Patent 6,759,241

LPS preparations, isolated from gram negative bacterial strains that contain at least one mutation in at least one of the htrB and msbB genes, and methods and therapeutics related thereto. The LPS preparations display both LPS antagonist and adjuvant activities.

SUMMARY OF THE INVENTION

The invention relates to methods and compositions comprising an adjuvant which is both an LPS antagonist and is non-pyrogenic. In a preferred embodiment, the non-pyrogenic LPS antagonist adjuvant is isolated from a gram negative bacterial strain that contains at least one mutation in at least one of the htrB and msbB genes.

In one aspect, the invention features an adjuvant comprising an LPS antagonist, wherein said LPS antagonist is isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes. In one embodiment, the LPS antagonist has reduced pyrogenicity. In a preferred embodiment, the LPS antagonist has substantially reduced pyrogenicity. In a more preferred embodiment, the LPS antagonist in non-pyrogenic. In a preferred embodiment, pyrogenicity is determined by measuring the levels of indicators of pyrogenicity or inflammation, such as, for example, IL-1.beta., IL-6 or TNF.alpha., in a cell, extracellular medium, or a subject. In a most preferred embodiment, the LPS antagonist elicits no detectable TNF.alpha. activity when contacted with a cell or administered to a subject.

The LPS antagonist may comprise an LPS derivative or a fragment thereof (e.g., a precursor component or derivative thereof), such as a lipid A precursor structure selected from the group consisting of lipid X, tetraacyl-lipid A (LA4), pentaacyl-lipid A (LAS), or a Rhodobacter sphaeroides diphosphoryl-lipid A structure (RsDPLA). The LPS antagonist can be purified from a gram negative bacteria is selected from the group consisting of Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp., and Vibrio spp.

In another embodiment, the gram negative bacterium defective in at least one of the msbB or htrB genes is also defective in at least one of the kdsA, kdsB, kdtA, lpxA, lpxB, lpxC lpxD or ssc genes.

In another embodiment, the invention provides a pharmaceutical preparation comprising a vaccine antigen, a pharmaceutically effective amount of an LPS antagonist, isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes, and a pharmaceutically acceptable carrier. The vaccine antigen may be any vaccine antigen, such as e.g., a polysaccharide, a protein or a nucleic acid. In an embodiment of the invention, the vaccine antigen is derived from a viral pathogen selected from the group consisting of orthomyxoviruses, retroviruses, herpesviruses, lentiviruses, rhabdoviruses, picornoviruses, poxviruses, rotavirus and parvoviruses. Exemplary antigens are influenza virus, RSV, EBV, CMV, herpes simplex virus, human immunodeficiency virus, rabies, poliovirus and vaccinia, human immunodeficiency virus antigens Nef, p24, gp120, gp41, Tat, Rev, and Pol; T cell and B cell epitopes of gp120; the hepatitis B surface antigen; rotavirus antigens, such as VP4 and VP7; influenza virus antigens such as hemagglutinin or nucleoprotein; and herpes simplex virus thymidine kinase.

In another embodiment the vaccine is derived from a bacterial pathogen selected from the group consisting of Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp., and Borellia burgdorferi. Exemplary vaccine antigens useful in the practice of the invention are the capsular polysaccharide of Neisseria meningitis; the Vi polysaccharide of Salmonella enterica serovar typhi; Shigella sonnei form 1 antigen; the O-antigen of V. cholerae Inaba strain 569; cholera toxin of V. cholerae; TCP of V. cholera; CFA/I fimbrial antigen of enterotoxigenic E. coli; the heat-labile toxin of E. coli; pertactin of Bordetella pertussis; adenylate cyclase-hemolysin of B. pertussis, and fragment C of tetanus toxin of Clostridium tetani.

In another embodiment of the invention the vaccine antigen is derived from a parasitic pathogen selected from the group consisting of Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Entamoeba spp., Eimeria spp., Leishmania spp., Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp. Exemplary parasitic vaccine antigen useful in the practice of the invention are the circumsporozoite antigen of P. berghei, the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp.; the galactose specific lectin of Entamoeba histolytica; gp63 of Leishmania spp.; paramyosin of Brugia malayi; the triose-phosphate isomerase of Schistosoma mansoni; the secreted globin-like protein of Trichostrongylus colubriformis; the glutathione-S-transferase of Frasciola hepatica, Schistosoma bovis and S. japonicum; and KLH of Schistosoma bovis.

In additional embodiments of the invention the vaccine antigen is derived from a tumor antigen selected from the group consisting of prostate specific antigen, TAG-72, carcinoembrionic antigen (CEA), MAGE-1, tyrosinase, and mutant p53 antigen; the CD3 receptor on T cells; an autoimmune antigen; or the IAS .beta. chain. Alternatively, the invention can be practiced with a vaccine antigen such as an immuno-stimulatory molecule selected from the group consisting of M-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IFN-.gamma..

An another aspect, the invention features combination therapies, comprising a pharmaceutical composition containing the LPS antagonist together with other agents that enhance the activity of the LPS antagonist (e.g., by increasing its adjuvant or antagonist activities or promoting stabilization of the LPS antagonist or other components of the pharmaceutical composition) or minimize deleterious effects of the LPS antagonist.

In another aspect, the invention features methods for preparing and using an adjuvant as described herein comprising an LPS antagonist, wherein said LPS antagonist is isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes.

In another aspect, the invention features methods for preparing and using a vaccine comprising a vaccine antigen and a pharmaceutically effective amount of an LPS antagonist isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes, and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

Overview of the Invention

Prior to the present invention, LPS antagonists had proven to be poor adjuvants. An unexpected and surprising element of the present invention, therefore, is the finding that LPS preparations isolated from gram negative bacterial strains that contain at least one mutation in at least one of the hirB and msbB genes display both LPS antagonist and adjuvant activities. Accordingly, one embodiment of the present invention provides a novel adjuvant comprising an LPS antagonist. In particular, the present invention provides compositions comprising an LPS antagonist isolated from a gram negative bacterial strain that contains at least one mutation in at least one of the htrB and msbB genes. The invention also provides methods for enhancing the efficacy of a vaccine in a subject, comprising administering to the subject one or more antigens, against which an immune response is desired, with one or more LPS antagonists.

5..3 Adjuvant Preparations Comprising an LPS Antagonist

5.3.1 Non-Pyrogenic LPS and Lipid A

We have found that gram negative bacterial strains, which contain a conditional mutation (or mutations) that result in the accumulation of lipid A precursors, are capable of exclusively producing non-pyrogenic LPS (i.e., LPS that is 10^-7 -fold less toxic than wildtype LPS) under specific growth conditions in supplemented culture medium (see PCT International Publication Nos. WO 97/18837 and WO 99/15162, the teachings of which are incorporated herein by reference).

Examples of such conditional mutations that affect the biosynthesis of lipid A and result in the accumulation of non-pyrogenic LPS include, but are not restricted to, mutations in htrB, msbB, kdsA, kdsB, and kdtA (Rick, et al., 1977, supra; Rick and Osborne, 1977, supra; Raetz, et al., 1985, supra; Raetz, 1990, supra; Raetz, 1993, supra; Schnaitman and Klena, 1993, supra; Lee, et al., 1995, Infect. Immun., 63:818; Karow and Georgopoulos, 1991, Molec. Microbiol., 5:2285; Karow, et al., 1991, supra). These mutations could be introduced alone. Alternatively, any combination of mutations in the kdsA, kdsB, lpxB, kdtA, lpxC (synonym is envA), lpxD (synonyms are firA and ssc), ssc, lpxA, htrB, and the msbB genes (Rick, et al., 1977, supra; Rick and Osborne, 1977, supra; Raetz, et al., 1985, supra; Raetz, 1990, supra; Raetz, 1993, supra; Schnaitman and Klena, 1993, supra; Lee, et al., 1995, supra; Karow and Georgopoulos, 1991, supra; Karow, et al., 1991, supra; Karow and Georgopoulos, 1991, supra), which may affect the biosynthesis of lipid A and result in the synthesis of non-pyrogenic lipid A structures, could be used.

5.3.2 Preparation of Mutants that Produce an LPS Antagonist

A mutation in a gene, e.g., of a gram negative bacterium, such as a mutation in a hirb and msbB gene, can be introduced using any well-known genetic technique. These include but are not restricted to non-specific mutagenesis, using chemical agents such as N-methyl-N'-nitro N-nitrosoguanidine, acridine orange, ethidium bromide, or non-lethal exposure to ultraviolet light (Miller (Ed.), 1991, In: A Short Course in Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Alternatively, the mutations can be introduced using Tn10 mutagenesis, bacteriophage-mediated transduction, lambda phage-mediated allelic exchange, or conjugational transfer, or site directed mutagenesis using recombinant DNA techniques (Miller (Ed.), 1991, supra; Hone, et al., 1987, J. Infect. Dis., 156:167; Noriega, et al, 1994, Infect. Immun., 62:5168; Hone, et al., 1991, Vaccine, 9:810; Chatfield, et al., 1992, Vaccine, 10:53; Pickard, et al., 1994, Infect. Immun., 62:3984; Odegaard, et al., 1997, J. Biol. Chem., 272:19688; Lee, et al., 1995, J. Biol. Chem., 270:27151; Garrett, et al., 1998, J. Biol. Chem., 273:12457). Any method for introducing mutations may be used and the mutations can be introduced in conjunction with one or more additional mutations, such as those described in PCT International Publication Nos. WO 97/18837 and WO 99/15162, the teachings of which are incorporated herein by reference.

The mutations can be either constitutively expressed or under the control of inducible promoters, such as the temperature sensitive heat shock family of promoters (Neidhardt, et al., supra), or the anaerobically-induced nirB promoter (Harborne, et al., 1992, Mol. Micro., 6:2805) or repressible promoters, such as uapA (Gorfinkiel, et al., 1993, J. Biol. Chem., 268:23376) or gcv (Stauffer, et al., 1994, J. Bact, 176:6159). Selection of the appropriate promoter will depend on the host bacterial strain and will be obvious to those skilled in the art.

5.3.3 Bacterial Strains

Any gram negative bacterial strain can be used as a source of LPS antagonist in the practice of the present invention. Examples of gram-negative bacteria include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp. and Vibrio spp.

The particular Escherichia strain used in the practice of the present invention is not critical. Examples of Escherichia strains which can be used include Escherichia coli (E. coli) strains DH5a, HB 101, HS-4, 4608-58, 1-184-68, 53638-C-17, 13-80, and 6-81 (Sambrook, et al., (Eds.), 1993, In: Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.); Grant, et al., 1990, Proc. Natl. Acad. Sci., USA, 87:4645; Sansonetti, et al., 1982, Ann. Microbiol. (Inst. Pasteur), 132A:351), enterotoxigenic E. coli (Evans, et al., 1975, Infect. Immun., 12:656), enteropathogenic E. coli (Donnenberg, et al., 1994, J. Infect. Dis., 169:831) and enterohemorrhagic E. coli (McKee and O'Brien, 1995, Infect. Immun., 63:2070).

The particular Shigella strain used in the practice of the present invention is not critical. Examples of Shigella strains which can be used include S. flexneri (ATCC No. 29903), S. sonnei (ATCC No. 29930), and S dysenteriae (ATCC No. 13313).

The particular Campylobacter strain used in the practice of the present invention is not critical. Examples of Campylobacter strains which can be used include but are not limited to C. jejuni (ATCC Nos. 43436, 43437, 43438), C. hyointestinalis (ATCC No. 35217), C. fetus (ATCC No. 19438) C. fecalis (ATCC No. 33709) C. doylei (ATCC No. 49349) and C. coli (ATCC Nos. 33559, 43133).

The particular Yersinia strain used in the practice of the present invention is not critical. Examples of Yersinia strains which can be used include Y. enzerocolitica (ATCC No. 9610) or Y. pestis (ATCC No. 19428), Y. enterocolitica Ye03-R2 (al-Hendy, et al., 1992, Infect. Immun., 60:870) and a Y. enterocolitica aroA mutant (O'Gaora, et al., 1990, Micro. Path., 9:105).

The particular Klebsiella strain used in the practice of the present invention is not critical. Examples of Klebsiella strains which can be used include K. pneumoniae (ATCC No. 13884).

The particular Bordetella strain used in the practice of the present invention is not critical. Examples of Bordetella strains which can be used include B. pertussis and B. bronchiseptica (ATCC No. 19395).

The particular Neisseria strain used in the practice of the present invention is not critical. Examples of Neisseria strains which can be used include N. meningitidis (ATCC No. 13077) N. gonorrhoea (ATCC No. 19424) and an N. gonorrhoea MS 11 aro mutant (Chamberlain, et al., 1993, Micro. Path., 15:51-63).

The particular Aeromonas strain used in the practice of the present invention is not critical. Examples of Aeromonas strains which can be used include A. salminocida (ATCC No. 33658), A. schuberii (ATCC No. 43700), A. hydrophila and A. eucrenophila (ATCC No. 23309).

The particular Francisella strain used in the practice of the present invention is not critical. Examples of Francisella strains which can be used include F. tularensis (ATCC No. 15482).

The particular Corynebacterium strain used in the practice of the present invention is not critical. Examples of Corynebacterium strains which can be used include C. pseudotuberculosis (ATCC No. 19410).

The particular Citrobacter strain used in the practice of the present invention is not critical. Examples of Citrobacter strains which can be used include C. freundii (ATCC No. 8090). The particular Chlamydia strain used in the practice of the present invention is not critical. Examples of Chlamydia strains which can be used include C. pneumoniae (ATCC No. VR1310).

The particular Haemophilus strain used in the practice of the present invention is not critical. Examples of Haemophilus strains which can be used include H. influenzae (Lee, et al., 1995, supra) and H. somnus (ATCC No. 43625).

The particular Brucella strain used in the practice of the present invention is not critical. Examples of Brucelia strains which can be used include B. abortus (ATCC No. 23448).

The particular Legionella strain used in the practice of the present invention is not critical. Examples of Legionella strains which can be used include L. pneumophila (ATCC No. 33156), and a L. pneumophila mip mutant (Ott, 1994, FEMS Micro. Rev., 14:161).

The particular Pseudomonas strain used in the practice of the present invention is not critical. Examples of Pseudomonas strains which can be used include P. aeruginosa (ATCC No. 23267).

The particular Helicobacter strain used in the practice of the present invention is not critical. Examples of Helicobacter strains which can be used include H. pylori (ATCC No. 43504) and H. mustelae (ATCC No. 43772).

The particular :Salmonella strain used in the practice of the present invention is not critical. Examples of Salmonella strains which can be used include S. typhi (ATCC No. 7251), S. typhimurium (ATCC No. 13311), S galinarum (ATCC No. 9184), S. enteridilis (ATCC No. 4931) and S. typhimurium (ATCC No. 6994), an S. typhi aroC, aroD mutants (Hone, 1991, et al., Vacc., 9:810-816) and an S. typhimurium aroA mutant (Mastroeni, et al., 1992, Micro. Pathol., 13:477491).

The particular Vibrio strain used in the practice of the present invention is not critical. Examples of Vibrio strains which can be used include Vibrio cholerae (ATCC No. 14035), Vibrio cincinnatiensis (ATCC No. 35912), a V. cholerae RSI virulence mutant (Taylor, et al., 1994, J. Infect. Dis., 170:1518-1523) and a V. cholerae ctxA, ace, zot, cep mutant (Waldor, et al., 1994, J. Infect. Dis., 170:278-283). Useful non-pyrogenic bacterial strains and their uses are disclosed in PCT International Publication Nos. WO 97/18837 and WO 99/15162, the teachings of which are incorporated herein by reference.

5.3.4 Bacterial Culture

The specific culture conditions for the growth of the mutant gram negative bacterial strains for the preparation of an adjuvant comprising an LPS antagonist are not critical to the present invention. For illustrative purposes, bacteria can be grown in any standard liquid medium suitable for bacterial growth, such a LB medium (Difco, Detroit Mich.), Nutrient broth (Difco), Tryptic Soy broth (Difco), or M9 minimal broth (Difco), using conventional culture techniques that are appropriate for the bacterial strain being grown (Miller, 1991, supra). As an alternative the bacteria can be cultured on solid media such as L-agar (Difco), Nutrient agar (Difco), Tryptic Soy agar (Difco), or M9 minimal agar (Difco).

The temperature at which the bacterial strains are cultured is not crucial to the present invention. However, individual bacterial strains may produce a non-pyrogenic LPS antagonist at one temperature and at other temperatures produce a pyrogenic LPS antagonist. For example, E. coli htrB msbB double mutants produce LPS that retains modest pyrogenic activity at 30^oC., but at temperatures above 33^oC. and below 44^oC., this strain produces a non-pyrogenic LPS antagonist (Hone, et al., 1998, supra).

A straight forward approach to identifying the optimal temperature for the culture of a particular bacterial strain is to grow the bacteria over a range of culture temperatures, isolate LPS from each culture (as described herein below) and measure the LPS antagonist activity of the LPS produced (as described in Hone, et al., 1998, supra). In this manner, culture temperatures can be identified that result in the production of non-pyrogenic or substantially lowered pyrogenic LPS antagonist by the bacterial strains.

Normally, exclusive expression of lipid A precursors is toxic to the bacterium. Thus, when these mutants are grown in non-permissive conditions, whereby lipid A precursors accumulate, the bacteria usually only undergo a single division before ceasing to grow. For example, in certain lipid A-defective mutants, expression of lipid IVa (a tetraacyl precursor of lipid A) can only reach levels of 30%-50% of the total LPS before growth of the strain ceases (Rick and Osborn, 1977, supra; Raetz, 1993, supra).

However, surprisingly, growth of the conditional mutants that produce lipid A precursors in non-permissive conditions, i.e., at 35^oC. to 44^oC., in the presence of quaternary cationic compounds, suppresses the conditional-lethal effect of these mutations and allows the accumulation of non-pyrogenic LPS/lipid A precursors (see below). Thus, under these culture conditions the bacteria continue to grow and accumulate substantially pure (>99%) non pyrogenic LPS.

The culture of htrB mutants at temperatures above 33^oC. can be enhanced by the presence of quaternary cationic compounds, which suppress the temperature-sensitive effect of this mutation (Karrow and Georgopoulos, 1992, J. Bacteriol., 174:702; Powell and Hone, 1995, U.S. Pat. No. 5,877,159). The particular quaternary cationic compound used is not critical to the practice of the present invention. Examples include tetraacyltetramethylammonium bromide (herein TTAB; Sigma, St Louis Mo., USA), polylysine (Sigma), polymyxin (Sigma), ethanolamine (Sigma) dimethyldictadecylammonium bromide (DDAB from ICN, Costa Mesa, Calif., USA), 1,2, diacyl-3-trimethylammonium-propane (TAP; Avanti Polar Lipids Inc, AL, USA), 2,-dioleyloxy-N-[2(perminecarboxamindo)-ethyl]-N,N-dimethyl-1-propanammoni umtrifluoroacetate (DOSPA; GibcoBRL, Gaithersburg, Md., USA), and N-[1-2,3 dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA; GibcoBRL) (Powell and Hone, 1995, supra).

The concentration of the quaternary cationic compound in the medium is not critical to the practice of the present invention but it is usually a concentration that is sublethal to the bacterial strain, ranging from 0.01 .mu.g/ml to 100 .mu.g/ml. The concentration of quaternary cationic compound selected will depend on the genotype of the bacterial host and can be identified by growing the mutant organism at non-permissive temperatures in the presence of quaternary cationic compound at concentrations in this range.

The optical density at 600 nm at which the bacteria are harvested is not critical, and can range from 0.1 to 5.0 and will be dependent on the specific strain, media and culture conditions employed.

The method used to extract LPS is not critical to the practice of the present invention and can be any one of the well-known methods for LPS extraction (Garrett, et al., 1997, J. Biol. Chem., 272:21855; Garrett, et al., 1998, J. Biol. Chem., 273:12457; Clementz, et al., 1996, supra; Clementz, et al., 1997, supra; Somerville, et al., 1996, supra; Lee, et al., 1995, supra; Sunshine, et al., 1997, supra; Galanos, et al., 1969, Eur. J. Biochem., 9:245; Westphal, et al., 1952, Naturforsch, 7b:155; Westphal and Jann, 1965, In: Methods in Carbohydrate Chemistry, Whistler (Ed.), 5:83).

For example, the LPS antagonist can be extracted using the well-known hot phenol-water extraction procedure (Galanos, et al., 1969, supra; Westphal, et al., 1952, supra; Westphal and Jann, 1965, supra). After culturing the bacteria as described above, the bacteria are harvested by centrifugation (4,500xg for 15 minutes) and washed once in endotoxin-free irrigation saline (0.85% w/v; Baxter). The weight of the bacterial pellets is determined and the bacteria are resuspended in endotoxin-free saline (Baxter) at a final density of 2% w/v and heated to 70^oC. An equal volume of pre-warmed H₂ O-equilibrate phenol (70^oC.) is added to each of the bacterial suspensions and mixed for 15 minutes at 70^oC. The mixtures are cooled to 25^oC. and centrifuged at 18,000xg for 15 minutes. Following centrifugation, the aqueous phases are removed, placed into dialysis tubing (SpectraPor) and dialyzed against running milli-Q H20 (Millipore) for 24 hours. The retentates are then placed into fresh 50 ml polypropylene tubes and treated first with RNaseA (100 [.mu.g/ml; Sigma) at 37^oC. for 1 hour, then with DNasel (50 .mu.g/ml and 5 mM MgCl₂ ; Sigma) at 37^oC. for 2 hours, and finally with Pronase (250 ;g/ml; Sigma) at 37^oC. for 4 hours. EDTA is then added to a final concentration of 5 mM and the hot-water phenol extraction procedure described above is repeated. Following dialysis against running milli-Q H₂ O (Millipore) for a further 48 hours, residual particulate material is removed by centrifugation at 20,000xg for 1 hour. Ultrapure LPS is subsequently separated from the resultant supernatants by centrifugation at 100,000xg for 1 hour. The resultant high-speed centrifugation supernatants are discarded and the 100,000xg LPS pellets are lyophilized, as described in Hone, et al., 1995, supra. The lyophilates are weighed and resuspended in endotoxin-free PBS (<0.001 EU/ml; Lifetechnologies) at a concentration of 1-10 mg/ml and stored at -20^oC.

5.3.6 Purity Determination of an LPS Antagonist

The purity of the LPS antagonist preparation (e.g., 10 .mu.g) can be assessed by SDS-polyacrylamide gel electrophoresis and silver staining procedures, using commercially available reagents (Bio-Rad, Hercules Calif.). In addition, the purity of the LPS antagonist preparations can be evaluated by UV spectrophotometry (Shimadzu UV-1201S UV-VIS Spectrophotometer), wherein no significant absorption maxima at 260 nm and 280 rim. should be detected in LPS preparations suspended in milli-Q HZO at 100 .mu.g/ml. The absence of absorption maxima indicates that the preparation does not contain substantial amounts of phenol, nucleic acids or polypeptides, i.e., the preparation contains less than 5 ng/ml of nucleic acids and less than 50 ng/ml of protein. In each instance, 10-fold serial dilutions of E. coli genomic DNA (50-0.5 ng/100 .mu.g LPS) and lysosyme (500-5 ng/100 .mu.g LPS; Sigma) mixed with the LPS antagonist preparations can be used as internal controls.

5.3.7 Purification of the Lipid A Fraction from an LPS Antagonist

While one embodiment of the current invention provides an adjuvant comprised of LPS antagonist isolated from mutant gram negative bacterial strains using the above extraction procedure, in another embodiment the adjuvant can be prepared by isolating the lipid A fraction of the LPS preparation.

Procedures for isolating the lipid A fraction from LPS are well known in the art (see, e.g., Garrett, et al., 1997, supra; Garrett, et al., 1998, supra; Clementz, et al., 1996, supra; Clementz, et al., 1997, supra). For illustrative purposes, the lipid A fraction can be isolated from an LPS antagonist preparation by mild acid hydrolysis in 1% SDS at pH 4.5. The lipid A fraction can then be isolated by conventional DEAE-cellulose column chromatography techniques (Garrett, et al., 1997, supra; Garrett, et al., 1998, supra; Clementz, et al., 1996, supra; Clementz, et al., 1997, supra).

Lipid A can be analyzed by a number of techniques, including conventional thin layer chromatography (TLC) procedures, using silica gel-20 TLC plates (Garrett, et al., 1997, supra; Garrett, et al., 1998, supra; Clementz, et al., 1996, supra; Clementz, et al., 1997, supra). Wild type lipid A, LA4 and LAS can be used as standards (Clementz, et al., 1996, supra; Clementz, et al., 1997, supra). In addition, the molecular mass the lipid A can be determined by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) and electronspray analysis (Somerville, et al., 1996, supra; Lee, et al., 1995, supra; Sunshine, et al., 1997, supra).

The latter analysis combined with the analysis of the pyrogenic activity of each species will determine the purity, structure and biological activity of the lipid A fraction and can provide a template for the development of synthetic lipid A partial structures that mimic the adjuvant activity of said LPS antagonist using procedures well known in the art (Galanos, et al., 1984, Eur. J. Biochem., 140:221; Manthey, et al., 1993, Infect. Immun., 61:3518; Homma, et al., 1989, Drugs Future, 14:645; Steutz, et al., 1990, In: Endotoxin research series, Nowotny, et al., (Eds.), 1:129; Van Dervort, et al., 1992, J. Immunol., 149:359; Perera, et al., 1993, Infect. Immun., 61:2015; Kotani, et al., 1985, Infect. Immun., 49:225; Kotani, et al., 1986, supra; Fagan, et al., 1994, J. Immunol., 153:5230; Wang, et al., 1991, supra; Christ, et al., 1994, Am. Chem. Soc., 116:3637; Rose, et al., 1995, Infect. Immun., 63:833; Lam, et al., 1991, supra; Marcher, 1987, Carbohydrate Res., 162:79).

5.3.8 Determination of the Molecular Structure of an LPS or Lipid A Molecule

Various methods well known in the art can be used to determine the molecular structure of an LPS or lipid A molecule isolated and purified from bacteria. Exemplary methods are described, e.g, in U.S. Pat. No. 5,648,343, in which the molecular structure of lipid A from Phizobium leguminosarum was determined. In particular, this patent describes methods for determining the glycosyl composition, fatty acid composition, glycosyl linkage analysis, and phosphate content of lipid A preparations. Such methods may involve NMR Spectroscopy and high resolution mass spectrometry, e.g., fast atom bombardment mass spectrometry (FAB-MS).

5.3.9 Synthetic LPS and Lipid A

Synthetic LPS and lipid A molecules with strong LPS antagonist properties and of reduced or absent pyrogenicity may be synthesized by a variety of organic chemistry synthetic techniques. In one embodiment of the present invention, the synthetic lipid A and LPS molecules are modeled after LPS molecules of reduced or absent pyrogenicity which occur in nature and molecules with strong LPS antagonist activities which occur in nature. Thus, the invention provides methods for preparing synthetic LPS and lipid A molecules that are identical to, analogs or derivatives of, naturally occurring LPS or lipid A molecules. As described in the previous section, methods for determining the structure of an LPS or lipid A molecule are well known in the art and can be used to first determine the structure of an LPS or lipid A molecule of interest, prior to synthesizing the same or similar LPS or lipid A molecules using organic chemistry methods.

For an overview of the synthesis of LPS and lipid A structures, see, e.g., Raetz, 1993, J. Bacteriology 175:5745-5753. (See also U.S. Pat. Nos. 5,593,969 and 5,191,072).

As set forth above, LPS is a complex polymer in four pats (see FIG. 1). Outermost is a carbohydrate chain variable length (called the O-antigen) which is attached to a core polysaccharide. The core polysaccharide is divided into the outer core and the backbone. These two structures vary between bacteria. Finally the backbone is attached to a glycolipid called lipid A. The link between lipid A and the rest of the molecule is usually via a number of 3-deoxy-D manno-octulosonic acid (KDO) molecules. The presence of KDO is often used as a marker for LPS (or outer membrane) even though it is not present in all bacterial LPS. The phosphate and 3-deoxy-D-mannooctulosomic acid (KDO) molecules (the presence of KDO is often used as a marker for lipopolysaccharide) are also substituted. Unsaturated and cyclopropane fatty acids which are common in other lipid types are absent from LPS.

Lipid A is composed of a disaccharide of glucosamines. The amino groups are substituted with 3-hydroxymristate while hydroxyl groups contain saturated (12-16 carbon) acids and 3-myristoxymyristate. LPS and lipid A may be obtained from commercial sources, e.g., from Sigma. However, by way of example, but not by way of limitation, LPS may be synthesized as follows: hydroxy acids and disaccharides are condensed followed by addition of saturated fatty acids. The hydroxy fatty acids may come from acetyl CoA whereas CMP-KDO may serve as the source of the second additional units. After the addition of saturated fatty acids, sugars are added from nucleotide diphosphate derivatives.

The O-antigen is may be synthesized in three stages. For example, but not by way of limitation, the oligosaccharide units are transferred from nucleotide diphosphate carriers to a galactose attached to another lipid carrier. The oligosaccharide units are then polymerized and lipid carriers are released in the process. Finally the complete O-antigen is transferred to the R core with the release of an isoprenoid carrier.

The polysaccharide unit may also be synthesized with donor saccharide moieties and acceptor moieties which are commercially available and/or may be synthesized through organic synthesis applying techniques known in the art. Activated saccharides generally consist of uridine or guanosine diphosphate and cytidine monophosphate derivatives of the saccharides in which the nucleoside mono and diphosphate serves as a leaving group. Thus, the activated saccharide may be a saccharide-UDP, a saccharide-GDP, or a saccharide-CMP. Nucleoside monophosphates are commercially available, may be prepared from known sources such as digested yeast RNA (see e.g., Leucks et al,. 1979, J. Am. Soc. 101:5829), or routinely prepared using known chemical synthetic techniques (see e.g., Heidlas et al., 1992, Ace. Chem. Res. 25:307; Kochetkov et al., 1973, Adv. Carbohydr. Chem. Biochem. 28:307). These nucleoside monophosphates may then be routinely transformed into nucleoside diphosphates by kinase treatment. For review, see Wong et al., 1994, Enzymes in Synthetic Organic Chemistry, Pergamon Press, Volume 12, pp. 256-264.

Glycosyltransferase enzymes for synthesizing the compositions of the invention can be obtained commercially or may be derived from biological fluids, tissue or cell cultures. Such biological sources include, but are not limited to, pig serum and bovine milk. Glycosyltransferases that catalyze specific glycosidic linkages may routinely be isolated and prepared as described in International Patent Publication No. WO 93/13198 (published Jul. 8, 1993). Alternatively, the glycosyltransferases can be produced through recombinant or synthetic techniques known in the art (For review, see Wong et al., 1994, Enzymes in Synthetic Organic Chemistry, Pergamon Press, Volume 12, pp. 275-279).

The compositions of the invention are preferably synthesized using enzymatic processes (see e.g., U.S. Pat. No. 5,189,674, and International Patent Publication No. 91/16449, published Oct. 31, 1991). Briefly, a glycosyltransferase is contacted with an appropriate.

The compositions of the invention are preferably synthesized using enzymatic processes (see e.g., U.S. patent application Ser. No. 5,189,674, and International Patent Publication No. 91/15449, published Oct. 31, 1991). Briefly, a glycosyltransferase is contacted with an appropriate activated saccharide and an appropriate acceptor molecule under conditions effective to transfer and covalently bond the saccharide to the acceptor molecule. Conditions of time, temperature, and pH appropriate and optimal for a particular saccharide unit transfer can be determined through routine testing, generally, physiological conditions will be acceptable. Certain co reagents may also be desirable; for example, it may be more effective to contact the glycosyltransferase with the activated sugar and the acceptor molecule in the presence of a divalent cation.

Optionally, an apparatus as described by U.S. Pat. No. 5,288,637, is used to prepare such compositions.

While glycosyltransferases are highly stereospecific and substrate-specific, minor chemical modifications are tolerated on both the donor and acceptor components. Accordingly, the oligosaccharide components of the invention may be synthesized using acceptor and/or donor components that have been modified so as not to interfere with enzymatic formation of the desired glycosidic linkage. The ability of such a modification not to interfere with the desired glycosidic linkage may routinely be determined using techniques and bioassays known in the art, such as, for example, labeling the carbohydrate moiety of the activated sugar donor, contacting the acceptor and donor moieties with the glycosyltransferase specific for forming the glycosidic linkage between the donor and acceptor moieties, and determining whether the label is incorporated into the molecule containing the acceptor moiety.

5.3.10 LPS and Lipid A Analogs and Derivatives

Also included within the scope of the present invention are LPS and lipid A molecules which are differentially modified during or after synthesis, or after isolation from bacteria, e.g., to reduce their pyrogenicity. In specific embodiments, the LPS and lipid A molecules are treated by alkaline hydrolysis or acyloxyacyl hydrolase. Any of numerous chemical modifications may be carried out by known techniques, such as acylation, deacylation, formylation, oxidation, reduction, etc.

It is also within the scope of this invention, to synthesize analogs of lipid a having one or mere acyloxyacyl groups removed. Lipid A, either chemically synthesized or isolated from a gram negative microorganism may be treated with acyloxyacyl hydrolase in order to achieve or enhance the non-pyrogenic properties of the preparation. Acyloxyacyl hydrolase hydrolyzes the ester bonds between non-hydroxylated fatty acids and the 3-hydroxy functions of 3-hydroxy fatty acids bound in ester or amide linkages to glucosamine disaccharide of lipid A.

It is further within the scope of this invention, to synthesize analogs of lipid A and LPS having one or more non-hydroxylated fatty acids removed. Lipid A or LPS either chemically synthesized or isolated from a gram negative microorganism may be deacylated in order to achieve or enhance the substantially reduced or absent pyrogenicity of the preparation.

5.3.11 Measurement of Biological Activity of an LPS Antagonist

Whether an LPS or lipid A preparation is an LPS antagonist and is devoid of pyrogenic activity can be assessed using any of a number of methods.

Various tests can be used to demonstrate that a compound is an LPS antagonist (see Examples section herein). For example, various amounts of the test compound can be incubated with a cell having LPS receptors and wildtype LPS, e.g., LPS from E. coli W3110, in conditions under which, but for the presence of the test compound, the wildtype LPS binds to the LPS receptor and induces LPS biological activities. One or more biological activities of LPS are then monitored and compared to those obtained when cells and wildtype LPS are incubated in the absence of the test compound. Optionally, the test comprises an LPS binding protein. The presence of a decrease in one or more biological activities of wildtype LPS in the presence of the compound relative to the absence of the compound indicates that the test compound is an LPS antagonist. A similar test in which the effect of a test compound on at least one biological activity of LPS is determined can also be performed in vivo, e.g., in a test animal such as a mouse. For example, the blood level of TNF can be measured in mice to which wildtype LPS and one of several doses of a test compound is administered (as described, e.g., in U.S. Pat. No. 5,158,939).

Another test that can be used to determine whether a compound is an LPS antagonist comprises contacting an LPS receptor, present on a cell surface or in a soluble form, with wildtype LPS in the presence of various amounts of the test compound. The extent of binding of the wildtype LPS to the soluble receptor can be detected, e.g., by labeling the LPS and/or the receptor, and by measuring the amount of label bound to either the receptor, the LPS, or both. LPS can be labeled with ³ H-acetate as described, e.g., in Munford et al. (1992) J. Immunol. Methods 148: 115 and can have a specific activity of at least 106 dpm/.mu.g.

In a specific embodiment, for determining the LPS antagonistic activity of a test compound, the assay comprises a cell which has been modified, e.g, transfected, to express an LPS receptor, such as CD14. CD14 cDNA can be cloned in the expression vector, e.g., pRc/RSV (Invitrogen, San Diego, Calif.) as previously described by Lee et al., J. Exp. Med., 175:1697 1705 (1992). CD14 expression can be detected by flow microfluorometry using FITC-MY-4 (Coulter, Hialeah, Fla.) and cells expressing CD 14 were selected by fluorescence-activated cell sorting with FITC-MY-4.

The pyrogenic activity of an LPS or lipid A can be determined by measuring, e.g., the production of pyrogenic cytokines by cells contacted with the LPS or lipid A preparation, e.g., by ELISA, as further described herein.

In another illustrative embodiment, a peripheral blood mononuclear cell (PBMC) activation assay can be used to assess the pyrogenic and LPS antagonist activities of an LPS antagonist preparation. These methods are well documented (Theofan, et al., 1994, J. Immunol., 152:3623; Fagan, et al., 1994, J. Immunol., 153:5230; Verhasselt, et al., 1997, J. Immunol., 158:2919; Colotta, et al., 1992, J. Immunol., 148:760; Mackensen, et al., 1992, Eur. Cyto. Net., 3:571; Eggesbo, et al., 1994, Cytokine, 6:521; Hone, et al., 1998, supra). Briefly, human PBMCs are isolated from whole blood and suspended at a density of 5x10⁵ per ml in complete medium (CM; RPMI containing 10 .mu.g/ml of pyruvate and glutamine, 100 .mu.g/ml of penicillin and streptomycin, and 10% (v/v) endotoxin-free human AB serum (Life Technologies)). The PBMCs are then placed into 48 well flat-bottom culture plates (Costar), and stimulated with an LPS antagonist preparation (to measure the pyrogenic activity) or mixtures of an LPS antagonist and Re LPS (Sigma) to measure LPS antagonist activity, as described (Kovach, et al., 1990, supra; Golenbock, et al., 1991, supra; Golenbock, et al., 1988, supra; Kitchens, et al., 1992, supra; Kitchens and Munford, 1995, supra; Munford and Hunter, 1992, supra; Rietschel, et al., 1994, supra; Ulmer, et al., 1992, supra; Ulmer, et al., 1992, supra; Wang, et al., 1990, supra; Wang, et al., 1991, supra; Qureshi, et al., 1991, supra; Qureshi, et al., 1991, supra; Zuckerman and Qureshi, 1992; Hone, et al., 1998, supra). In positive control wells the PBMCs are stimulated with comparable doses of E. coli Re LPS (Sigma) in place of the LPS antagonist and in negative wells the PBMCs remain unstimulated. Culture supernatants are collected 8, 24 or 48 hours after addition of the LPS and TNF-.alpha. IL-1 .beta. and/or other cytokine levels in the culture supernatants are quantitated by commercially available capture ELISAs (R & D Systems).

The efficacy of the adjuvant to enhance an immune response against an antigen can be determined, e.g., by examining the presence and/or the extent of a humoral (antibody) response and/or cell mediated immunity. Assays for measuring humoral and cell mediated responses are known in the art and are also described in Example 3 and, e.g., in PCT/US98/26291 (WO 99/29728). For example, a composition of the present invention containing an LPS antagonist may be tested in mice for the ability to enhance an antibody response to an antigen and the delayed-type hypersensitivity (DTH) response, measured by an increase in footpad swelling after inoculation in the footpad of the test animal, as compared to the measurements in an animal to which the same composition without the LPS antagonist was administered. Each animal in the test group may receive the amount of antigen combined with different amounts of the LPS antagonist. Serum samples are then obtained from each animal after the final inoculation, and the serum is analyzed for the presence of antibodies against the antigen using methods known in the art, e.g., ELISA. DTH responses to the antigen can be measured after the final inoculation (e.g., within 1-7 days). An increase in serum antibodies against the antigen and/or an increase in footpad swelling in the animals having received the antigen together with an LPS antagonist relative to the responses in animals having received the antigen alone indicates that the LPS antagonist is an adjuvant.

5.4 Preparations Comprising a Vaccine Antigen and a Pharmaceutically Active Amount of an LPS Antagonist

5.4.1 Formulation

To formulate preparations comprising a vaccine antigen (for example, a polysaccharide, protein or nucleic acid vaccine) and an LPS antagonist, the optimal dose of each component must first be ascertained. By way of illustration, the optimal dose of vaccine and an LPS antagonist can be determined using a "checkerboard" approach, wherein the dose of LPS and vaccine antigen are varied over a broad range and the optimal dose of each determined by identifying the combination that results in maximal stimulation of host immunity (See Examples herein).

These preparations can be administered by any one of a number of well-known routes of vaccination, including but not restricted to the intradermal, transdermal, topical, subcutaneous, intramuscular, intraperitoneal, intranasal, parenteral (e.g., intravenous) or oral routes. To determine the immunogenicity of the antigen in the absence of an LPS antagonist, groups of laboratory animals are vaccinated with the individual doses of the vaccine antigen formulated alone. The development of humoral and cell-mediated immune responses against the vaccine antigen are measured using standard immunological techniques before vaccination and on days 10, 20, and 30 after vaccination, as described (Coligan, et al., (Eds.) 1994, In: Current Protocols in Immunology, John Wiley and Sons Inc, NY).

The LPS antagonist and the antigen are preferably administered simultaneously as a single composition. The LPS antagonist or antigen can, however, also be administered separately, e.g., consequentially, provided that separate administration results in an increased immune response against the antigen relative to an immune response resulting from administration of the antigen without the LPS antagonist.

The exact amount of such LPS antagonists required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein and optimization procedures known in the art. Generally, dosage will approximate that which is typical for suitable LPS antagonist activity and activation of target cells (generally in the ng/kg range), preferably in the range of about 0.0001 mg/patient to 600 mg/patient. More preferable ranges are about 0.001 mg/patient to 350mg/patient. Most preferable ranges are from 0.01 to 100 mg/patient. One skilled in the art, however, could readily elucidate other dosage ranges and regimens and the above are expressly intending to be non limiting.

5.4.2 Vaccine Antigens

As mentioned above, it is envisaged that preparations containing an LPS antagonist can be combined with any vaccine antigen. For the purposes of this invention, the vaccine antigen may comprise, e.g., a polysaccharide, amino acid, peptide, nucleic acid (e.g., DNA or RNA or derivative thereof), or combinations thereof.

In instances in which the vaccine antigen is composed of DNA, the DNA may be either closed circular or linear. The DNA may be derived from a commercially available plasmid (e.g., pCEP4 or pRc/RSV obtained from Invitrogen Corporation (San Diego, Calif.), pXTI, pSG5, pPbac or pMbac obtained from Stratagene (La Jolla, Calif.), pPUR or pMAM obtained from Clontech (Palo Alto, Calif.), or pSV-.beta.-gal obtained from Promega Corporation (Madison, Wis.)), or synthesized either de novo or by adaptation of a publicly or commercially available eukaryotic expression system. The DNA may encode a eukaryotic expression cassette, consisting of a promoter (e.g., the human actin promoter (Morishita, et al., 1991, Biochim. Biophys. Acta, 1090:2216), the HLA class I promoter (Koller and Orr, 1985, J. Immunol., 134:2727), the CMV intermediate early promoter (Thomsen, et al., 1984, Proc. Natl. Acad. Sci. USA, 81:659; Rotondaro, et al., 1996, Gene, 168:195), the SV40 early region and late promoters (Fromm and Berg, 1982, J. Mol. Appl. Genet., 1:457; May, et al., 1987, Nucl. Acids Res., 15:2445; Huang, et al., 1990, Genes Dev., 4:287); sequences encoding at least one gene from a viral, bacterial or parasitic pathogen as described below; transcriptional and translational enhancer sequences (See e.g., May, et al., 1987, supra; Banedji, et al., 1981, Cell, 27:299; Lewin (Ed.), 1998, In: Genes, John Wiley and Sons, NY); and sequences encoding poly-adenosine (Lewin, 1998, supra).

In instances in which the vaccine antigen is composed of RNA, the RNA may contain sequences encoding at least one gene from a viral, bacterial or parasitic pathogen; translational enhancer sequences (e.g., 5' cap translation enhancer (Lewin (Ed.), 1998, supra); a cap independent translation enhancer (CITE) sequence, such as those derived from encephalomyocarditis virus (Duke, et al., 1992, J. Virol., 66:1602); and sequences encoding poly adenosine (Lewin, 1998, supra). Alternatively, the RNA may be composed of an RNA molecule encoding a recombinant Semiliki forest virus vector (Berglund, et al., 1999, Vaccine, 17:497) that expresses a passenger viral, bacterial or parasitic gene; or a recombinant VEE virus vector (Pushko, et al., 1997, Virology, 239:389) that expresses a passenger viral, bacterial or parasitic gene.

The vaccine antigen may be derived from any viral, bacterial or parasitic pathogen which is pathogenic for a human, domestic animal or wild animal, and may be any molecule that is expressed by any viral, bacterial, or parasitic pathogen prior to or during entry into, colonization of, or replication in its host. The vaccine antigen may be given alone or in combination with one or more viral, bacterial, or parasitic antigens.

Alternatively, the vaccine antigen may be a tumor-, transplantation-, or autoimmune specific antigen. These latter vaccine antigens may be given alone or in combination with one or more tumor-, transplantation-, or autoimmune-specific antigens.

The viral pathogens, from which the viral antigens are derived, include, but are not limited to, Orthomyxoviruses, such as influenza virus; Retroviruses, such as RSV, Herpesviruses, such as EBV; CMV or herpes simplex virus; Lentiviruses, such as human immunodeficiency virus; Rhabdoviruses, such as rabies; Picomoviruses, such as poliovirus; Poxviruses, such as vaccinia; Rotavirus; and Parvoviruses.

Examples of vaccine antigens of viral pathogens include the human immunodeficiency virus antigens Nef; p24; the Env proteins gp 120, gp 160, and gp41; Tat; Rev; Gag; Vif; and Pol (Hahn, et al., 1985, Nature, 313:277-280) and T cell and B cell epitopes of gp120 (Palker, et al., 1989, J. Immunol., 142:3612-3619). Other vaccine antigens include the hepatitis B surface antigen (Wu, et al.: 1.989, Proc. Natl. Acad. Sci., USA, 86:4726-4730); rotavirus antigens, such as VP4 (Mackow, et al., 1990, Proc. Natl. Acad. Sci., USA, 87:518-522) and VP7 (Green, et al., 1988, J. Virol. 62:1819-1823), influenza virus antigens such as hemagglutinin or nucleoprotein (Robinson, et al., supra, Webster, et al., supra) and herpes simplex virus thymidine kinase (Whitley, et al., In: New Generation Vaccines, p.825-854).

The bacterial pathogens, from which the bacterial antigens are derived, include but are not limited to, Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp., and Borellia burgdorferi.

Examples of vaccine antigens of bacterial pathogens include the capsular polysaccharide of Neisseria meningitis (Goldblatt, 1998, Med. Microbiol., 47:563; Nieminen, et al., 1998. Vaccine, 16:630; Rennels, et al., 1998, Pediatrics, 101:604), the Vi polysaccharide of Salmonella enterica serovar typhi (Robbins and Robbins, 1984, J. Infect. Dis., 150:436; Klugman, et al., 1996, Vaccine, 14:435; Teddy, et al., 1999, Vaccine, 17:110), Shigella sonnei form 1 antigen (Formal, et al., 1981, Infect. Immun., 34:746-750); the O-antigen of V. cholerae Inaba strain 569B (Forrest, et al., 1989, J. Infect. Dis., 159:145-146); cholera toxin of V. cholerae (Finkelstein, 1992, In D. Barua and E. B. Greenough III (Eds.) Current Topics in Infectious Disease: Cholera. Plenum Pub. Co NY); TCP of V. cholerae (Finkelstein, 1992, supra); CFA/I fimbrial antigen of enterotoxigenic E. coli (Yamamoto, et al., 1985, Infect. Immun, 50:925-928); the heat-labile toxin of E. coli (LT; Clements, et al., 1984, 46:564-569); pertactin of Bordetella pertussis (Roberts, et al., 1992, Vaccine, 10:43-48); adenylate cyclase-hemolysin of B. pertussis (Guiso, et al., 1991, Micro. Path., 11:423-431); fragment C of tetanus toxin of Clostridium tetani (Fairweather, et al., 1990, Infect. Immun., 58:1323-1326).

The parasitic pathogens, from which the parasitic antigens are derived, include but are not limited to, Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Entamoeba spp., Eimeria spp., Leishmania spp., Schistosome spp., Brugia spp., Fascida spp., Dirofalaria spp., Wuchereria spp., and Onchocerea spp.

Examples of vaccine antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp. (Sadoff, et al., 1988, Science; 240:336-337), such as the circumsporozoite antigen of P. besghei or the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp. (Spetzler, et al., 1994, lnt. J. Pept. Prot. Res., 43:351-358); the galactose specific lectin of Fntamoeba histolylica (Mann, et al., 1991, Proc. Natl. Acad. Sci., USA, 88:3248-3252), gp63 of Leishrnnia spp. (Russell, et al., 1988, J. Immunol., 140:1274-1278), paramyosin of Brugio rnalayi (Li, et al., 1991, Mol. Biochem. Parasitol., 49:315-323), the triose-phosphate isomerase of Sclristosorna rnansoni (Shoemaker, et al., 1992, Proc. Natl. Acad. Sci., USA, 89:1842-1846); the secreted, lobin-like protein of Trichostrongylus colubrqjcrmis (Frenkel, et al., 1992, Mol. Biochem. Parasitol., 50:2 7-36); the glutathione-S-transferase of F'rasciola hepatica (Hillyer, et al., 1992, Exp. Parasito:., 7 5:176-186), Schistosoma bovis and S. japonicum (Bashir, et al., 1994, Trop. Geog. 1led 46:255-258); and KLH of Schisiosoma bovis and S. japonicum (Bashir, et al., 1994, supra).

Examples of tumor specific antigens include but are not restricted to prostate specific antigen (Gattuso, et al., 1995, Human Pathol., 26:123), TAG-72 and carcinoembrionic antigen (CEA) (Kris, et al., 1999, Cancer Res., 59:676; Guadagni, et al., 1994, Int. J. Biol. Markers, 9:53), MAGE-1 and tyrosinase (Coulie, et al., 1993, J. Immunother., 14:104); mutant p53 antigen (Mayordomo, et al., 1996, J. Exp. Med., 183:1357). Recently it has been shown in mice that immunization with non-malignant cells expressing a tumor antigen provides a vaccine effect by inducing an immune response that clears malignant tumor cells displaying the same antigen (Koeppen, et al., 1993, Anal. N.Y. Acad. Sci., 690:244).

Examples of transplant antigens include the CD3 receptor on T cells (Alegre, et al., 1995, Digest. Dis. Sci., 40:58-64). Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and reverse most rejection episodes (Alegre, et al., 1995, supra).

Examples of autoimmune antigens include IAS .beta. chain (Topham, et al., 1994, Proc. Natl. Acad. Sci., USA, 91:8005-8009). Vaccination of mice with an 18 amino acid peptide from IAS .beta. chain has been demonstrated to provide protection and treatment to mice with experimental autoimmune encephalomyelitis (Topham, et al., 1994, supra).

Alternatively, the present invention allows for the inclusion of immune-stimulatory molecules in the above described preparations, such as growth factors and cytokines. These immunostimulatory molecules include, but are not limited to, growth factors, such as M-CSF, UM-CSF and cytokines, such as IL-2, IL-4, IL-5, IL-6. IL-10, IL-12 or IFN-.gamma. (Nossal, 1999, supra; Vogel and Powell, 1995, supra; Nash, et al., 1993, supra; Pardoll, 1995, supra; Kurane, et al., 1997, supra; Tagliabue and Boraschi, 1993, supra; Lofthouse, et al., 1995, supra; Pasquini, et al., 1997, supra; Jankovic, et al., 1997, supra).

5.4.3 Combination Therapy

According to a specific embodiment of the present invention, an LP S antagonist, may optionally be used in combination with other therapeutic agents to enhance the antiviral effect achieved.

In an exemplary embodiment, an LPS antagonist is used in combination with an antiviral agent. Such antiviral agents which may be used with a preparation of an LPS antagonist include but are not limited to those which function on a different target molecule involved in viral replication, e.g., reverse transcriptase inhibitors (e.g., azidothymidine (AZT), lantivudine (3TC), deoxyinosine (ddI), and dideoxycytidine (ddC)), viral protease inhibitors, glycosylation inhibitors; those which act on a different target molecule involved with viral transmission; those which act on a different loci of the same molecule; and those which prevent or reduce the occurrence of viral resistance. One skilled in the respective art would know of a wide variety of antiviral therapies which exhibit the above modes of activity for a given virus.

An LPS antagonist can also be used in combination with retrovirus inhibitors, such as nucleoside derivatives. Nucleoside derivatives are modified forms of purine and pyrimidine nucleosides which are the building blocks of RNA and DNA. Many of the nucleoside derivatives under study as potential anti-HIV medications, for example, result in premature termination of viral DNA replication before the entire genome has been transcribed. These derivatives lack 3' substituents that can bind to subsequent nucleosides and result in chain termination. Nucleoside derivatives such as 3' azido-3'-thymidine (AZT) and dideoxyinosine (ddI) have been exploited as inhibitors of HIV-1 replication, both in vitro and in vivo. Nucleoside analogs are currently the only licensed therapeutics for the treatment of HIV infection and AIDS (Fischl, et al., 1987, N. Engl. J. Med., 317:185; Mitsuya and Broder, 1987, Nature, 325:773). This class of compounds works by inhibiting reverse transcriptase resulting in a block in cDNA synthesis (Mitsuya and Broder, 1987, supra), these inhibitors work early in the infectious cycle of HIV-1 and inhibit integration into T-cell genome.

Further, a preparation of reduced or absent pyrogenicity of LPS or lipid A can be used in combination with nucleoside derivatives which include but are not limited to, 2',3' dideoxyadenosine (ddA); 2',3'-dideoxyguanosine (ddG); 2',3'-dideoxyinosine (ddI); 2',3' dideoxycytidine (ddC); 2',3'-dideoxythymidine (ddT); 2',3'-dideoxy-dideoxythymidine (d4T) and 3'-azido-2',3'-dideoxythymidine (AZT). Alternatively, halogenated nucleoside derivatives may be used, preferably 2',3'-dideoxy-2'-fluoronucleosides including, but not limited to, 2',3'-dideoxy 2'-fluoroadenosine; 2',3'-dideoxy-2'-fluoroinosine; 2',3'-dideoxy-2'-fluorothymidine: 2',3'-dideoxy 2'-fluorocytosine; and 2',3'-dideoxy-2',3'-didehydro-2'-fluoronucleosides including, but not limited to 2',3'-dideoxy-2',3'-didehydro-2'-fluorothymidine (Fd4T). Preferably, the 2',3'-dideoxy-2' fluoronucleosides of the invention are those in which the fluorine linkage is in the beta configuration, including, but not limited to, 2'3'-dideoxy-2'-beta-fluoroadenosine (F-ddA), 2',3' dideoxy-2'-beta-fluoroinosine (F-ddI), and 2',3'-dideoxy-2'-beta-fluorocytosine (F-ddC). Such combinations allow one to use a lower dose of the nucleoside derivative thus reducing the toxicity associated with that agent, without loss of antiviral activity because of the use of the LPS antagonist. Moreover, such a combination reduces or avoids viral resistance.

According to the present invention, preparations of LPS antagonist can also be used in combination with uridine phosphorylase inhibitors, including but not limited to acyclouridine compounds, including benzylacyclouridine (BAU); benzyloxybenzylacyclouridine (BBAU); aminomethyl-benzylacyclouridine (AMBAU); aminomethyl-benzyloxybenzylacyclouridine (AMB-BAU); hydroxymethyl-benzylacyclouridine (HMBAU); and hydroxymethyl benzyloxybenzylacyclouridine (HMBBAU).

According to the present invention, preparations of LPS antagonist can be used in combination with viral protease inhibitors, including but not limited to, Invirase (saquinavir, Roche), ABT-538 (Abbott, CAS Reg. No. 155213-67-5), AG1343 (Burroughs Wellcome/Glaxo, CAS Reg. No. 161814-49-9). Protease inhibitors are generally thought to work primarily during or after assembly (i.e., viral budding) to inhibit maturation of virions to a mature infectious state. For example, ABT-538 has been shown to have potent antiviral activity in vitro and favorable pharmokinetic and safety profiles in vivo (Ho, et al., 1995, Nature, 373:123). Administration of ABT-538 to AIDS patients causes plasma HIV-1 levels to decrease exponentially and CD4 lymphocyte counts to rise substantially. The exponential decline in plasma viraemia following ABT-538 treatment reflects both the clearance of free virions and the loss of HIV-1 producing cells as the drug substantially blocks new rounds of infection. ABT-538 treatment reduces virus mediated destruction of CD4 lymphocytes. Combining this treatment with a preparation of LPS antagonist, which inhibits at an earlier stage of HIV infection, viral fusion, would be likely to have synergistic effects and have a dramatic clinical impact.

In order to evaluate potential therapeutic efficacy of U IS antagonist in combination with the antiviral therapeutics described above, these combinations may be tested for antiviral activity according to methods known in the art.

A compound of the invention can be administered to a human patient by itself or in pharmaceutical compositions where it is mixed with suitable carriers or excipients at doses to treat or ameliorate various conditions involving viral infection. A therapeutically effective dose further refers to that amount of the compound sufficient to inhibit viral infection. Therapeutically effective doses may be administered alone or as adjunctive therapy in combination with other treatments for viral infection or associated diseases. Techniques for the formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, Pa., latest addition.

In another embodiment, viral infection is treated or prevented by administration of a Therapeutic of the invention in combination with one or more chemokines. In particular, the Therapeutic is administered with one or more C-C type chemokines, especially one or more from the group consisting of RANTES, MIP-1.alpha. and MIP-1.beta. (see, e.g, WO 99/2978 by Gallo et al.).

5.4.4 Monitoring Effects of an LPS Antagonist During Clinical Trials

The treatment of an individual with an LPS antagonist can be monitored by determining LPS antagonist characteristics, such as LPS antagonist level or activity (e.g., fever, IL-1 and/or IL-6 production, acute phase reactant production), IL-1, TNF .alpha. and/or IL-6 mRNA levels, and/or transcription levels. Clinical tests useful for testing the efficacy of the treatment are well known in the art and include ELISA, Northern blot, RT-PCR, etc. These measurements indicates whether the treatment is effective or whether it should be adjusted or optimized.

In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a preadministrarion sample from a subject prior to administration of the LPS antagonist; (ii) detecting the LPS antagonist or an activity thereof in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of the LPS antagonist or an activity thereof in the post-administration samples; (v) comparing the level of the LPS antagonist or activity thereof in the preadministration sample with that in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the level of LPS antagonizing activity or to increase the immune response to a co-administered antigen, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease LPS antagonist activity, i.e., to decrease the effectiveness of the agent.

Cells of a subject may also be obtained before and after administration of an LPS antagonist to detect the level of expression of genes that respond to an LPS antagonist, to verify that the LPS antagonist does not increase or decrease the expression of genes which could be from cells exposed in vivo to an LPS antagonist and mRNA from the same type of cells that were not exposed to the LPS antagonist could be reverse transcribed and hybridized to a chip containing DNA from numerous genes, to thereby compare the expression of genes in cells treated and not treated with an LPS antagonist. If, for example, an LPS antagonist turns on the expression of a proto-oncogene in an individual, use of this particular LPS antagonist may be undesirable.

5.4.5 Kits

The invention further provides kits for administration of an adjuvant of the invention with a vaccine antigen to a subject and/or for monitoring the level of adjuvant or its toxicity in a subject.

In one embodiment, the kit comprises an LPS antagonist. The kit can further comprise a vaccine antigen. The kit can also comprise an agent for detecting the LPS antagonist, such as an antibody. In another embodiment, the kit comprises an agent which detects an activity of an LPS antagonist, such as the level of a cytokine, e.g, TNF-.alpha., IL1 or IL-6. A kit may also contain standard solutions or graphics (e.g., charts), including positive and negative controls.

A kit of the invention may also provide reagents for detecting ablionnal levels, form or activity of an LPS antagonist, or a breakdown product of an LPS antagonist. In an embodiment of the invention the kit detects autoantibodies specific for an LPS antagonist. These reagents may be labeled.

The components of the kit can be packaged in a suitable container. The kit can further comprise instructions for using the reagents of the kit.

5.4.6 Methods of Treatment

The methods and compositions of the invention may be used as a vaccine in a subject in which immunity for the antigen(s) is desired. Such antigens can be any antigen known in the art to be useful in a vaccine formulation. The methods and compositions of the present invention can be used to enhance the efficacy of any vaccine known in the art. The vaccine of the present invention may be used to enhance an immune response to infectious agents and diseased or abnormal cells, such as, but not limited to, bacteria, parasites, fungi, viruses, tumors, and cancers. The compositions of the invention may be used to either treat or prevent a disease or disorder amenable to treatment or prevention by generating an immune response to the antigen provided in the composition. In one preferred embodiment, the antigen(s) are proteins, fragments or derivatives, including truncation isoforms thereof, encoded by any genes of the HIV genome including the env, gag, pol, nef, vif, rev, and tat genes. In a more preferred embodiment, the antigen is an HIV-associated gp120 protein.

Diseases and disorders that can be treated according to the invention include viral infections, such as an infection by HIV, CMB, hepatitis, herpes virus, measles virus; bacterial infections; fungal and parasitic infections; cancers; autoimmune diseases; and any other disease or disorder amenable to treatment or prevention by eliciting an immune response against a particular antigen or antigens.

The methods and compositions of the present invention may be used to elicit a humoral and/or a cell-mediated response against the antigen(s) of the vaccine in a subject. In one specific embodiment, the methods and compositions elicit a humoral response against the administered antigen in a subject. In another specific embodiment, the methods and compositions elicit a cell med;;zted response against the administered antigen in subject. Preferably both a humoral and a cell-mediated response is triggered.

The subjects to which the present invention is applicable may be any mammalian or vertebrate species, which include, but are not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, monkeys, rabbits, chimpanzees, and humans. In a preferred embodiment, the subject is a human.

5.4.7 Effective Dose

Toxicity and therapeutic efficacy of the LPS antagonist can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀. LPS antagonists which exhibit large therapeutic indices are preferred. While LPS antagonists that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

5.4.8 Formulation and Use

Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, eye drops, injection, inhalation or insufflation either through the mouth or the nose, or oral, buccal, parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Rentmington's Pharmaceutical Sciences. Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. The agent can be alternatively administered intravascularly, such as intravenously (IV) or intraarterially.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

For example, for solid compositions, conventional nontoxic solid carriers include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administratable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as described herein and optional pharmaceutical adjuvants is an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences (latest edition).

The LPS antagonist may be administered alone or in combination with other molecules known to have a beneficial effect, such as to enhance the immune response to an antigen (e.g., adjuvant activity), molecules capable of tissue repair and regeneration and/or inhibiting inflammation. Examples of useful cofactors include chemokines (see WO 99/29728). Other useful cofactors include symptom-alleviating cofactors, including antiseptics, antibiotics, antiviral and antifungal agents and analgesics and anesthetics. In addition, substances that enhance the stability and/or activity of the LPS antagonist may be co-administered, either together or sequentially, with the LPS antagonist. In yet another embodiment, the co-factor is an antagonist to any deleterious activity or side-effect of the LPS antagonist.

The LPS antagonist also may be associated with means for targeting the LPS antagonist to a desired tissue. For example, an antibody or other binding protein that interacts specifically with a surface molecule on the desired target tissue cells may be used. Such targeting molecules further may be covalently associated to the LPS antagonist, e.g., by chemical crosslinking, or by using standard genetic engineering means to create, for example, an acid labile bond such as an Asp-Pro linkage. Useful targeting molecules may be designed, for example, using the simple chain binding site technology disclosed, for example, in U.S. Pat. No. 5,091,513.

Preparations, e.g., those for oral administration, may be suitably formulated to give controlled release of the active compound and can be embedded in a slow release matrix for that purpose. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Other suitable delivery systems include microspheres which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology utilizes microspheres of precapillary size which can be injected via. a coronary catheter into any selected part of the body, without causing inflammation or ischemia. The administered therapeutic is slowly released from these microspheres and taken up by surrounding tissue cells.

Systemic administration can also be by transmacosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used ire the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

In clinical settings, a gene delivery system for the gene encoding a therapeutic or vaccine antigen can be introduced into a patient in conjunction with the LPS antagonist by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter or by stereotactic injection.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient and an LPS antagonist. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The practice of the present invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

Claim 1 of 55 Claims

What is claimed is:

1. A vaccine preparation comprising

(i) a pharmaceutically effective amount of a substantially pure LPS antagonist isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes, or an analog or derivative thereof, wherein the LPS antagonist has reduced pyrogenicity relative to an LPS antagonist isolated from the wildtype bacterium,

(ii) a vaccine antigen, which is not isolated from the gram negative bacterium, and

(iii) a pharmaceutically acceptable carrier.

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