Title:  Vaccine against lipopolysaccharide core

United States Patent:  6,749,831

Issued:  June 15, 2004

Inventors:  Bennett-Guerrero; Elliott (New York, NY); Barclay; George Robin (Midlothian, GB); Poxton; Ian Raymond (Edinburgh, GB); McIntosh; Thomas James (Durham, NC); Snyder; David Scott (Durham, NC)

Assignee:  Medical Defense Technology, LLC (New York, NY)

Appl. No.:  423546

Filed:  April 25, 2000

PCT Filed:  May 15, 1998

PCT NO:  PCT/US98/09988

PCT PUB.NO.:  WO98/51217

PCT PUB. Date:  November 19, 1998

Abstract

Compare core LPS (lacking O-polysaccharide side chains) from Gram-negative bacteria are incorporated into a vaccine typically in liposomes. The complete core of E. coli K 12 is particularly useful. Upon administration to a mammal the vaccine stimulates synthesis of antibodies which are cross-protective against smooth and rough forms of LPS from at least two different Gram-negative bacterial strains having different core structures.

SUMMARY OF THE INVENTION

Vaccination (active immunization) with complete core rough LPS antigen particularly from E. coli K12 provides both strain-specific protection and cross-core protection without unacceptable toxicity or other side effects. An antigen is considered a complete-core, rough LPS in that it includes, at a minimum, Lipid A, heptose and 3-deoxy-D-manno-2-octulosonate (KDO) residues, as well as the outer core galactose and glucose residues. Typically, it also includes the outer core N-acetyl-D-glucosamine residues. For example, it includes the outer core structure of Rb and typically also the structure of Ra, as shown in FIG. 2. It does not include the O-polysaccharide outer region (also called O-polysaccharide side chain).

Thus, one aspect of the invention generally features a method of reducing the adverse effects of endotoxemia in a warm-blooded animal (a mammal, typically a human patient), by administering an effective amount of a composition comprising complete-core, rough, lipopolysaccharide (LPS) antigen (e.g., an Ra LPS) of a Gram-negative bacterium, particularly E. coli K12. Preferably, the immunizing composition is a cocktail of complete-core, rough, lipopolysaccharide (LPS) antigen from other Gram-negative bacterium. Useful rough LPSs are those from E. coli and Salmonella, particularly from each of the five known chemotypes of E. coli: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12 (Jansson et al., Eur. J. Biochem. 115:571 (1981)). See FIG. 3. Only one core structure accounts for all known Salmonella species, and any Ra Salmonella strain can be used, for example Salmonella minnesota R60. Rietschel et al. Infect. Dis. Clin. N. Am. 5:753 (1991). Complete core LPS lacking polysaccharide side chains from other Gram-negative bacteria that may be useful include those from the family Enterobacteriaceae (i.e. the genera Escherichia, Salmonella, Klebsiella, Citrobacter, Shigella, Proteus, Edwardsiella, Enterobacter, Hafnia, Serratia, Providencia, Morganella, Yersinia, Erwinia), the family Pseudomonadaceae, e.g., Pseudomonas aeruginosa and the family Bactoroides, e.g., B. fragilis. See, generally, Essentials of Medical Microbiology, 31'rd Ed., Volk, et al., pp. 397 and 416 (J/P. Lippencott Co. Philadelphia, Pa. (1986) for a compilation of Gram-negative bacteria. The composition may include a complete-core, rough, LPS antigen from several (two, three, four or more) Gram-negative bacteria, each of which is different (e.g., different species or at least different strains of the same species) from the other. In such mixtures, the core antigen from each of the four bacteria may be present in functionally equal amounts (e.g., in amounts which are intended to maximize the expression of the common core epitope(s)).

Desirably, vaccines should cause the patient to produce an antibody that binds to an epitope in the core region of the LPS core of at least one Gram-negative bacterial strain whose LPS is not part of the composition, thereby providing for cross-reactivity and cross-protection. It is difficult to achieve genuine vertical and genuine horizontal cross-reactivity and cross-protection against smooth and rough gram negative LPS, in particular in E. coli, the species most commonly isolated from surgical and intensive care unit patients. Cross-reactivity is of two kinds, which may be described as horizontal and vertical. Vertical cross-reactivity refers to an antibody's reaction with LPS's within the same strain that are different sizes, i.e., having different degress of substitution or length of the O-specific side chain. Horizontal cross-reactivity refers to an antibody's reaction with core structures that are different--i.e., different strains, species, etc. In particular, the patient's antibody response desirably will bind and protect against smooth as well as rough forms of LPS. Without wishing to bind ourselves to any particular theory, we believe that the epitope of the immunogen used in the vaccine according to the invention is accessible in both smooth and rough forms of LPS.

It may be particularly useful to include the antigen in a liposome structure. For example, the ratio (weight:weight)of lipid in the liposome to the LPS antigen is between 1:1 and 5000:1 (more typically between 10:1 and 1000:1). The liposome may include a component to provide stability or alter the compound's charge, selected from the group consisting of: phospholipid, cholesterol, positively charged compounds, negatively charged compounds, amphipathic compounds. Multilamellar type liposomes (MLV) in particular may be used. Small or large unilamellar liposomes (SUVs and LUVs) also may be used.

The composition may be administered intramuscularly intravenously, subcutaneously, intraperitonealy, via the respiratory tract, or via gastrointestinal tract. The dose of antigen can be readily determined by standard dosage trials which correlate dosage with titer and/or protection. A functional dosage may be between 0.01 ng and 1000 ng per kilogram of patient body weight, but further optimization may indicate that higher dosages (up to 100 .mu.g/kg of body weight) are desirable consistent with safety and avoiding untoward side effects. IgM antibodies can provide suitable protection, and, were the goal is generation of IgM antibodies, the composition may be administered sufficiently in advance to permit IgM antibodies to be produced (at least 2 days more typically longer) prior to potential endotoxin exposure. Also in that case, the composition would not be administered so far in advance that the IgM response deteriorates substantially--e.g., less than 14 days prior to exposure. The composition may be administered in multiple doses, the first of which is administered at least 2 days prior to potential endotoxin exposure.

Antigen in the composition may be present as part of bacteria that have been killed e.g., by heat or formaldehyde. Alternatively, the antigen may be separated from the bacterium before formulation of the composition. Alternatively the LPS antigen can be in the form of purified LPS or complexed to an acceptable carrier. Appelmelk et al., J. Immunol. Meth., 82:199 (1985).

The antigen may be chemically detoxified. The bacterium may be genetically engineered for various reasons, e.g., to reduce toxicity. The composition may also include an adjuvant, e.g., alum.

The invention also features vaccine compositions described above in connection with the method. Thus, the vaccine is comprised of an effective amount of one or more complete-core, rough, LPS of a Gram negative bacteria. Upon administration to a warm-blooded animal the compositions stimulate the synthesis of antibodies which recognize an epitope in the core region of the LPS molecule and which are cross-protective against endotoxemia caused by at least two different Gram-negative bacterial strains having different core structures. In particular, the antibodies synthesized in response to the vaccine are cross-protective against smooth LPS as well as complete core rough LPS (lacking O-polysaccharide side chains). In E. coli, the antibodies induced by the vaccine preferably react with all common smooth strain isolates, and preferably also with rough forms of all five core types (R1, R2, R3, R4, and K12). Preferably the antibodies induced by the vaccine are also reactive with both smooth and rough forms of LPS of different strains of Salmonella.

Furthermore, the vaccine described in this invention preferably causes no unacceptable toxicity following its administration to mammals. Toxicity may be controlled by incorporating the LPS into liposomes, by detoxifying the LPSs lipid A component and/or by alteration of the lipid A component by genetic manipulation of the above mentioned bacterial strains.

The vaccine composition can be used to immunize a donor, from whom antibodies are harvested for administration to a patient. Preferably the antibodies harvested comprise a substantial percentage of IgM class antibody.

Another aspect of the invention features a method of quantitating lipopolysaccharide incorporated into liposomes (PAS method). This method, unlike e.g., typical radiolabelling methods, does not require conversion of the lipopolysaccharide to a form which is unsuitable for clinical use.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Medical Indications

The patients to be treated with the vaccine include those at risk for endotoxin exposure. Specific candidates for active immunization include patients scheduled for surgery, patients subjected to chemotherapy or radiation therapy as well as burn patients, trauma patients, dialysis patients and hospitalized (particularly ICU) patients, whether or not they exhibit sepsis or septic shock. Other potential candidates for vaccination include members of the military, fireman, and policeman, as well as endurance athletes and livestock such as horses or cows.

LPS Component

As described above, the LPS antigen to be included in the vaccine can be any complete core LPS lacking O-polysaccharide side chains, preferably from E. coli K12, as described below. Although Rb chemotypes may be used, the preferred embodiment is Ra or complete core chemotypes. LPS can be purified from cultured bacteria or purchased commercially, e.g., from Difco, Sigma, List Biologicals, in Campbell, Calif. The organisms in question are widely available from depositories, including the National Culture Type Collection in England (NCTC); the University of Edinburgh collection in Edinburgh Scotland,¹ and the Forschungsinstitut in Borstell (FB), Germany D-2061. Examples of specific bacterial include, but are not limited to, the following strains: E. coli K12--e.g., Edinburgh #MPRL2320; FB W3100 or List Biologicals; E. coli R1--e.g.Edinburgh #MPRL2316 or FB F470; E. coli R2--e.g., Edinburgh #MPRL2317 or FB F576; E. coli R3--e.g., Edinburgh #MPRL2318 or FB F653; E. coli R4--e.g., Edinburgh #MPRL2431 or FB F2513; Ra S. minnesota R60 Edinburgh #MPRL1265 or List Biologicals; S. typhimurium Ra (e.g. TV119,1542), P. aeruginosa PAC611 (e.g., Edinburgh #MPRL1091) and K. aerogenes M10B (e.g., Edinburgh #MPRL0954), S. minnesota Rb chemotype (e.g. Edinburgh #MPRL1091) R345); Bacteroides fragilis--NCTC 9343; B. vulgatis NCTC 10583; B. thetaiotaomicron NCTC 10582.

¹ University of Edinburgh Medical School (Edinburgh, 3Scotland), attention Ian Poxton, Ph.D.

Without wishing to bind ourselves to a single theory by which the invention operates, we note that the inner core region may contain an important epitope in terms of stimulating the synthesis of cross-reactive and cross-protective anti-LPS antibodies. However, sufficient outer core structures may be necessary to maintain the inner core epitope in a three-dimensional structure which is similar to that encountered in clinically significant LPS isolates (i.e. smooth and rough forms of complete core LPS). The absence of polysaccharide side chain (i.e. rough LPS) allows the core epitope to be the dominant epitope. In smooth forms of LPS, the polysaccharide side chain is a much more dominant epitope than the core thus significantly reducing the relative amount of anti-core antibody produced. In other words, vaccines containing smooth LPS elicit primarily a serotype specific (i.e. anti-polysaccharide side chain) antibody response as opposed to the anti-core response which is the focus of our invention.

E. coli K12 may be particularly useful because it is not generally present in the patient population. Therefore, K12 is less likely to provoke a memory response to the outer core, and more likely to provoke a cross-reactive memory response to the inner core.

If whole bacteria are to be included in the vaccine the bacterium will be killed by a technique well known to those in the art, such as heat killing or formaldehyde killing. In this case, the entire LPS of rough mutant bacterium will be included as part of the killed bacterium. It is desirable to avoid bacterial killing methods which can alter the core.

Alternatively, complete core LPS can be isolated from the desired bacteria according to standard techniques as outlined by Hancock et al., Bacterial Cell Surface Techniques, pp. 91 (John Wiley & Sons 1988). As noted, it is preferable to include all of the core LPS, without the O-polysaccharide outer LPS structures, i.e. use R-mutant bacteria expressing full LPS core.

Patient Response

As noted, the desired patient response is cross-protective antibodies that bind to the core of rough and smooth LPS of Gram-negative bacteria generally, regardless of whether their outer LPS structures are similar.

For example, in E. coli, the antibodies induced by this vaccine preferably react with all common smooth strain isolates, and preferably also with rough strain LPSs of all five core types (R1, R2, R3, R4, and K12). Preferably the antibodies induced by this vaccine are also reactive with different smooth and rough LPSs of Salmonella.

It is possible to achieve a vigorous and effective antibody response using compositions with acceptable levels of (or no) toxicity. The vaccine stimulates the synthesis of antibodies which recognize an epitope in the core region of the LPS molecule and which are cross-protective against endotoxemia caused by at least two different Gram-negative bacterial strains having different LPS structures and in particular are cross-protective against smooth strains as well as complete core rough strains.

Typically, the vaccine will be a cocktail of the purified LPS from different strains of bacteria, preferably rough strains having a complete core, for example a mixture of LPS from K12 with LPS from R1 and R3 rough strains of E. coli, or with the Ra strain of Salmonella minnesota R60. E. coli R2 and R4 are less important but also candidates. Preferred cocktails (depending on the breadth of protection desired) include K12 with R1; K12 with Pseudomonas (e.g., P. aeruginosa) and Klebsiella (e.g., K. aerogenes). Since the Bacteroides are a particularly significant population in the gut, it may be important to protect specifically against Bacteroides endotoxin by including Bactoeroides in the cocktail. e.g., together with K12 or together with K12, Pseudomonas and Klebsiella.

Alternatively, the purified LPS from one of these strains, a mixture of any combination of these strains, or a different strain of bacteria may be used in any ratio of the individual strains in the case of use of more than one LPS type.

The route of administration is preferably subcutaneous or intramuscular, although any alternative route which results in these immunogens reaching the antigen presenting cells and antibody producing cells is acceptable. Some other examples include but are not limited to intravenous, intraperitoneal, and via the respiratory or gastrointestinal tract.

The dose of this composition should stimulate the host to produce increased quantities of cross-reactive and cross-protective antibodies levels, consistent with avoiding toxicity, as described above.

The composition is administered before endotoxin exposure. To the extent that the vaccine works in part by stimulating the host to synthesize antibodies of the IgM class, the vaccine is preferably given between 2 to 14 days prior to potential endotoxin exposure. Alternatively, additional doses of any of the possible permutations of this vaccine may allow for greater effectiveness and increases in desired antibody levels or even further reduced toxicity. It is anticipated that in most vaccinees the antibody response to inner core determinants will be a secondary (i.e. memory) response as opposed to a primary (i.e. naive) response. This is because most vaccinees will have been exposed at some time in their lifetime to the LPS core epitopes, presumably from LPS that has leaked through the gut barrier into the bloodstream. In other words, an important function of our method of vaccination is to cause an increase in the serum concentration of antibodies which may already be present, but at levels which do not allow for sufficient protection from a toxic exposure of LPS during periods of stress and trauma. The above in no means suggests that there are not patients who will also benefit from vaccination with this invention by means of a primary (i.e. naive) antibody response.

A vaccine with the LPS mentioned above is preferably rendered non-pyrogenic and non-toxic by incorporation of the LPS into liposomes. The liposome (exclusive of the LPS component) may contain a combination of (1) a phospholipid and cholesterol or (2) a phospholipid, cholesterol and a negatively or positively charged (lipophilic) amphipathic compound. The phospholipid component may be selected from the group comprising any lipid capable of forming liposomes, including, but not limited to: any phosphatidyl-choline derivative, glycerophosphatides, lysophosphatides, sphingomyelins, and mixtures thereof. The negatively charged (lipophilic) amphipathic compounds may be selected from the group comprising di(alkyl)phosphates, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, dicetyl phosphate, or any other similar negatively charged amphipathic compound that can impart a negative charge to a liposome surface. When positively charged (lipophilic) amphipathic compounds are employed; they are selected from the group comprising alkyl amines, such as stearylamine and hexadecylamine. The ratio in the constituents of the liposomes (exclusive of the LPS) will effect the liposomes' charge, rigidity, stability and may vary greatly while still allowing for reduced toxicity and increased immunogenicity of the LPS they contain. Polyethylene glycol lipids (PEG) may be incorporated into the liposomes, for example at approximately 10 to 20 mole %, in order to increase the amount of time that the liposomes remain in the systemic circulation, thus affecting their immunogenicity. Alternatively, very rigid bilayers may be made by using lipids which are gel phase at body temperature (37 degrees C.), for example distearoyl phosphatidylcholine or distearoyl phosphatidylserine. The type of liposomes used is preferably multilamellar liposomes (MLV) but alternatively upon sonication, or by alternative methods of manufacture, small or large unilamellar liposomes (SUVs and LUVs) of varying sizes can be employed. Different salt forms of LPS may alter the degree of incorporation of LPS into the liposomes, for example, the acid salt form, magnesium salt form, and calcium salt form may allow for increased incorporation due to their increased hydrophobicity.

Liposomes are defined as closed vesicles, or sacs, which contain phospholipids (examples of which are lecithin and sphingomyelin) and which may contain other lipids (examples of which are cholesterol and other steroids; charged lipids such as dicetyl phosphate and octadecylamine; glycolipids; fatty acids and other long-chain alkyl compounds; hydrophobic glycoproteins; and lipid soluble vitamins and lipoidal surfactant-like molecules). When shaken in the presence of an excess amount of water, the lipid mixture is formed into discrete particles consisting of concentric spherical shells of lipid bilayer membranes which are referred to as multilamellar liposomes (MLV). Upon sonication, or by alternative methods of manufacture, small or large unilamellar liposomes (SUV or LUV, respectively) can be formed.

Upon injection into animals and man, liposomes are taken up rapidly by cells of the reticuloendothelial system, particularly those of the liver. Because of the relative impermeability of liposomes and their speedy removal from the circulatory system, substances such as lipid A and certain forms of LPS remain incorporated within the liposomes and are less likely to be exposed to cells and/or receptors through which they can exert potentially toxic effects. Moreover, liposomes may allow for a prolonged effectiveness through slow biodegradation of the multilamellar membrane structure of the liposomes.

The toxicity of the lipid A component of the above mentioned complete core rough mutant strains also can be reduced or eliminated by chemical detoxification as described in Bhattacharjee A et al., WO 95/29662. The preferred method for this detoxification maintains the LPS configuration such that it still stimulates the synthesis of antibody/ies which recognize an epitope in the core region of the LPS molecule and which is cross-protective against endotoxemia caused by at least two different Gram-negative bacterial strains having different core structures. In particular, the antibodies synthesized in response to this vaccine are cross-protective against smooth strains as well as complete core strains. The detoxified LPS may be administered in the form of purified LPS, or alternatively can be incorporated into liposomes or complexed to an acceptable carrier.

Alternatively the toxicity of the lipid A component of the above mentioned strains of bacteria can be reduced or eliminated by genetic alteration of the bacterial strains as described in Somerville J E et al, J Clin Invest 1996; 97:359-365. The resulting LPS from these cells (in the form of heat killed cells) is reduced in toxicity while still affording immunogenicity to LPS core. The preferred method for this genetic alteration maintains the LPS in a sufficient three-dimensional shape that it still acts sufficiently as an immunogen in a host to stimulate the synthesis of antibody/ies which recognize an epitope in the core region of the LPS molecule and which is cross-protective against endotoxemia caused by at least two different Gram-negative bacterial strains having different core structures. In particular, the antibodies synthesized in response to this vaccine are cross-protective against smooth strains as well as complete core rough strains. At the same time, this genetic process preferably renders the LPS non-pyrogenic and non-toxic in the warm-blooded animal. The LPS from these genetically altered bacterial strains are preferably administered in form of purified LPS incorporated into liposomes. LPS from these altered strains can alternatively be administered in the form of killed cells. Alternatively, the detoxified LPS may be administered in the form of purified LPS, or alternatively can be complexed to an acceptable carrier.

Toxicity of any of the LPS rough antigen compositions described in this invention may also be reduced by other methods, for example, competitive detoxification of lipid A by synthetic anti-endotoxin peptides. Rustici et al. Science 259:361 (1993). An alternative method of reducing toxicity is to administer the LPS antigen with or at around the same time as an anti-inflammatory agent, e.g., anti-TNF-alpha monoclonal antibody. Fisher C J et al. N Engl J Med 334:1697 (1996).

Claim 1 of 32 Claims

What is claimed is:

1. A method of reducing adverse effects of endotoxin in a warm-blooded animal, which comprises administering to the warm-blooded animal an effective amount of a composition comprising rough, complete-core lipopolysaccharide (LPS) antigens of at least two Gram negative bacterial strains, each of said strains having a classification independently selected from the following classifications: E. coli; Pseudomonas; and Bacteroides, said antigens being separated from cells of said bacterial strains.

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