JP2001527562A - Vaccine against lipopolysaccharide core - Google Patents

Abstract

  (57) [Summary] Complete core LPS from Gram-negative bacteria (lacking the O-polysaccharide side chain) is incorporated into vaccines, typically in liposomes. The complete core of the E. coli K12 strain is particularly useful. The vaccine, when administered to mammals, facilitates the synthesis of cross-protective antibodies against smooth and rough LPS from at least two different Gram-negative strains with different core structures.

Description

DETAILED DESCRIPTION OF THE INVENTION                       Vaccine against lipopolysaccharide core                         Cross-references for related applications   This application was filed on May 16, 1997, which is hereby incorporated by reference. No. 60 / 046,680 of the inventors of the present invention.                                Field of the invention   The present invention relates to the general field of reducing the harmful effects of endotoxins from Gram-negative bacteria. It is                                Background of the Invention   Endotoxins (also called lipopolysaccharides [LPS]) have toxic effects after entering the bloodstream. It is thought to express much. Endotoxin blood where endotoxin is present in the blood The disease can occur in various situations, for example, during periods of stress. For example, in Toxemia has undergone certain types of surgery, anticancer chemotherapy, radiation therapy and immunosuppressive therapy. May occur in patients who have suffered various injuries, burns or wounds It can happen. In addition to military personnel, police officers and firefighters, endurance athletes, horses and homes It can also occur in livestock. After immunosuppressive therapy and patients with sepsis or septic shock As well as those suffering from stress or trauma as described above .   One way in which endotoxins reach the blood is from the patient's brain, which can cause infection, The intestine loses its ability to contain LPS in the event of injury or trauma. Normally, the intestinal flora contains large amounts of endotoxins from Gram-negative bacteria. Human colon It is estimated to contain an average of 25 billion nanograms of endotoxin, with endotoxin concentrations of 10^TwoOh no This is an enormous amount, considering that toxic substances are toxic to humans. I lived Leakage of bacterial cells into the bloodstream can cause infection as the bacteria grow . Many bacteria in the intestine are dead bacteria, and endotoxins contained inside the dead cell membrane fragments May enter the bloodstream. In this case, no infection occurs by itself. But instead In addition, endotoxin that has flowed into the blood from the dead bacteria is responsible for tumor necrosis factor and various interrogations. Triggers a systemic inflammatory response by activating macrophages that release kins It is thought to happen. Exposure to endotoxin and the resulting systemic inflammatory response Is Of the body organs, including the lungs, kidneys, heart, blood vessels, digestive tract, blood / coagulation system and nervous system May cause failure. This pro-inflammatory response is severe and can result in organ failure and sometimes death. May bring.   LPS is considered a major cause of septic shock. The ratio of this systemic inflammation Less severe forms, unlike organ failure, have been shown to cause organ dysfunction. Is being recognized. In the mildest form, endotoxemia may occur in postoperative patients or for other reasons. Causes fever, nausea, and malaise, which are common symptoms in patients hospitalized for These symptoms may occur in athletes after intense activity.   If the host is exposed to high levels of endotoxin or is sensitive to its effects In that case, a higher inflammatory response can occur. For example, many post-surgical patients Develops pulmonary dysfunction requiring feeding. They have blood or kidney complications Sometimes. These complications are less likely to cause death, but cause pain, Prolong the length of hospital stay, thereby increasing medical costs. 28 million surgical patients in the United States At least 10% of people are systemic as a result of exposure to endotoxin from Gram-negative bacteria It is thought to cause inflammation and is presumed to have the potential for complications. Introduction For Bennett-Guerrero (B ennett-Guerrero) et al., Crit Care Med, 25: A112, 1997 and Bennett-Guerrero. J. et al. Am. Med. Ass. 277: 646-650, 1997.   Bacteria and their endotoxins are thought to cause high frequency of complications , Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus, Enterobacter, Salmonet There are La, Serratia, and Shigella. These bacteria produce lipopolysaccharide (LPS) Gram-negative bacteria, a class characterized by specific types of outer membranes, including as major components It is. The components of LPS vary by bacterial species, but are generally described with reference to Figure 1. As shown, a) lipid A, b) core, and c) O-polysaccharide outer region (O-polysa ccharide outer region). Lipid A lipid region is outer membrane Is embedded in the outer leaflet. Polysaccharide core region is lipid A Located between the O-polysaccharide external region. Lipid A is effective against virtually all Gram-negative bacteria. It has the same basic structure and is a major endotoxin determinant. In the LPS core area There is a high degree of similarity between bacterial genera. For example, the inner core region contains heptose and And 3-de Oxy-D-manno-2-octulosonate (KDO) residues The region contains galactose, glucose or N-acetyl-D-glucosamine residues It exhibits various modes depending on the bacterial species. O-polysaccharide external region (O-specific antigen or O-specific (Also called heterologous side chains) are highly variable and may have one or more It is composed of sugar repeating units.   The presence of the O-polysaccharide side chain results in a smooth appearance in cultures of wild-type bacteria, Wild-type bacteria with polysaccharide side chains usually contain O-polysaccharide and (optionally) core regions. Mutants that have a rough appearance due to lack of part of the bacteria). For example, a rough mutant of Salmonella The various chemotypes are usually named Ra, Rb, Rc, Rd and Re. I have.   As can be seen from FIG. 2, each type of LPS contains a lipid A structure. Ra Chemotay The Rb chemotype features an N-acetyl-D-glucosamine residue. Rc chemotype is characterized by lack of N-acetyl-D-glucosamine and Characterized by lacking lactose residues, the Rd chemotype is an optional component of the outer core The Re chemotype is the only KDO region attached to lipid A. It is characterized by.   Figure 3 shows all monkeys in addition to the five known complete core chemotypes of E. coli. 1 illustrates one known complete core chemotype for Monella species.   All LPS molecules present on any cell or in a homogeneous population of cells It does not have oligosaccharide side chains. For example, a single strain from a population of smooth bacterial strains Cells contain rough LPSN, LPS not substituted by any polysaccharide side chains Some seem to be included.   Various treatments for the toxic effects of LPS have been proposed or attempted. Against endotoxin One of the defenses of mammals that bind is to bind to and neutralize endotoxins carried in the blood Antibodies are present in the blood to prevent or control this type of infection. Or alternatives to antibiotic therapy to reduce the toxic effects of endotoxins. Alternatively, immunological methods have been proposed as additional therapies. For example, conventional polyclones Antiserum and hyperimmune serum increase opsonization or phagocytosis of bacterial cells. strength Alternatively, assuming neutralization of the biological activity of LPS, the patient's innate Attempts have been made to reinforce defenses. However, the effectiveness of antisera, for example, Large, depending on a number of factors that are not easily standardized, including the composition and titer of the Change. The use of these antisera also implies a risk of transmitting viral infections. There is also a disc.   To enhance the production of cross-reactive anti-endotoxin antibodies by the host, Of vaccines with various immunogens to healthy or hyperimmune serum donors Species (ie, active immunization) have been attempted. Over the last 20 years, various types of wakuchi Compositions and methods of immunization are being investigated. For example, Bhatcharlie (Bhat tacharjee A), WO 95/29662, McCabe WR, J Inf ec Dis 1988; 158: 291, Greisman SE, Proc Soc Exp Bio Med.  1978, 158: 482, Goto M, Res Comm Chem Path Pharm 1992; 76:24 9. DeMaria A, J Infec Dis 1988; 158: 301, Baumgartner ( Baumgartner JD), J Infec Dis 1991; 163: 769, Cross A, J Infec  Dis 1994; 170: 834, Cryz SJ, U.S. Patent No. 4,755,381, Mylar (Miler JM), J Med Microbiol 1977; 10:19, Ashton (Ashton FE), Mic rob Pathog 1989; 6: 455; Dorner F, U.S. Patent No. 4,946,677; Liz (Cryz SJ), US Patent No. 4,771,127, Collins MS, US Patent No. 4,693,891, Pier GB, US Pat. No. 4,285,936, Cryz SJ , J Infec Dis 1991; 163: 1040; Cryz SJ; J Clin Invest 1987; 80 See: 5LPCT WO 92/06709, U.S. Patent No. 5,641,492.   The vaccine can be used in animals and humans to be vaccinated, for example, in Ziegler. (Ziegler) et al., NewEng. J. Med., 307: 1225 (1982), DeMaria, Infec. Dis. Common side effects as reported in 158: 301 (1988)-fever, Fatigue and other types of toxicity must not occur. These side effects are Pre-operative elderly and many medical problems are important target populations for vaccines It may be exacerbated in high-risk individuals, such as affected patients.   The anti-endotoxin core antibody concentration was measured in 301 patients before cardiac surgery, and surgery was performed. Test results have been published that examine the relationship with subsequent results. Bennet-Guerrero tt -Guerrero) et al. Am. Med. Ass. 277: 646 (1997). These anti endotoxin core anti The body has been measured using an ELISA that seems to detect antibodies against the core of LPS . In this study, patients with higher levels of core-specific antibodies were attributed to endotoxemia. High mortality or prolonged hospital stay. this Publications describe the control and treatment of endotoxemia in these patients. Is not described.   Regarding the toxicity of LPS, there are several approaches to certain lipid A or LPS Some evidence has been obtained that it may reduce the toxicity of liposomes.   Liposomes have been proposed as carriers for formulations containing lipid A. For example, In an attempt to create a vaccine specific for meningococcal LPS, LPS was Incorporation into the media reduces the toxicity of LPS from meningococci Have been. Petrov et al., Infect. Immunity 60: 3897 (1992). That In another prior art, a chemically modified monophosphoryl lipid A (MPLA) Incorporating a modified form of lipid A into liposomes reduces the toxicity of MPLA components This has been shown to be reduced. Richards et al. Vaccine 7: 506 (19 89). By incorporating MPLA and malaria immunogens into liposomes, It was considered that the Subant effect also occurred. That is, incorporation into liposomes Increased the immunogenicity of the malaria immunogenic component of this antimalarial vaccine .   Methods for detoxifying the lipid A component of LPS have also been described. Locust Bhattacharjee et al. Infec. Dis. 173: 1157 (1996).   Genetic modification of the bacterial strain and the resulting bacterium is responsible for the lipid A component There have also been reports of including LPS, which is less biotoxic. Somerville (S omerville) et al. Clin. Invest. 97: 359 (1996).                                Summary of the Invention   Complete core rough LPS antigen, especially E. coli Vaccination with K12 strains (active immunization) may result in unacceptable toxicity or Strain-specific protection and cross- core) provides both protection. Antigens are composed of the outer core galactose and glucose. Residues, plus lipid A, heptose and 3-deoxy-D-manno-2-octulo Sone Is considered to be a complete core, rough LPS, containing at least the amino acid (KDO) residues. Typically , Which also include the outer core N-acetyl-D-glucosamine residue. For example, this is shown in Figure 2. It includes the outer core structure of Rb as shown, and typically also includes the structure of Ra. this Does not include the O-polysaccharide outer region (also called the O-polysaccharide side chain).   Accordingly, one aspect of the present invention generally relates to Gram-negative bacteria, particularly E. coli K12 strains. Effective amount of a composition containing a complete core rough lipopolysaccharide (LPS) antigen (eg, Ra LPS) In warm-blooded animals (mammals, typically human patients) by administering It features a method of reducing the harmful effects of endotoxemia. Preferably, the immune composition is , Mixed with other gram-negative bacteria complete core rough lipopolysaccharide (LPS) antigen (cockt ail). Useful rough LPS are from E. coli and Salmonella, In particular, the five known chemotypes of Escherichia coli: Escherichia coli R1, E. coli B2, E. coli R3, and colon It is derived from each of bacteria R4 and Escherichia coli K12 (Jansson et al., Eur. Biochem. 115: 571 (1981)). See FIG. All known Salmonella species The core structure is occupied by only one type, for example, Salmonella minnesota (Salmonella minnesota) Use any Ra-type Salmonella strain such as R60 strain (Rietschel et al. Infect. Dis. Clin. N. Am. 5: 753 (199 1)). Complete core L except for polysaccharide side chains from other Gram-negative bacteria that may be useful PS include Enterobacteriaceae (ie, Escherichia, Salmonella, Klebsiella) , Citrobacter, Shigella, Proteus, Edward Sierra, Entero Bacter, Hafnia, Serratia, Providencia, Morganella, El Pseudomonas spp., Pseudomonas aeruginosa, Bacteroides -Includes those derived from Bacteroides such as fragilis. About the introduction ESSENTIALS OF MEDICAL MICROBIOLOGY, Third Edition, Volk et al., Pp. 397 and 416 (J / P. Lippencott Co. Philadelphia, P. See A (1986) Gram-negative bacterial population. The compositions are each other Different (eg different species or at least different strains of the same species), some (2, 3, 4 or more) complete core-rough LPS antibodies from Gram-negative bacteria May contain the original. In such a mixture, each of the four bacteria Core antigens can be expressed in functionally equivalent amounts (e.g., So that To the extent that they are present).   The vaccine is desirably at least one graph whose LPS is not part of the composition. Produce antibodies that bind to an epitope within the core region of the LPS core of To produce cross-reactivity and cross-protection. Sum Purely vertical and purely horizontal for coarse and rough gram-negative LPS Some cross-reactivity and cross-protection is difficult to achieve, and Especially in E. coli, the species most frequently isolated from intensive care units Have difficulty. There are two types of cross-reactivity, each labeled horizontal and vertical. Can be manifested. Vertical cross-reactivity means that the antibodies are different strains within the same strain. Reacts with LPS, which differs in the degree of substitution or O-specific side chain length To do. Horizontal cross-reactivity means that the antibodies are different, Reacts with the core structure of the strain, species, etc. In particular, the antibody response of patients It is desirable to combine with the smooth type LPS in addition to the type to provide protection. The inventor Do not wish to be bound by any particular theory, The epitope of the immunogen used in the vaccine according to the present invention is smooth and We believe that it is accessible to both types of LPS.   It is believed that encapsulating the antigen inside the liposome structure is particularly useful. For example, the ratio of lipid to LPS antigen (weight to weight) in the liposome is from 1: 1 to 5000: 1. (More typically between 10: 1 and 1000: 1). Liposomes increase stability To obtain or alter the charge of the compound, from the group consisting of: May contain selected ingredients: phospholipids, cholesterol, positively charged compounds, Negatively charged compounds, amphiphilic compounds. Especially using multilamellar liposomes (MLV) It is possible. Using small or large unilamellar liposomes (SUV and LUV) You may.   The composition can be administered intramuscularly, intravenously, subcutaneously, intraperitoneally, or by respiratory or gastrointestinal tract. Can be given. Antigen dosage correlates dose with titer and / or protection Can be readily determined by standard dose testing to determine Functional dose is 1k patient weight It is considered to be between 0.01 ng and 1000 ng per g, but further optimization will improve safety Higher doses (up to 100 μg / kg body weight) without harm and avoid harmful side effects ) May also be indicated to be desirable. IgM antibodies can provide adequate protection, If the target is the production of IgM antibodies, the endotoxin should be used to allow the production of IgM antibodies. Before the expected point of exposure (at least two days before, typically before) The composition may be sufficiently administered in advance. Also, in this case, the IgM response is substantially Should be administered before it is significantly reduced, for example, 14 days prior to exposure It is thought that there is no. First dose at least 2 days before expected endotoxin exposure The composition may be administered multiple times in such a form as to be administered to a subject.   The antigens in the composition are those of bacteria killed by heat or formaldehyde, etc. It may be present as a part. Alternatively, isolate antigens from bacteria before formulating the composition May be. Alternatively, the LPS antigen is a complex with purified LPS or an acceptable carrier (Appelmelk et al., J. Immunol. Met). h., 82: 199 (1985)).   The antigen may be chemically detoxified. Bacteria for various reasons, such as reducing toxicity May be subjected to genetic manipulation. The composition may also include an adjuvant such as alum You.   The invention also features a vaccine composition as described above in connection with the method. Therefore The vaccine contains an effective amount of one or more complete core rough LPSs of Gram-negative bacteria. No. When administered to a warm-blooded animal, the composition will contain an epitope located within the core region of the LPS molecule. At least two different gram shades that recognize loops and have different core structures Of Antibodies with Cross-Protection against Endotoxemia Caused by an Infectious Strain Prompt. In particular, antibodies synthesized in response to vaccines are completely core rough LPS (O -Lacks polysaccharide side chains) and has cross-protection against smooth LPS. Cerebral fungus In this case, the antibodies induced by the vaccine are preferably all common smooth Reacts with isolates of the strains, preferably five rough types (R1, R2, R3, R4, etc.) And K12). Preferably, the vaccine-induced anti- The body responds to both smooth and rough LPS of different strains of Salmonella I do.   Further, the vaccine described in the present invention is preferably licensed after administration to a mammal. Causes no unacceptable toxicity. Toxicity incorporates LPS into liposomes Detoxifying the lipid A component of LPS and / or inheriting said bacterial strain Target It can be controlled by modifying the lipid A component by manipulation.   The vaccine composition may be used by a donor to collect antibodies for administration to a patient. Can be used for immunization. Preferably, the collected antibodies are IgM class antibodies. In a substantial ratio.   Another aspect of the invention is to quantify lipopolysaccharide incorporated in liposomes. (PAS method). This method is different from typical radiolabeling methods. And there is no need to convert the lipopolysaccharide to a form that is not suitable for clinical use.   Other features and aspects of the present invention will become apparent from the following description of specific embodiments. It is considered to be.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic diagram of a smooth type and a rough type LPS.   FIG. 2 is a schematic diagram of the chemical structure of a Salmonella R mutant.   Figure 3 shows the known identities found in Salmonella and E. coli complete core rough LPS. It is a schematic diagram of a chemical structure of a type.                            Description of the preferred embodiment Medical applications   Patients treated with this vaccine are at risk for exposure to endotoxin Patients are included. Specific candidates for active immunity include patients scheduled for surgery , Chemotherapy or radiation therapy, burn patients, trauma patients, dialysis Patients and hospitalized (especially ICU) patients present with sepsis or septic shock Whether or not included. Other possible vaccination candidates Are soldiers, firefighters and police officers, endurance athletes, Which livestock is included.LPS component   As mentioned above, the LPS antigen to be included in the vaccine is any that lacks the O-polysaccharide side chain. It may be a complete core LPS, preferably from E. coli K12, as described below. Things. The Rb chemotype may be used, but the preferred embodiment is Ra or complete core It is a type chemotype. LPS may be purified from cultured bacteria, and Difco (Difco co), Sigma, Campbell, California pb ell) from List Biologicals. You may purchase something. The microorganism is a national culture typeco Collection (National Culture Type Collection) (NCTC), Scotland Collection of the University of Edinburgh in Edinburgh¹, And Forschungsinstitut in Borstell (FB) in Germany and D-2061 (FB) It is widely available from contract facilities, including. Examples of specific bacteria include the following strains: Restrictedly included: Escherichia coli strain K12-eg Edinburgh # MPRL2320, FBW3100 or List Biologicals, E. coli R1 strain-eg Edinburg h # MPRL2316 or FB F470, E. coli R2 strain-e.g. Edinburgh # MPRL2317 or FB  F576, cerebral fungus R3 strain-For example, Edinburgh # MPRL2318 or FB F653, E. coli R4 strain- For example, Edinburgh # MPRL2431 or FBF2513, Ra Salmonella minnesota (S. min. nesota) R60 strain Edinburgh # MPRL1265 or List Biologics (List Bio) logicals), S. typhimurium Ra type (eg, TV119, 1542), P. aeruginosa PAC611 (eg Edinburgh # MPRL1091) and Klebsi E. aerogenes strain M10B (eg, Edinburgh # MPRL0954), Salmonella minnesota (S. minnesota) Rb chemotype (eg Edinburgh # MPRL1 091) R345 strain, Bacteroides fragilis-NCTC 934 3. Bacillus vulgatis NCTC 10583, Bacillus citay Automicron (B. thetaiotaomicron) NCTC 10582.   ¹University of Edinburgh Medical School (Edinb urgh, Scotland), Ian Poxton, Ph.D.   We wish to be bound by a single mentor with respect to the operation of the present invention. Although we do not have anti-LP in which the inner core region is cross-reactive and cross-protective, It is specifically mentioned that epitopes important for promoting the synthesis of S antibodies may be included. I However, internal core epitopes can be identified as clinically significant LPS isolates (ie, And to maintain a three-dimensional structure similar to that produced by rough and complete core LPS) Requires a sufficient outer core region. Due to the absence of polysaccharide side chains (Ie, rough LPS), the core epitope can be the dominant epitope. S Polysaccharide side chains are a much more dominant epitope in Mews-type LPS than in core Because of the production The relative amount of anti-core antibody used is significantly reduced. In other words, smooth LP Vaccines containing S are in contrast to the anti-core response, which is the central subject of our invention Elicit primarily serotype-specific (ie, anti-polysaccharide side chain) antibody responses.   Escherichia coli K12 is considered particularly useful because it is not generally present in patient populations. Can be For this reason, K12 is unlikely to trigger a memory response to the outer core, It is likely to elicit a cross-reactive memory response to the inner core.   If the vaccine is to include whole bacteria, heat-sterilize or form Can be killed by techniques well known to those skilled in the art, such as aldehyde sterilization . In this case, the whole LPS of the rough mutant bacteria is considered to be included as a part of the dead bacteria. available. It is desirable to avoid sterilization methods that alter the core.   Or, Hancock et al., BACTERIAL CELL SURF ACE TECHNIQUES), pp. As outlined in 91 (John Wiley & Sons 1988) The complete core LPS can also be isolated from the desired bacteria according to standard techniques . As mentioned above, include all core LPS except O-polysaccharide external LPS structure, i.e. It is preferable to use an R mutant bacterium that expresses all of the LPS core.Patient response   As noted above, the desired patient response is whether their external LPS structures are similar Whether with or without rough and smooth LPS cores of Gram-negative bacteria The production of binding cross-protective antibodies.   For example, in the case of E. coli, the antibodies induced by this vaccine are preferably all Reacts with all common smooth strain isolates, preferably five types of rough LPS Reacts with all of A (R1, R2, R3, R4 and K12). Preferably this wax Antibodies elicited by the chins include various smooth and rough forms of Salmonella. Also reacts with LPS.   With compositions having an acceptable level of toxicity (or no toxicity at all), It is possible to achieve a strong and effective antibody response. This vaccine is LPS Recognizes an epitope within the core region of the molecule and has a different LPS structure Against endotoxemia caused by at least two different Gram-negative strains Cross protection, especially against fully core strains and smooth strains. Even Promotes the synthesis of cross-protective antibodies.   Typically, the vaccine is a coctail of purified LPS from bacteria of different strains. ), Preferably a rough strain having a complete core, such as L from K12 A mixture of PS and E. coli R1 and R3 rough strains, or Salmonella minnesota ( Salmonella minnesota) It is a mixture with R60 strain Ra type. Escherichia coli R2 and R4 are important It falls in gender, but is also a candidate. Preferred mixture (depending on the desired range of protection) K12 and R1, K12 and Pseudomonas (for example, P. aeruginosa) And Klebsiella (eg, K. aerogenes). Bacteroides in the intestine Are particularly important populations in, for example, with or with K12, Pseudomonas Both Bacteroides and Klebsiella contain Bacteroides in the mixture, It is important to provide specific protection against Lloides endotoxin.   Alternatively, when multiple types of LPS are used, one of these strains, A mixture of any combination, or purified LPS from different strains of bacteria Can be used in any ratio for individual strains.   The route of administration is preferably subcutaneous or intramuscular, but these immunogens are presented as antigens Any alternative route to cells and antibody-producing cells is acceptable. Other In some cases, intravenous, intraperitoneal, and respiratory or gastrointestinal Limitedly included.   Administration of the composition, as described above, does not interfere with avoidance of toxicity, And the host must be stimulated to produce large amounts of cross-protective antibodies.   The composition is administered prior to exposure to endotoxin. Part of the action of the vaccine is the host Vaccine is preferably endotoxin, as long as it stimulates the synthesis of IgM class antibodies by It is administered 2 to 14 days before expected exposure to the element. Or this vaccine Additional efficacy in any possible combination of Higher antibody levels or significantly reduced toxicity. Big In some vaccines, the antibody response to the inner core determinant is primary (ie, Unlike the naive) response, it is considered to be a secondary (ie, memory) response. This This is most likely due to LPS leaking into the bloodstream through the intestinal barrier. Kuching binds to the LPS core epitope at some point during its lifetime. Exposure Because it seems to be done. In other words, our method of vaccination Important functions of stress and trauma, although they may already be present To a level that does not provide sufficient protection against toxic exposure to LPS The purpose is to increase the serum concentration of indispensable antibodies. The above is the primary (Naive) Patients who also benefit from the vaccination according to the invention due to the antibody response It does not imply that no one is present.   The above-mentioned vaccine using LPS is obtained by incorporating LPS into liposomes. Preferably, it is non-pyrogenic and non-toxic. Liposomes (excluding LPS components) (1) phospholipids and cholesterol or (2) phospholipids, cholesterol and And combinations of negatively and positively charged (lipophilic) amphiphilic compounds . The phospholipid component may be any phosphatidylcholine derivative, glycerophosphatide Lysophosphatide, sphingomyelin and mixtures thereof, without limitation Can be selected from the group consisting of all lipids capable of forming liposomes . Negatively charged (lipophilic) amphiphilic compounds are di (alkyl) phosphates, Phosphatidic acid, phosphatidyl serine, phosphatidyl glycerol, phos Fatidylinositol, dicetyl phosphate or Any other similar negatively charged parents that can impart a negative charge to the liposome surface It can be selected from the group consisting of the amphiphilic compounds. Positively charged (lipophilic) amphiphilic compound If used, they may be stearylamine and hexadecylamine. Selected from the group consisting of alkylamines. Structure of liposomes (excluding LPS) The ratio of the components is thought to affect the charge, stiffness and stability of the liposome, They reduce the toxicity of LPS and maintain their immunogenicity, It seems that it can be changed sharply. Prolongs the life of liposomes in systemic circulation To increase their immunogenicity, and (PEG) can also be incorporated into liposomes, for example at about 10-20 mole%. . Or, for example, distearoyl phosphatidyl choline or distearoyl pho The use of lipids in the gel phase at body temperature (37 ° C), such as sphatidylserine, Alternatively, a very rigid double layer can be produced. Types of liposomes used Are preferably multilamellar liposomes (MLV), but are alternatively sonicated or Is an alternative Depending on the method of construction, small or large unilamellar liposomes (SUV and L UV) can also be used. Incorporation of LPS into liposomes for various salt forms of LPS The extent of this may vary, for example, acid salt form, magnesium salt form, and potassium. Increased incorporation may be possible in the form of lucium salts due to increased hydrophilicity It is.   Liposomes are phospholipids (examples include lecithin and sphingomyelin). And other lipids, such as cholesterol and other steroids C: charged lipids such as dicetyl phosphate and octadecylamine, glycolipids, Fatty acids and other long-chain alkyl compounds, hydrophilic glycoproteins, and fat-soluble As a closed vesicle or sac that may also contain Tamine and lipoidal surfactant-like molecules) Is defined. When shaken in the presence of excess water, this lipid mixture forms a lipid bilayer. Formed dispersive particles consisting of concentric spherical shells, which are multilamellar liposomes. (MLV). Smaller or smaller by sonication or alternative manufacturing methods Large unilamellar liposomes (SUV or LUV, respectively) can be formed.   When injected into animals and humans, liposomes are transformed into cells of the reticuloendothelial system, especially those of the liver. As a result, it is rapidly captured. Are liposomes relatively impermeable and circulatory? Substances such as lipid A and certain forms of LPS Cells that remain incorporated into the system and may cause toxic effects And / or is unlikely to be exposed to the receptor. In addition, multiple layers of liposomes Because biodegradation of membrane structure is slow, liposomes can have sustained efficacy .   The toxicity of the lipid A component of the complete core-rough mutant strain was (Bhattacharjee A) et al., Chemicals as described in WO 95/29662. It can also be reduced or eliminated by detoxification. Preferred for this detox According to the method, the configuration of LPS recognizes an epitope within the core region of the LPS molecule. By at least two different Gram-negative strains, both with different core structures Preservation of cross-protective antibody synthesis against induced endotoxemia Will be maintained. In particular, antibodies synthesized in response to this vaccine It also has cross-protection against smooth strains in addition to core strains. Detoxified LPS Is the spirit It may be administered in the form of a manufactured LPS or, alternatively, it may be incorporated into liposomes. Or a complex with an acceptable carrier.   Alternatively, the toxicity of the lipid A component of the above bacterial strain was determined by Somerville JE. , J Clin Invest 1996; 97: 359-365 for genetic modification of bacterial strains. Therefore, it can be reduced or eliminated. These cells (in the form of heat-sterilized cells) LPS) resulted in LPS with reduced toxicity, but Immunogenicity is still retained. According to a preferred method for this genetic modification, LPS recognizes epitopes in the core region of the LPS molecule and differentiates them in the host. Caused by at least two different Gram-negative strains with different core structures Sufficient as an immunogen to stimulate the synthesis of cross-protective antibodies against endotoxemia It is maintained in a sufficient three-dimensional shape to work. Especially in response to this vaccine Antibodies synthesized are cross-protected against smooth core strains in addition to complete core rough strains. It has control. At the same time, this genetic process preferably reduces LPS in warm-blooded animals. Make it pyrogenic and non-toxic. LPS from these genetically modified bacterial strains Administered, preferably in the form of purified LPS incorporated into liposomes. It is. Alternatively, LPS derived from these modified strains can be administered in the form of dead cells. Can also be. Alternatively, detoxified LPS may be administered as a purified LPS form. Alternatively, a complex may be formed with an acceptable carrier.   The toxicity of any LPS rough antigen composition described in the present invention can be Mitigated by other methods, such as competitive detoxification of lipid A with anti-endotoxin peptides You can also. Rustici et al. Science 259: 361 (1993). toxicity One alternative method to reduce the Before and after administration of anti-inflammatory drugs such as anti-TNF-α monoclonal antibody. Fisher CJ et al., N Engl J Med 334: 1697 (1996)).                                  Example   The following examples are presented for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. It is not meant to limit the enclosure. These examples are likely to be performed experiments And that all or any of the experiments were actually performed It does not mean.Materials for liposome preparation   Synthetic dimyristoylphosphatidylcholine (DMPC), dimyristoylphospha Tidylserine, and dimyristoyl phosphatidylglycerol (DMPG) It was purchased from Avanti Polar Lipids. Choles Terol, 3- (N-morpholino) propanesulfonic acid (MOPS), periodic acid and The Schiff reagent based on paralozanillin was purchased from Sigma. Destruction Bacterial saline and wash water are purchased from Abbottlabs . 3MEmpore solid phase extraction discs are Fisher (Fisher). Limulus amebocyte lysate ) (LAL) Standards and reagents are from Associates of Cape Cod.  Cape Cod).   Materials and reagents may have pyrogen removal by one or more of the following procedures: Perform: 1) Heating-Heat glassware at about 180 ° C for more than 16 hours, 2) Base treatment- Immerse in isopropanol / concentrated potassium hydroxide for 2 hours or more. 3) Hydrogen peroxide After immersing in concentrated hydrogen peroxide at 70 ° C for 1 hour or more, remove water with pyrogen 4) Ultrafiltration-Amicon Centriprep iprep) Filter the solution through an ultrafiltration membrane (molecular weight 3000).   Endotoxin contamination in glassware, plasticware, solutions and buffers The standard LAL assay (Associates of Cape Cod, Cape Cod , MA, USA).   Store MOPS salt buffer and 0.5% periodate solution at room temperature until use. Fat Stock solution is stored at -20 ° C. LPS is dissolved in washing water or 0.1% TEA and Or store in a polystyrene container (Evergreen) at 4 ° C. Liposome preparation Store at 4 ° C. The Schiff reagent based on paralozanillin is kept at 4 ° C until use. save.                                   Method Lipopolysaccharide   Maintain established strains of the following bacteria according to standard procedures: E. coli rough strain K12 , R1, R2, R3 and R4, E. coli smooth strain 018, 06, 0157, 012, 015, Bini Ra type of Salmonella minnesota R60 is obtained as above it can. Smooth and rough LPS are based on Hancock, cited above. Purified according to the established methods described in pp 91-92. Cerebral fungus J5 (Rc Chemo Type), E. coli, S. Minnesota (S. minnesota) R595 (Re chemotype) and S . LPS from Minnesota (wild type) and Salmonella typhimurium (wild type) List Biological Laboratories Inc. ( Campbell, Calif). In addition to native LPS, Electricity of native LPS using the established method described in Hancock et al., Pp 93-95. LPS in acid form (deionized) produced by dialysis may be used .LPS Of liposomes into liposomes   Liposomes are prepared by standard procedures. Multilamellar vesicles (MLVs) As described in Method # 1 or # 3, Dijkstra et al., J Immu nol Methods 1988; 114: 197-205 with minor modifications or new Prepare according to standard procedure (Method # 2).   For example, in method # 1, 1 ml of a 5 mg / ml native K. 1 ml of water containing lipids (DMPC: DMPG: cholesterol, 4: 1: 4, mol / mol) It was added to the dispersion system, that is, the lipid: LPS ratio was 10: 1 (wt / wt). next Add 2 minutes to this mixture at 40-50 ° C for 2 minutes with an interval of 2 minutes between each sonication. Five sonications were performed. The solution is subsequently rotovapped. Dried and resuspended in a buffer consisting of 4 mM MOPS) 153 mM NaCL, pH 7.8. Lipo LPS that is not incorporated into the lysosome (free LPS) can be prepared by adding the preparation to Komp Spin KA 21.5 Centrifuge three times for 10 minutes in a centrifuge, and decant all supernatants. And the pellet was removed by resuspension in the same volume of buffer as originally . This procedure was repeated three times, but the concentration of unincorporated LPS was significantly reduced did. This method for reducing free LPS is based on most liposome preparation methods described below. Can be used.   In method # 2, 5 ml of a chloroform: methanol (2: 1, v / v) solution was added to 2 ml of 0.1 M Vortexed with HCl. Then the lower organic phase is separated from the upper one And used to dissolve 5 mg of the LPS in acid form. Then 50mg of lipid (DMPC : DMPG: cholesterol, 4: 1: 4, mol / mol) was dissolved in this LPS solution. LP The solution in which S and lipid are co-dissolved is dried by rotary evaporation, and the same as in method # 1 And resuspended.   In Method # 3, briefly, 4 mM dimyristoyl phosphatidylcholine: 1 mM Dimyristoyl phosphatidylserine: 1 ml of undiluted lipid consisting of 4 mM cholesterol Was dried at 50 ° C. by rotary evaporation. E. coli strains K12, R1, R2, R3, R4 and And the same weight of LPS from Salmonella minnesota R60 150 μl of water to 50 μl of a 0.1 mg / ml solution of the mixed LPS suspended in 0.1% TEA . Subsequently, the water tank was sonicated for 5 minutes or more in a water tank type sonicator containing hot tap water. The preparation was vortexed and sonicated vigorously. The preparation is then steamed After drying (or freeze-drying), 4mM MOPS, 153mM physiological diet The suspension was vigorously vortexed and resuspended in a buffer solution consisting of saline and pH 7.8.   Incorporation of LPS into liposomes requires the use of a single type of LPS. In some cases (for example, E. coli K12 (Ra) 5 mg), the same amount of different LPS may be used. (Eg, 0.83 mg of LPS from six complete core rough mutants).   Large unilamellar vesicles (LUVs) can be pyrolyzed before use by the hydrogen peroxide treatment described above. Avestin Liposofa Extruder with Agen Removal a pair of 100 nm polycarbonates with pyrogen removal Prepared from MLV by repeated (minimum 15) passage through carbonate membrane .   LPS incorporated into the liposome by Method # 3 contains 4 mM MOPS / 153 mM physiological diet. Using saline as a running buffer and a flow rate of 0.7 ml / min, a 1.1 x 28.5 cm bio Purified on a gel (BioGel) A15M column. Free LPS almost completely (> 99.9%) lipo As a result of its incorporation into the This step is likely because no difference was observed in the cell lysate (LAL) activity. It seems unnecessary.LPS Vitro quantification of the toxicity of   Determination of the biological activity of the toxic lipid A component in all and control samples of the vaccine Was performed using a standard LAL assay according to the manufacturer's instructions (Associate Pyrotell and Pyrochrome from s of Cape Cod, Cape Cod, MA, USA). Free LP S's The incorporation ratio into liposomes is generally a significant increase in LAL activity after effective incorporation. Reflected in the decline. LPS to liposomes using traditional methods including those mentioned above Is typically above 90% and in some cases above 99%.LPS Periodic Acid / Schiff Base (PAS) Staining   An aliquot of LPS containing either MLV or LUV was diluted with water to 2 ml and 2 ml Extract with toluene. Vortex in a Sorvall GLC-2 centrifuge tube After centrifugation at 2800 rpm for 5 minutes, remove the upper toluene phase, The phases are re-extracted with 2 ml of fresh toluene. Subsequently, the aqueous and intermediate phases are collected and Acidify with 5050 μl concentrated hydrochloric acid and add 5 ml chloroform: methanol (2: 1) Extract. The aqueous phase is re-extracted once with chloroform, and the combined organic phases are mixed at 50-60 ° C. Dry underneath. Next, the residue was dissolved in chloroform: methanol (2: 1), Aligned with LPS standard curve dripped from 50% ethanol, Empore C8 extraction data Drop a spot on the disc. After vacuum drying the disc, in 0.5% periodic acid Incubate at room temperature for about 30 minutes. Take them out and rinse with distilled water Place in a test tube containing Schiff's reagent based on pararosaniline and close the lid. You. Next, warm the test tube with heated tap water until color develops. So the disk Remove, rinse and dry again. Agfa Arcus II us II) desktop scanner with Adobe Photoshop and And use in combination with NIH Image Software to define the resulting spots. Quantify.Immunization and pyrogenicity testing   The determination of pyrogenicity and toxicity of vaccines containing LPS is well-established and Rabbit outlined in USP (USP 23, <151>, 1995, Rockville, MD) It is best to use a thermal model. In this established procedure, the experimental substance is administered If the substance is not pyrogenic in rabbits compared to control animals, the substance is non- It is defined as pyrogenic and may be fever after administration to other mammals, especially humans. Or toxicity is unlikely. Rabbits and humans respond to endotoxin This procedure is an ideal model because it has comparable sensitivity.   Briefly, mature female New Zealand whitela weighing 1.8 to 3.0 kg Bits (New Zealand White rabbit) are checked for suitability for exothermic testing Two sham tests were performed to confirm. All test materials are as specified Either intravenous (IV) in a volume of 1 ml or intramuscular (IM) in a volume of 0.6 ml Was administered. 3 rabbits per group to complete the test in compliance with regulatory requirements Is required. However, in the examples described below, free LPS Significant difference in pyrogenicity with LPS incorporated in liposomes Two rabbits per group were sufficient. Check for baseline stability The body temperature of the rabbit was measured and the animals were observed at 15-minute intervals for 3 hours after administration of the test sample. Measured. C) Individuals whose body temperature rises more than 0.5 ° C above the baseline value In the absence of herons, the material is considered non-pyrogenic.Assess the effectiveness of various immunogens   A widely accepted test for the effectiveness of an immunogen is After administration to rabbits or human subjects, the It is to collect blood. Various days before the test and after intramuscular (thigh muscle) inoculation At any time, a blood sample is collected from the marginal ear vein and placed in a red top tube (red top tu). be). Some intramuscular inoculations involve the administration of alum, an adjuvant More. In these cases, 0.3 ml (equivalent to 1.50 mg) of myo prior to administration Uban (Sergeant Co., Clifton, NJ diluted 2% stock solution of Alhydrogel to 0.5% Was mixed well with 0.3 ml of vaccine immunogen. Centrifuge blood and remove serum Collected and stored at -80 ° C.ELISA Of immunized rabbit serum with LPS   Cross-reactivity of sera from immunized rabbits was measured by enzyme-linked immunosorbent assay (ELISA). Blood loss was determined by standard methods for binding to purified LPS. Various thoughts Serum was collected from rabbits before and after inoculation of the immunogen. Serum horizontal crossing Multiple sera of this serum were used to determine the extent of reactivity and vertical cross-reactivity. A test on LPS made was conducted. After the method of active immunization described in the present invention And rabbit serum were used for smooth and complete core rough LPSN of Escherichia coli and monkeys. Combined with LPS of Monella smooth and complete core rough type. Extensively studied Escherichia coli Rc J5 mutant, Salmonella minnesota ) Rabbits that have been vaccinated with prior art immunogens such as Re mutants and lipid A Serum from Excellent binding properties were shown.LPS- Polymyxin complex   The molecular weight of LPS was calculated as previously described (Scott BB et al., SerodiagI. mmunother Infect Dis; 4:25 (1990)). 0.2mM dissolved in pyrogen-free water 0.4 mM polymyxin B sulfate (Sigm) in which purified LPS (5 ml) was dissolved in pyrogen-free water a) Mix with (5 ml) and sonicate by applying about 10 short (5 seconds) ultrasonic stimuli I went together. Place the resulting milky suspension in a 2000 MVVCO membrane Dialysis overnight against fresh distilled water containing 0.05% sodium azide (w / v) Uncomplexed polymyxin B was removed. The permeability often present as a coherent precipitate The precipitated material is recovered as a 10 ml suspension containing a putative 0.1 mM LPS. And put in a polypropylene minisorb tube (Nunc) at -40 ° C. Saved in.LPS coating of microplate   LPS-polymyxin complex was resuspended by sonication. LPS-polymyxin The conjugate was prepared using freshly prepared distilled water containing 0.05% sodium azide. Diluted 1:80 with 05M carbonate-bicarbonate buffer, pH 9.6. With continuous rapid stirring, The diluted conjugate (containing 1.25 μM LPS) is evenly distributed in the coating buffer. Etc. and maintain in a 96-well microtiter plate or 8-well 100 per well for crotiter strips (in a 96-well frame) μl was added. The microtiter plates and strips used were ELISA Moderate polystyrene (Greiner) moderately bonded great, flat bottom well): Yes Microtiter plates from some other grades and some other manufacturers Seems to be unsuitable for this assay. The plates are stacked and a synthetic resin (Clingf ilm) and incubated overnight at 37 ° C. 0, as previously described Plate using phosphate buffered saline (PBS) supplemented with .05% (v / v) tween-20 Was washed. 5% bovine serum albumin (BSA) PBS containing 0.05% sodium azide The solution was added at 120 μl per well. Stack plates and wrap with synthetic resin And incubated overnight at 37 ° C. Wash plate and distill fresh as above Rinsing with water, blotting by transfer to absorbent paper, 37 Dried at ℃ Was. Place the dried plate in a plastic bag and seal (1 plate per bag). Stored at -40 ° C until use.LPS ELISA   The test serum or plasma sample was used as an ELISA diluent [PBS / tween-20 (0.05% v / v) / Polyethylene glycol 8000 (4% w / v) / BSA (1.0% w / v) / Natriazide (0.05% w / v)] in a 1: 200 dilution of the LPS-coated plate. 100 μl was added per well to every three wells. Place plate under static atmosphere (blower Incubate at 37 ° C for 5 hours in an incubator Later washed (PBS / tween). Other dilutions, incubation times, and And test replicates may be used, but are identical for any set of experimental controls and experimental groups. Condition must be set. For measuring the amount of bound immunoglobulin of each class Is a species-specific immunoglobulin heavy chain-specific antibody to which alkaline phosphatase is bound. The body was used. IgM antibody is measured using a μ-chain-specific complex, and IgG antibody is γ-chain-specific. It was measured using the complex. Heavy chain-specific species-specific antibodies (eg, Harlan Sera-La b Anti-rabbit Ig antibody purchased from (UK) was diluted 1: 1000 with ELISA dilution buffer. Using). Add 100 μl of the diluted complex per well and plate at 37 ° C For 120 minutes. Wash plate with PBS / tween and rinse with distilled water Wash, blot, and use freshly prepared pNPP alkaline phosphatase. A substrate substrate solution (Sigma N-2770) was added in an amount of 100 μl per well. 30 at room temperature After a few minutes, the color is developed and an automated ELISA plate reader (Molecular Devices T hermomax) (405 nm and 650 nm as reference) Test results were expressed as net absorbance at 405 nm and 650 nm (reference). Also Reads the test results as described above and uses the software (Molecula r Standards on the same plate using automatic curve fitting by Devices Softmax It can also be expressed as a ratio. 3 wells of each microplate Down from one column at a time with ELISA diluent, eg from a 1:50 dilution The test samples were placed in triplicate by arranging three of each of the eight 2-fold dilution series It can also be compared with the sera.Binding of immunized rabbit serum to LPS by Western blotting   The cross-reactivity of sera from immunized rabbits was The clarification was also determined by standard methods for binding purified LPS. Various possible Serum was collected from rabbits before and after inoculation of the immunogen. Serum horizontal cross anti Multiple purifications of this serum were used to determine the degree of responsivity and vertical cross-reactivity. A test for LPS was performed. After the method of active immunization described in the present invention, Rabbit serum is composed of smooth and complete core-rough LPSN of cerebral bacteria and salmo Combined with smooth and complete core-rough LPS of Nera. Extensively considered Rabbits vaccinated with prior art immunogens such as the Rc J5 mutant of Escherichia coli Showed better binding than sera from Eggs and LPs derived from Salmonella minnesota Re mutants It is expected to show better binding than serum from rabbits immunized with S I was imagined.   Laemmli (Laemm) except that SDS was omitted from the stacking and separation gel buffer. li) Using a buffer system of (Nature; 227: 680-85 (1990)), 12% (w / v) PAGE analysis on a lylamide gel was performed. LPS (5-10 μg for rough LPS, For a loose LPS, mix 20-25 μg of the sample with the Laemmli sample buffer). 60 volts until the sample reaches the separation gel, after which the dye tip Electrophoresis was performed at 150 volts until the gel moved 7.5 cm. Oxidation point for 15 minutes Except for Hancock and Poxton (Bacterial cell surface technology) Surgery (Bacterial Cell Surface Techniques), pub. Wiley, p. 281 (1988)) The separated LPS was stained by a modified silver staining method. For immunoblotting, tow Towbin et al., Tris from Proc Natl Acad Sci USA; 76: 4350-54 (1979)). Apply 10-12 volts for 16 hours at 4 ° C using glycine, methanol buffer This LPS was converted to a nitrocellulose membrane having a pore size of 0.2 μm (Schleicher and S chuell, Germany). Immunostaining of transferred LPS yields optimal results Choose the incubation time and serum dilution so that Hancock and Poxton except blots were rinsed (Poxton) (Bacterial Cell Surface Techniques), pub. Wiley, p. 204-5 (1988)).   The LPS content extracted from the smooth-type bacteria was analyzed by electrophoresis for O-specific side chains. of There are several bands corresponding to LPS molecules with various molecular weights depending on the size. Released. These LPS molecules are LPS molecules without any O-specific side chains (complete core ( Ra) (equivalent to the size of the rough mutant)), with 40 or more units in the side chain. LPS was varied.Based on inhibition of LPS-induced stimulation of LAL assay by sera from vaccinated rabbits Yamago Defense   Limulus amebocyte lysate (LAL) assay It is an established test for the biological activity / toxicity of Pido A (the toxic component of LPS). Book The assay quantifies the activity of biologically active LPS, but the presence of protective anti-LPS antibodies. In the presence, the LAL test is significantly inhibited due to competitive binding and neutralization of LPS. LA There are a number of ways to demonstrate protection from anti-LPS antibodies using the L assay. LAL inspection Test kit is from Coesteet of Chromogenix of Sweden. Can be purchased from manufacturers such as Coatest Endotoxin. Different types of LAL assays, such as gel clot or chromogenic, can be used. it can.   One example of this method used a dynamic chromogenic LAL assay. This assay Before and after vaccination with complete core LPS incorporated in liposomes. After sera, serum was collected from rabbits. Escherichia coli R1 LPS (1000μg / ml) Dilute 1/5 with pyrogen-free water on plate, then use whole plate 5 times so that the final volume of the 5 wells is 40 μl (1000 μg / ml to 1.6 μg / ml). Dilution of LPS was due to 1) reduced LAL activation in the presence of serum. Only wells that do not have enough LPS to wake up; 2) wells with excess LPS This is in order to avoid the fact that there will be only a well. Each serum or Prepared a dilution series for the control (3 rows in total). Row A-pyrogen removal water Row B-rabbit serum on day 0 (complete core immunogen) Row C-Rabbit serum from day 63 (complete core immunogen) 20 μl of water or rabbit serum was added to each well and left at room temperature for 30 minutes. continue Add 20 μl of LAL / substrate to each well using a multitip pipettor (reaction opening). Start), immediately load the reader, and read every 20 seconds for 120 minutes. Was . The time (seconds) from the start of the reaction until the absorbance (OD) reaches 0.5 depends on the activation of LAL. Reflects degree / speed. In the presence of water and LPS, the LAL reaction proceeds rapidly, 0.5 was reached quickly. Preimmune serum is nonspecific for LPS in addition to some anti-LPS antibodies Contains inhibitors (such as lipoproteins). Since this serum partially neutralizes LPS, LAL Slow down the reaction and extend the time until the OD reaches 0.5. Complete core Postimmune sera from rabbits immunized with this are required to reach an OD of 0.5 Significant neutralization / protection of R1 LPS, as indicated by a marked increase in time Brought. Similar results were obtained with other types of irritating LPS. However, this was consistent with the LPS binding data in the ELISA described above.   Another method using inhibition of LAL activity involves both before and after vaccination. A known amount of LPS was added to several dilutions of serum from the subject, The LAL activity (EU) for each is compared. Serum, especially blood after vaccination Protective anti-LPS antibodies in serum result in reduced LAL activity compared to preimmune values.Cross protection based on inhibition of LPS-induced IL-6 secretion by mouse peritoneal macrophages   Some monokines, including tumor necrosis factor (TNF), IL-1 and IL-6, are Mediates many of the pathophysiological events associated with bacterial negative endotoxemia. this These monokines respond to LPS both in vitro and in vivo to monocytes and Secreted by macrophages. Serum containing protective anti-LPS antibody is LPS-induced macrophage or monocyte sting as shown in the Block the fierce. This type of assay uses established mouse cell lines (eg, J774.2 ), Established human cell lines (eg, THP-1), freshly harvested mouse peritoneal cells (C3H / He N) or using freshly harvested human monocytes / macrophages. Up In Say, TNF or IL-6 levels after stimulation with LPS can be examined. Cellular The amount of any type of LPS (eg, E. coli R1) used for stimulation must be determined in preliminary experiments. It is necessary. Too little LPS results in poor stimulation and monokine in the control group. Undetectable values, but too much LPS also counteracts the effects of large amounts of protective antibodies There is fear. Monokines produced in serum containing protective antibodies after stimulation with LPS Values (eg, TNF values) are lower compared to serum controls.   In one example, the assay is performed by Delahooke DM et al., Infection. a nd Immunity, 1995, p840-46. In this assay, LPS A human cell line that secretes TNF after stimulation (THP1) is used. In this assay, Serum after vaccination with whole core antigens shows significant inhibition of TNF induction Was.   In another example, peritoneal lavage with a 0.34M sucrose solution in distilled water To collect mouse peritoneal cells (C3H / HeN). Peritoneal cells in 0.2 ml serum-free medium (IMDM-ATLN Schreier and Tees, Immunological Methods, Vol. II, Acad. Pr 5 × 10 during ess (1981): 263)^Five(1) E. coli R3 (0.05 ng / m l) or E. coli 018 (0.05 ng / ml) or S. Minnesota (S. minnesota) wild Smooth type (0.05ng / ml) or S.D. LPS derived from Minnesota R60 (0.05ng / ml) In the presence or absence of LPS, such as (2) the composition according to the present invention or Is E. coli Rc J5 mutant or S. Inoculated with various immunogens such as Minnesota Re mutant 37 ° C in the presence or absence of diluted or undiluted serum from rabbits For 4 hours. The supernatant was collected, followed by the IL-6 dependent hybridoma cell line B13.2 9 (Aarden et al., Eur. J. Immunol. 1987, 17, 1911) as follows. Measure the amount of IL-6 present in: B13.29 cells in serum-free medium 2.5 × 10^FourCells / ml and in the absence of IL-6 In addition, the cells are cultured for 72 hours in the presence and absence of the culture supernatant. Ants in culture Dispense the coat (200 μl / well) into a flat-bottomed microtiter plate. The IL-6 concentration in the supernatant is calculated using a standard curve of IL-6. Post-immune serum brings IL-6 secretion should be low compared to pre-immune serum.Cross protection against lethal doses of endotoxin   Another indicator of the effectiveness of the present invention is its ability to confer cross protection against LPS. There is a point. The invention, a suitable control, or E. coli Rc J5 mutant LPS or Any of the previously described immunogens, such as LPS of Salmonella minnesota Re mutant Using a mouse lethal model in which mice were intraperitoneally inoculated on day 0 You. A second dose of antigen is given on days 7 and 14. Between days 19 and 21, 6 A 95% lethal dose of a specific LPS was given intravenously to a group of 6 female C57BL / 6 mice 〜8 weeks old Can be administered. Galactosamine (D-GalN) (800mg / kg) Sometimes the abdominal cavity To be administered within. Minimum static LPS required to kill approximately 95% of animals in preliminary experiments Intravenous dose (LD₉₅). Survival is recorded for up to 24 hours. This procedure Now, the ability to indicate which immunization with a particular immunogen resulted in protective antibodies Alternative methods can be used without substantially altering. For example, Other strains of mice may be suitable and the galactosamine dose may vary. Vaccination regimens to allow for more doses. Can be changed. Serum was isolated from rabbits inoculated with the experimental immunogen and Administering this serum to mice before injecting galactosamine and LPS , An experiment can also be performed. This method using passive immunization is Can be used to indicate sex.                                 Example 1   Liposomes containing the complete core LPS of E. coli K12 were prepared according to method # 2 above. Lipo containing a mixture of 6 complete core LPSs (Escherichia coli R1-R4, K12, S. Minnesota R60 Ra) Sosomes were made according to the method described above.   Groups of three mature rabbits received alum (as described above) on days 0, 14, and 56. Immunization was performed intramuscularly with 0.5 mg of the antigen. Nos. 0, 14, 21, 56 and Blood was collected on day 63 and processed as described above.   Using the ELISA method described above, in rabbits immunized with K12, E. coli And IgG for smooth and rough LPS from Salmonella and Salmonella It was shown that both antibody levels increased.   Using the Western / immunoblot method described above, both groups of immunized rabbits And rough LPs from Escherichia coli and Salmonella typhimurium with serum Enhanced binding to S was shown. Obtained from rabbits immunized with K12 only Serum, smooth LPS from E. coli (serotypes 018, 012 and 015) and murine The binding to LPS derived from the wild type of S. typhi was determined by a mixture of six complete LPS cores. The binding was equivalent to that of serum obtained from rabbits that had undergone chlorination.                                 Example 2   R1, R2, R3, R4 and K12 of Ra E. coli, a rough bacterial strain with a complete core Strains and derived from h strain of Salmonella minnesota R60 same Amount of purified lipopolysaccharide alone or after incorporation into liposomes as described above Argue. Evaluate only heat sterilized cells and liposomes. The composition is as described above And materials according to the method described in the above section.group 1. 100 μg MLV (without LPS) 2． Purified LPS from Escherichia coli R1 (total 3ng) 3． Purified LPS (0.3 ng total) (E. coli K12, R1, R2, R3 and R4 and S. minneso) 0.05 ng each from R60) Four. Purified LPS (3 ng total) (E. coli K12, R1, R2, R3 and R4 and S. Minnesota R) 0.5ng each from 60) Five. Purified LPS (30 ng total) (E. coli K12, R1, R2, R3 and R4 and S. minnesota) 5ng each from R60) 6． Purified LPS (300 ng total) (E. coli K12, R1, R2, R3 and R4 and S. minneso) 50ng each from R60) 7． Six of the above, incorporated into MLVs in a 1: 1000 ratio by weight (LPS: lipid) Of LPS (3 ng in total) (E. coli K12, R1, R2, R3 and R4 and S. Minnesota) 0.5ng each from R60) 8． Six of the above, incorporated into MLVs in a 1: 1000 ratio by weight (LPS: lipid) LPS mixture (total 30 ng) (E. coli K12, R1, R2, R3 and R4 and S. mineso) 5ng each from R60) 9． Six of the above, incorporated into MLVs in a 1: 1000 ratio by weight (LPS: lipid) LPS mixture (total 300 ng) (E. coli K12, R1, R2, R3 and R4 and S. mineso) 50ng each from R60) Ten. Six of the above, incorporated in LUV in a ratio of 1: 1000 by weight (LPS: lipid) Of LPS (3 ng in total) (E. coli K12, R1, R2, R3 and R4 and S. Minnesota) 0.5ng each from R60) 11． Six of the above, incorporated in LUV in a ratio of 1: 1000 by weight (LPS: lipid) LPS mixture (total 30 ng) (E. coli K12, R1, R2, R3 and R4 and S. mineso) 5ng each from R60) 12． Six of the above, incorporated in LUV in a ratio of 1: 1000 by weight (LPS: lipid) Of LPS (300 ng total) (cerebral bacteria K12, R1, R2, R3 and R4 and S. minneso) 50ng each from R60) 13. Heat-sterilized cells derived from Escherichia coli R1 (the amount having the same LAL activity as 3 ng of Escherichia coli R1 LPS) )

[Procedural amendment] [Date of submission] December 17, 1999 (December 17, 1999) [Content of amendment] Claims 1. A method for producing a medicament for reducing the harmful effects of endotoxin in a warm-blooded animal, comprising an effective amount of a composition containing a rough complete core lipopolysaccharide (LPS) antigen of Gram-negative bacteria . 2． Composition The following Gram-negative bacteria: Escherichia coli, Pseudomonas, Klebsiella, Sa Rumonera and at least two rough among Bacteroides complete Koaripo containing polysaccharide P S) antigen, The method of claim 1, wherein. 3． 3. The method of claim 2, wherein the composition comprises at least the rough complete core lipopolysaccharide (LPS) antigen of E. coli, Pseudomonas and Bacteroides . Four. 2. The method of claim 1, wherein the composition comprises dead whole cells. Five. Composition comprises dead cells all cells of E. coli K12 strain containing rough complete core LPS antigens,請 Motomeko 4 The method according. 6． Composition, the following Gram-negative bacteria species: including Escherichia coli K12, cerebral bacteria R1 strain, Bakuteroi Death fragilis, at least three Ra chemotype mutants dead bacteria all cells of a mixture of Pseudomonas aeruginosa, claims Method according to 2. 7． Composition, the following Gram-negative bacteria species: Escherichia coli K12 strain, E. coli R1 strain, Bakuteroi Death fragilis, including each of Ra LPS mutants dead bacterial whole cells of Pseudomonas aeruginosa The method of claim 6 wherein. 8． 2. The method of claim 1, wherein the composition comprises purified detoxified Ra LPS complexed with a protein . 9． Ra LPS complex is Ra LPS cerebral bacteria K12 was complexed, methods who claim 8. Ten. 9. The method of claim 8, wherein the composition comprises a mixture of Ra LPS from multiple species of Gram-negative bacteria, complexed with the protein . 11． Composition, the following Gram-negative bacteria species: Escherichia coli K12 strain, E. coli R1 strain, Bakutero Idesu fragilis, comprising a complex of Ra LPS derived from at least three of the Pseudomonas aeruginosa The method of claim 10, wherein . 12． Composition comprises Ra LPS incorporated into liposomes, methods who claim 1. 13. 13. The method of claim 12, wherein the composition comprises Ra LPS of E. coli K12 strain in the liposome . 14. Composition comprises a mixture of Ra LPS you derived from multiple species of gram-negative bacteria which is incorporated in a liposome, method of claim 12, wherein. 15． Mixture is the following gram-negative bacteria species: Escherichia coli K12 strain, E. coli R1 strain, Bakuteroi Death fragilis, and Ra LPS derived from at least three of the Pseudomonas aeruginosa The method of claim 14, wherein. 16． 2. The method of claim 1, wherein the composition comprises the rough complete core lipopolysaccharide (LPS) antigen of E. coli K12 strain . 17 ． 17. The method of claim 16, wherein the composition further comprises a rough complete core lipopolysaccharide (LPS) antigen of a second bacterium other than E. coli K12 strain. 18 ． 18. The method according to claim 17 , wherein the animal is a mammal. 19 . 19. The method of claim 18 , wherein the animal is a human patient. 20 . 17. The method of claim 16 , wherein the composition comprises LPS of Ra rough E. coli K12 strain. 21 . 18. The method according to claim 17 , wherein the second bacterium is Escherichia coli or Salmonella. 22 . 18. The method according to claim 17 , wherein the second bacterium is Bacteroides. 23 . 18. The method of claim 17 , wherein the composition comprises a complete core rough LPS antigen from a third gram-negative bacterium different from the first and second gram-negative bacteria. 24 . 24. The method of claim 23 , wherein the composition comprises a complete core rough LPS antigen from a fourth gram-negative bacterium different from the first, second and third gram-negative bacteria. 25 . 18. The method according to claim 17 , wherein the second Gram-negative bacterium is E. coli R1 strain. 26 . 18. The method of claim 17 , wherein the second gram-negative bacterium is Salmonella. 27 . 24. The method of claim 23 , wherein the second bacterium is Klebsiella and the third bacterium is Pseudomonas. 28 . 25. The method of claim 24, wherein the second bacterium is Klebsiella, the third bacterium is Pseudomonas, and the fourth bacterium is Bacteroides. 29 . Salmonella is Salmonella minnesota (Salmonella minnesota), 請 Motomeko 21 or claim 26 A method according. 30 . 25. The method of claim 24 , wherein the complete core antigen from each of the four bacteria is present in generally equal amounts by weight. 31 . 24. The method of claim 23 , wherein the composition comprises LPS antigens from at least two different Gram-negative strains of the same species. 32 . 17. The method of claim 16 , wherein the antigen causes the patient to produce an antibody that binds to an epitope within the core region of the LPS of at least one Gram-negative strain whose LPS is not part of the composition. 33 . 33. The method of claim 32 , wherein the patient's antibody binds to at least one LPS of a smooth Gram-negative strain. 34 . 33. The method of claim 32 , wherein the composition comprises the antigen in a liposome. 35 . 35. The method of claim 34 , wherein the ratio of lipid to LPS antigen (weight to weight) in the liposome is between 1: 1 and 5000: 1. 36 . 35. The method of claim 34 , wherein the ratio (weight to weight) is between 10: 1 and 1000: 1. 37 . 35. The method of claim 34 , wherein the liposome comprises a component selected from the group consisting of phospholipids, cholesterol, positively charged compounds, negatively charged compounds, and amphiphilic compounds. 38 . 35. The method according to claim 34 , wherein the liposome is a multilamellar liposome (MLV). 39 . 35. The method of claim 34 , wherein the LPS in the acid salt form has been incorporated into the liposome. 40 . 35. The method of claim 34 , wherein the liposomes are small or large unilamellar liposomes (SUV and LUV). 41 . 17. The method of claim 16 , wherein the composition is administered intramuscularly, intravenously, subcutaneously, intraperitoneally, transtracheally, or transgut. 42 . 17. The method of claim 16 , wherein the dose of the antigen is greater than 0.01 ng / kg of patient weight. 43 . 43. The method of claim 42 , wherein the dose of the antigen is less than 1000 ng / kg of patient weight. 44 . 43. The method of claim 42 , wherein the dose of the antigen is less than 100 μg / kg of patient weight. 45 . 17. The method of claim 16 , wherein the composition is administered multiple times, the first of which is administered at least two days before the expected exposure to endotoxin. 46 . 17. The method of claim 16 , wherein the antigen is present in killed bacteria. 47 . 17. The method of claim 16 , wherein the antigen has been separated from the bacteria. 48 . 17. The method of claim 16 , wherein the antigen has been chemically detoxified. 49 . 48. The method of claim 16 or claim 47 , wherein the bacterium is genetically engineered. 50 . 17. The method of claim 16 , wherein the composition further comprises an adjuvant. 51 . 51. The method of claim 50 , wherein the adjuvant is alum. 52 . A vaccine composition for reducing an adverse effect of endotoxemia in a human patient, comprising an effective amount of a composition comprising a purified complete core rough lipopolysaccharide antigen of Escherichia coli K12, wherein the antigen comprises A composition further comprising a liposome comprising: 53 . A method for reducing an adverse effect of endotoxemia in a warm-blooded animal, comprising administering to a warm-blooded animal an effective amount of a composition comprising a rough lipopolysaccharide (LPS) antigen of a gram-negative bacterium, The method wherein the LPS antigen comprises a component of Escherichia coli Rb chemotype LPS or its equivalent in another species. 54 . A method for quantifying lipopolysaccharide incorporated in liposomes by performing periodic acid / Schiff base staining. 55 . 55. The method of claim 54 , wherein the test is performed on a vaccine lot intended for clinical use. 56 . A method for reducing the adverse effects of endotoxemia in a warm-blooded animal, comprising administering to the warm-blooded animal an effective amount of an antibody produced by immunization with the composition of claim 1 or 16 . 57 . 57. The method of claim 56 , wherein the antibody comprises a substantial proportion of an IgM antibody. 58 . A method for reducing the adverse effects of endotoxemia in a warm-blooded animal, comprising administering to the warm-blooded animal an effective amount of a composition comprising a rough complete core lipopolysaccharide (LPS) antigen of a gram-negative bacterium.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/02 A61K 47/02 A61P 39/02 A61P 39/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, S, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD , MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, U Z, VN, YU, ZW (72) Inventor Poxton Ian Raymond Edinburgh Teobio Places Department of Medical Microbiology University of Edinburgh Medical School (72) Inventor Mackintosh Thomas James Darham Room, North Carolina, United States 443 Sands Building Department Of Cell Byology Duke You Bar City Medical Center (72) inventor Cedar debit Scott United States, North Carolina da Hamu Room 443 Sands Birudi packaging department of cell-by-rheology Duke University Medical Center

Claims (1)

[Claims] 1． A method for reducing the deleterious effects of endotoxin in a warm-blooded animal, comprising the steps of Escherichia coli K Effective amount of composition containing 12 rough complete core lipopolysaccharide (LPS) antigens in warm-blooded animals A method comprising administering. 2． A composition comprising a rough complete core lipopolysaccharide (LPS) antigen of a second bacterium other than E. coli K12 strain 2. The method of claim 1, further comprising: 3． 2. The method according to claim 1, wherein the animal is a mammal. Four. 3. The method according to claim 2, wherein the mammal is a human patient. Five. 2. The method according to claim 1, wherein the composition comprises LPS of Ra rough type K12 strain. 6． 3. The method according to claim 2, wherein the second bacterium is Escherichia coli or Salmonella. 7． 3. The method according to claim 2, wherein the second bacterium is Bacteroides. 8． The composition is derived from a third gram-negative bacterium different from the first and second gram-negative bacterium 3. The method of claim 2, comprising a complete core rough LPS antigen. 9． A fourth gram-negative bacterium, wherein the composition is different from the first, second and third gram-negative bacteria 9. The method according to claim 8, comprising a complete core rough LPS antigen from the subject. Ten. 3. The method according to claim 2, wherein the second Gram-negative bacterium is E. coli R1 strain. 11． 3. The method according to claim 2, wherein the second Gram-negative bacterium is Salmonella. 12． Wherein the second bacterium is Klebsiella and the third bacterium is Pseudomonas Item 8. The method according to Item 8. 13. The second gram-negative bacterium is Klebsiella and the third bacterium is Pseudomonas. 10. The method of claim 9, wherein the fourth bacterium is Bacteroides. 14. Salmonella is Salmonella minnesota strain R60 12. The method according to claim 6 or claim 11, wherein 15. core antigens from each of the four bacteria are generally equal in weight, The method according to claim 9. 16． The composition comprises LPS antigens from at least two different Gram-negative strains of the same species. The method according to claim 7, wherein 17． The antigen is at least one gram-negative bacterium whose LPS is not part of the composition; The patient is required to produce antibodies that bind to an epitope within the core region of the LPS of the strain. Request Method according to 1. 18． Wherein the patient's antibody binds to at least one LPS of a smooth Gram-negative strain, The method of claim 17. 19. 2. The method of claim 1, wherein the composition comprises the antigen in a liposome. 20. The ratio of lipid to LPS antigen in the liposome (weight to weight) is between 1: 1 and 5000: 1 20. The method of claim 19, wherein there is. twenty one. 21. The method of claim 20, wherein the ratio (weight to weight) is between 10: 1 and 1000: 1. twenty two. Liposomes are phospholipids, cholesterol, positively charged compounds, negatively charged Compound comprising a component selected from the group consisting of: the method of. twenty three. 20. The method according to claim 19, wherein the liposome is a multilamellar liposome (MLV). twenty four. 20. The method of claim 19, wherein the LPS in the acid salt form is incorporated into the liposome. . twenty five. The liposomes are small or large unilamellar liposomes (SUV and LUV), The method according to claim 19. 26. The composition is administered intramuscularly, intravenously, subcutaneously, intraperitoneally, via the respiratory tract or the gastrointestinal tract 2. The method of claim 1, wherein the method is performed. 27. 2. The method of claim 1, wherein the dose of the antigen is greater than 0.01 ng / kg of patient weight. 28. The method of claim 27, wherein the dose of the antigen is less than 1000 ng / kg of patient weight. . 29. The method of claim 27, wherein the dosage of the antigen is less than 100 μg / kg of patient weight. . 30. The composition is administered multiple times, the first of which is expected to be exposed to endotoxin 2. The method of claim 1, wherein the method is administered at least two days prior to the time of administration. 31. 2. The method of claim 1, wherein the antigen is present in killed bacteria. 32. 2. The method of claim 1, wherein the antigen has been separated from the bacteria. 33. 2. The method of claim 1, wherein the antigen has been chemically detoxified. 34. 32. The method of claim 1 or claim 31, wherein the bacterium has been genetically engineered. 35. 2. The method of claim 1, wherein the composition further comprises an adjuvant. 36. 34. The method of claim 33, wherein the adjuvant is alum. 37. Composition containing purified complete core rough lipopolysaccharide antigen of Escherichia coli K12 Vaccine for reducing the adverse effects of endotoxemia in human patients, including effective doses set A composition, wherein the composition further comprises a liposome comprising the antigen. 38. An effective amount of a composition containing a rough lipopolysaccharide (LPS) antigen of Gram-negative bacteria To reduce the adverse effects of endotoxemia in warm-blooded animals, including administering to animals The method, wherein the LPS antigen is a component of Escherichia coli Rb-type LPS or an equivalent thereof in another species. A method that includes 39. Incorporation into liposomes by periodic acid / Schiff base staining A method for quantifying the obtained lipopolysaccharide. 40. Claims wherein the test is performed on a vaccine lot intended for clinical use The method of 39. 41. An effective amount of an antibody produced by immunization with the composition of claim 1 Reduce the adverse effects of endotoxemia in warm-blooded animals, including administration to animals Method. 42. 42. The method of claim 41, wherein the antibody comprises a substantial proportion of an IgM antibody. 43. Effective amount of composition containing rough complete core lipopolysaccharide (LPS) antigen of Gram-negative bacteria Mitigating the adverse effects of endotoxemia in warm-blooded animals, including administering How to reduce.