp 13C NMR spectra This is consistent with previous reports [30, 47]. Each spectrum is reported as this 10 major signals with the same or nearly the same chemical shifts as those of (Figure 3).

Example 2: Preparation and characterization of DeA-LPS-cholera toxin conjugate DeA-L Binding of PS and protein is accomplished using one of two methods. direction In method 1, the covalent attachment of LPS to the protein is linked to pneumococcal cell wall polysaccharides As described in [52], 5PDP was used to analyze proteins and De This is achieved by thiolating both A-LPS. DeA-LPS ( 3mg/m+) or protein (1.0mg/ml) in 0.15M HEPE S, dissolved in 2mM EDTA, pH 7.5. 5PDP (2 in ethanol 0mM) is added dropwise at a weight ratio of 0.5 DeA-LPS and 0.2 protein. . The reaction mixture was stirred for 1 hour at ambient temperature and 5X35 cm sephade in H2O and PH3 for proteins. Pass through a G-25 column. Lyophilize DeA-LPS-5PDP, Proteins were concentrated by membrane filtration (Amicon, YMIO) do. Induction by DeA-LPS or 5PDP in aliquots of protein. After reduction of N-pyridyl disulfide bonds by 40 mM DTT Determined by spectrophotometer assuming molar extinction coefficient at 0 nm as 8.08xlO' [53] 4 N-pyridyl disulfide on DeA-LPS-8 PDP G-25 Sephadex in 0.2M NaCl reduced with 0mM DTT. the void volume fraction (void volume fraction). lume fractions) with the 5PDP derivative of the protein.

The reaction mixture was stirred at room temperature for 2 hours and then treated with S-300 Seph in 0.2M NaCl. Pass through a 5xlOOcm column of Allyl and pool the void volume fractions. Ko D It is called eA-LPS-CTI. Aliquot of DeA-LPS-CTI in saline solution was treated with 0.05M EDAC for 1 hour at room temperature at pH 6.0. Crosslink the conjugate. Unreacted EDAC is removed by extensive dialysis against water do.

In method 2, Haemophilus influenzae (Haemophi l) DeA-LPS was added to ADH as described for type b. [9.48] DeA-LPS in saline, 10 mg/ml IN pH 10.5 with NaOH and an equal amount of CNBr (1 g in acetonitrile) /ml). pH: IN Na0Hl: O, 0-11. Oi l: Maintain for 3 minutes. Equal volume of 0.5M ADH in 0.5M NaHCOg and adjust the pH to 8.5. The reaction mixture was heated at room temperature for 1 h, followed by 3 to Stir overnight at 8°C, then pour into a 5 x 35 cm Sephadex G-25 column in H,O. Pass it through the ram. Fractions from the void volume are pooled and lyophilized. DeA- Dissolve LPS-AH derivative to 10 mg/m+ in 0.15 M NaCl . Add an equal volume of 9:/bacterium (approximately 10 mg/ml) and adjust the pH to O, LM. Add 6°EDAC adjusted to 5.51:i with HCI to a final concentration of 0.05M and adjust the pH. Maintain the temperature at 5.5-60 for 1 hour. The reaction mixture was dissolved in S-3 in 0.2 M NaCl. Pass through a 2.5 x 90 cm column of 00 Ceph acrylic and remove the image in the void volume. pool the minutes. CT (lot 582) and CT (lot rst) DeA-LPS-CTI I and D It is called eA-LPS-CTIII.

As in Example 1, in vitro and in vivo methods of conjugate characterized by. Induction of DeA-LPS by adipic acid hydrazide The degree is trinitrobenzene sulfonic acid (TNBS) using ADH as standard. [9]. Protein standard is bovine serum albumin [17]. Hexose is o-8P [55]. The endotoxin concentration is It is estimated in the same way as in the first embodiment. The double immunodiffusion method was also carried out in the same manner as in Example 1. Let's do it. The in vitro cytotoxicity of CT was determined by observing the elongation of CHO cells. [15] The pyrogenicity of the conjugate was determined in rabbits using the method of Hofstein et al. [25 ].

Table 1 shows the results of conjugate characterization.

Table 1. Characterization of hydrazine-treated lipopolysaccharide-protein conjugates of V. cholerae *0. 05M further processed with EDAC (Materials & Marys (MAT) ERIALS AND METHODS).

NA: Inappropriate Polysaccharide [] measured by anthrone reaction with DeA-LPS as standard. Collection The percentage is the saturation in the conjugate compared to the starting weight of the adipic acid hydrazide derivative. Calculated based on the weight of the id.

The extent of induction of DeA-LPS by 5PDP or ADH is similar.

The DeA-LPS/protein (wt/wt) ratio is higher than that of 5PDP due to ADH. The CT conjugate produced by DeA-L, PS-CTI was slightly lower; The range is from 72 for DeA-LPS-CTI to 1.5 for DeA-LPS-CTI. all combinations The yield for According to the calculation, it is about 80%. Similar results were obtained for tetanus toxoid conjugates. Obtained by method [1] for use as a protein component. Typical double immunodiffusion In the experiment, Inaba serotype hyperimmune antiserum was used to detect DeA-LPS and DeA-LPS- It is shown that an identical sedimentation line occurs for CTll (FIG. 2). Similarly, CT and and LPS antiserum have a line of identity with respect to DeA-LPS-CTI and CT. occurs. A thin spur with CT antihematology exceeds LPS antiserum and conjugate. spread, suggesting the presence of a small amount of unbound CT in this preparation. Up CT and D in the conjugate estimated by the in vitro and in vivo assays described below. The residual toxicity of eA-LPS is extremely low. In thermal induction tests, DeA-LPS , was not pyrogenic when injected at 1 mg/Kg (rabbit body weight). combination The endotoxin content was approximately 2 EU/mg by LAL assay. CT is CHO fine 0.4 ng/ml resulted in elongation in cells. Although it produces the same amount of elongation The amount of CT in bound form required was greater than 103-101°. Coming DeA-LPS-CTI IT, which is a standard product intended for floor use, is a CHO specification. No toxicity was detected in the cell assay at 1.0 mg/ml, and it also complies with U.S. regulations. 10 human doses listed in the provisions of Law C (DeA-LP 325 mg/dose) passed general safety testing in guinea pigs.

Example 3: Comparative efficacy of cell-mediated vaccine and LPS-CT conjugate vaccine V. cholerae LPS antiserum was administered to adult female BALB/c mice using heat-killed Vibrio cholerae strain 2524. Prepared by injecting [41]. Donkey CT antiserum as described [13] Prepare. To assess immunogenicity, 6-week-old BALB/c or General purpose mouse (NIH) 2.5mg or 10mg of DeA-LPS alone Inject subcutaneously or as a conjugate in saline. injected into mice at two-week intervals, Blood was collected 7 days after each immunization. 4th dose after 3rd injection At 4 weeks, mice are bled after 7 days and 6 rows. group of mice , 0.125 mg or 1.25 mg/dose of aluminum hydroxide. Combination also immunizes. 4xl each of Inaba and Ogawa serotypes Cellular cholera vaccine containing O@ (Wye Purchased from tb-Ayerst Laboratories), Marietsuta, PA. (possible) is used as a control. Mice were immunized with 1 ml or 0.2 ml of vaccine O. sensitize.

Complement-mediated vibriocidal antibodies are measured against Inaba and Ogawa strains [1 9.20]. Add a 10-fold serum dilution to approximately 1 equal volume of cells diluted with guinea pig serum. 000 cells/ml and incubate for 1 hour at 37°C. hyperimmune serum Used as a standard in each assay. Serum titers yielded 50% vibriocidal activity. It is expressed as the reciprocal of the highest dilution of serum. Some serum, Inaba serotype bacteria These blood samples were assayed for vibriocidal antibodies against strains 569B and 075. The titer of the supernatant was the same for both strains. Therefore, the vibriocidal activity of the serum The sex is assayed by strain 569B. Inhibition of vibricidal activity was performed before adding bacteria. LPSSDeA-LPS, 0-3P or CT 100mg/mI was added to various dilutions. It is assayed by mixing with diluted antiserum for 1 hour at 37°C [20].

LPS and protein antibody concentrations were determined using Immuno Ion4. using plates (Dynatech, Nyantilly, VA). Determined by enzyme-linked immunosorbent assay (ELISA). plate, LPSL in phosphate buffered saline (PBS), Omg/ml or CT5mg/m 100 ml/well. LPS antibody concentration was determined using hyperimmune serum as a control. It is expressed in ELISA units. CT antibody concentrations are typical in the art. Prepared by repeated immunization of mice with CT using methods known in the art. Expressed in EL units using hyperimmune mouse pool standard sera obtained. antibody concentration is expressed as the geometric mean. Antibody concentrations below the sensitivity of ELISA are 2 minutes below that concentration. The value is 1. Comparisons of geometric means are performed using two-tailed tests and Wilcoxon (Wilcoxon) tests. coxon) test.

After either injection, immunization was performed using 2.5 mg or 10 mg of DeA-LPS alone. LPS antibodies were not detected in the commercially produced or BALB/c mice. stomach. Table 2 shows the results of immunization with DeA-LPS-CTI and DeA-LPS-CTI I. The concentration of antibodies against LPS in sensitized commercial mice is shown.

Table 2. General purpose mice immunized with DeA-LPS alone or as a conjugate. Serum 1gM and IgG LPS antibody (ELISA) a3 approx. 6 Week-old female commercial mice were subcutaneously injected with the antigen saline solution every week for the third time. Blood was shed.

h vs. fSh vs. dSp=NS, h vs. gSp=0.002, d vs. cSp=o, 08 ; f vs. eSp = 0.06, h vs. i, NS0. Does not elicit LPS antibodies. DeA-LPS-CTII has no effect on Ig after the second injection. Elicit G and IgM antibodies. Both conjugates were tested after the third and fourth injections. Significantly increases IgG antibodies (p<0.01). IgG concentration after 4th injection was similar in mice injected with LPS or DeA-LPS-CTII. It is. The 2.5 mg or 10.0 mg LPS dose is elicits IgG antibodies. The IgG concentration was determined at the fourth time of DeA-LPS-CTI I. The same is true in serum collected on day 7 or column 6 after injection. The same antibody A similar concentration of EDAC-treated DeA-L with a 10 mg dose of conjugate by PS-CTI and by the conjugate adsorbed on alum. It will be done.

Table 3 shows the LPS antibody concentrations produced by the conjugates in BALB/c mice. This indicates that the degree of the mouse is lower than that of a general-purpose mouse.

Table 3. Immunized with DeA-LPS-CT conjugate or cellular cholera vaccine Serum 1 gM and IgG LPS anti-L drawn in BALB/c mice PS antibody *Mice whose blood was collected 5 months after the 33rd immunization.

e vs. a, P=0.0004, e vs. P=NS, e vs. d, P=0.0001, d vs c, P=0.02° In BALB/c mice injected with conjugate after first and second dosing. IgM antibodies are low and IgG antibodies are not detected. Low concentration after third injection Detected. Antibody concentrations remain similar 5 months after the last injection.

Cellular vaccines induce high concentrations of both IgG and 1 gM after the second injection. , and induces a booster effect by IgG antibody concentration after the third injection. throw Dose volumes of 0.1 ml and 0.2 ml result in similar concentrations. Injection of cellular vaccine IgG concentrations in injected mice dropped to about 1 of their optimal value 5 months after the last injection. p=0. oool) And DeA-LPS-CTI I It is similar to the value provided.

Both 2.5 mg and 10 mg of DeA-LPS or PBS (control) Does not elicit vibriocidal antibodies against Nava and Ogawa serotypes. Shown in Table 4 As such, DeA-LPS-CTI I was found to be effective after the first injection in commercial mice. , eliciting low concentrations of vibriocidal antibodies against Inaba strains.

Table 4. Pooled blood from NIH commercial mice immunized with conjugate or LPS Vibriocidal antibody titer of serum *ND - Not tested.

2.5 μg of DeA-LPS was subcutaneously injected into NIH commercial mice, and their blood The supernatants were pooled in equal amounts for each group.

Both DeA-LPS-CTI and DeA-LPS-CTI I are Elicit a booster response after one injection. LPS produces the highest concentration of vibriocidal antibodies. Pull out. In BALB/c mice, both conjugates were bibicidal after the first injection. eliciting antibodies; only DeA-LPS-CTI I was used for the second and third Elicit a booster response after injection (Table 5).

Table 5. DeA-LPS conjugated to cholera toxin (CT) alone or whole cell cholera Vibriocidal activity of serum from BALB/c mice immunized with vaccine None of the known conjugates elicits IgG antibodies after the first injection. capsular polysaccharide This apparently small size of the DeA-LPS-CT conjugate compared to that by [48] The immunogenicity is determined by two factors: (1) the smaller o-sp of Vibrio cholerae 01; The immunogenicity is due to its simple structure 4-amino-4,6-sideoxy-I,-gly7 by a linear homopolymer of verosamine acylated by rotetronic acid). [30.47]; (2) relatively low molecular weight 0-8P of Vibrio cholerae 01 [3 6, 46].

The inventors believe that the disease is caused by non-invasive microorganisms, and its symptoms are endotoxic. Cholera, which is transmitted by the virus and does not involve inflammation, can be treated with blood-killing vibrioviruses. We propose the following mechanisms by which antibodies protect.

First, serum antibodies, particularly class IgG antibodies, enter the intestinal lumen [28. 59]. Complement proteins are also believed to be present. Second, because the intestinal wall is spiral come into contact with each other. Third, the inoculum that survives the acidic conditions of the stomach is probably about 10 Vibrio cholerae [22.31. Fourth, V. cholerae has short polynucleotides on its LPS. sugar; this feature is related to its high susceptibility to the complement-dependent effects of serum antibodies. There are [42 children. These factors, i.e., pressure on each other's surfaces and Low inoculum at mucosal surfaces stirring up organisms, blood killing vibrio antibodies and complement shows how blood-killing vibrioantibodies protect against cholera, i.e., when ingested. This section explains how Vibrio cholerae is lysed on the intestinal mucosal surface.

The conjugate is more effective against the homologous serotype (Inaba) than against the heterologous serotype (Ogawa). Provides high vibricidal activity. Cellular vaccines with both serotypes are It induces higher levels of vibricidal antibodies against Ogawa than against barflies. Inaba serotype The level of vibricidal activity against and in high titer. After the third injection, whole cell and conjugate vaccines The levels of vibricidal activity against Inaba produced by Killer Bibb Rio activity was determined by Inaba serotype LPS, DeA-LPS, or 0-8. After adsorption with P, it is removed from the conjugate-induced antibody. By Inaba LPS Furthermore, the adsorption of vibriocidal activity from the serum of mice injected with the cell-mediated vaccine Remove all. In contrast, DeA-LPS and o-sp Approximately 90% of the vibricidal activity is removed. Adsorption by Ogawa LPS is due to Inaba bacteria. It removes about 90% of the vibricidal activity against the strain. Is adsorption by CT a conjugate? or alter vibriocidal titers from serum of mice injected with cell-mediated vaccines. I won't let you. DeA-LPS-CTI, DeA-LPS”CTI or other Adsorption of the conjugates onto alum does not affect their immunogenicity.

Table 6 shows cholera toxin antibodies induced by conjugate immunization. CT Significant increases in antibodies to both conjugates after each injection in all mice of both strains Therefore, it is brought about.

Table 6. Serum IgG cholera in mice immunized with DeA-LPS conjugate Toxin antibody count average (25-75 centiles) 8 whole cell cholera ELISA antibody concentration of mice immunized with vaccine, DeA-LPS or PBS The degree was <0.01.

b vs. aSP = <0.001; d vs. c, P = area 0001; e vs. d, P = 0.0 4. hSg vs. f, P<0.01; b vs. eSP=0.01: Lk vs. i, P＜0゜01゜ Example 4: Diagnostic and therapeutic applications The LPS-protein conjugate of the present invention provides a human or animal subject with the LPS-protein conjugate. Treat infections caused by microorganisms containing S and/or protein antigens It can be administered in the form of a vaccine for protection or prevention purposes. This kind of work Cutin can contain about 5-100 μg of LPS-protein conjugate.

These vaccines can be administered subcutaneously or intramuscularly. against the conjugate Antibodies generated from sterile-filtered or radiation-sterilized milk or goats) and administered orally. The conjugate is alum can be suspended in salt water, saline, buffer solutions or oil-water emulsions; Subjects receive a series of 1 injection, preferably 1 to 5 injections over a 12 month period. The vaccine can be injected by

Monoclonal or polyclonal antibodies of human or animal origin can be used in the vaccines mentioned above. can be manufactured by using. These antibodies are effective against animals and humans. Thus, the LPS and protein components of the LPS-protein conjugate of the present invention are obtained. For the prevention or treatment of infections caused by one or more microorganisms The conjugate vaccine can be administered alone or in combination with the conjugate vaccine. these Antibodies can be used alone or as an adjunctive therapy with the LPs of the invention for purposes of passive immunization. in combination with S-protein conjugate vaccines to subjects in need of them. It can be administered to Such antibodies can be obtained from serum or serum containing the antibody in question. It can be obtained in the form of gamma globulin.

Of particular interest are antibodies against the LPS and CT antigens of V. cholerae. this Their antibodies are directed against related bacterial toxin antigens, particularly E. coli and Campylobacter jejuni. (Campylobaceter jejeuni) and Aeromonas hid Secreted by Aeromonas hydrophilia demonstrated neutralizing activity against the toxins.

Monoclonal or polyclonal vaccines produced by the use of the conjugate vaccines of the present invention. Antibodies can also be used for diagnostic purposes or as t, ps-protein conjugates, etc. LPS alone or its polysaccharide fragments or their A method for developing microorganisms expressing derivatives of , which can be used for research in pathogenesis, prevention, and immunopathology. be able to. Antibodies can also be used to study immunological responses to the above antigens. I can be there.

Such antibodies can be used to induce or react with other substances for disease diagnosis. or for the identification of microorganisms containing LPS or proteins used in conjugates. Kits can be manufactured.

Although the invention has been thus described, the same may be modified in many ways. is clear. Such modifications shall be considered a departure from the spirit and scope of the invention. No such changes should be made and would be obvious to a person skilled in the art. These are also meant to be included within the scope of the claims.

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,'1. ′″・ Holding capacity (ml) Submission of translation of written amendment (Article 184-7, Paragraph 1 of the Patent Act) July 181, 1994

Claims (1)

[Claims] 1. An isolated and purified bacterial strain that has been detoxified and exhibits low pyrogenicity in mammals. A vaccine comprising popolysaccharide (LPS); and a pharmaceutically acceptable carrier. 2. The vaccine according to claim 1, wherein LPS is detoxified by reaction with hydrazine. hmm. 3. The vaccine according to claim 1, wherein the LPS is detoxified by acid hydrolysis. 4. Proteins isolated from or secreted by bacterial strains A linkage comprising the LPS of claim 1 covalently bonded to the LPS by a bifunctional linker. Coalescing compound. 5. The conjugate according to claim 4, wherein the protein is a cell surface localized protein. . 6. 5. The conjugate of claim 4, wherein the protein is a bacterial toxin. 7. 7. The method according to claim 6, wherein said LPS and said bacterial toxin are obtained from the same microorganism. Combined body. 8. The conjugate according to claim 6, wherein the microorganism is Vibrio cholerae. 9. the toxin is cholera toxin of Vibrio cholerae, and the symptomatic organism is Vibrio cholerae. The conjugate according to claim 7. 10. The covalent bond is adipic acid dihydrazide, diaminohexane, amino- ε-caproic acid and N-hydrosuccinimidic anhydride-based heterobifunctional is achieved by reaction with a bifunctional linker selected from the group consisting of The conjugate according to claim 4. 11. The N-hydrosuccinimidic anhydride-based heterobifunctional linker is N -Succinimidyl-3-(2-pyridyldithio)propionate 11. The conjugate according to 10. 12. The adipic acid dihydrazide reaction component is 11, further reacted with a conjugate according to claim 10, further reacted with Covalent aggregates of LPS-protein conjugates. 13. A method for producing a covalent aggregate of an LPS-protein conjugate, the method comprising: (1) The LPS component of the conjugate is detoxified, and (2) the detoxified PS component of step (1) is adipied. (3) detoxified LPS derived from step (2). mixed with the protein component of the conjugate; (4) Add 1-ethyl-3(3-dimethylaminopropylene) to the mixture of step (3). ) add carbodiimide, and (5) The above method comprising collecting a covalent aggregate of LPS-protein conjugate. 14. In step (1), LPS is detoxified by reaction with hydrazine. The method according to claim 13. 15. 2. The vaccine of claim 1, further comprising a second vaccine. 16. 15. The second vaccine is a commonly available DTP vaccine. Vaccines described in. 17. A vaccine comprising the conjugate of claim 4 and a second vaccine. 18. 17. The second vaccine is a commonly available DTP vaccine. Vaccines described in. 19. A method of immunizing a patient against cholera, which provides protection against cholera. administering to a patient an amount of the vaccine of claim 1 sufficient to The above method including. 20. A method of immunizing a patient against cholera, which provides protection against cholera. administering to a patient an amount of the conjugate of claim 4 sufficient to provide The above method. 21. A method of immunizing a patient against cholera, which provides protection against cholera. 10. administering to a patient an amount of the conjugate of claim 9 sufficient to provide The above method.