CA2064194A1 - Lipid a analog/immunogenic carrier conjugates and the use thereof as vaccines - Google Patents

Lipid a analog/immunogenic carrier conjugates and the use thereof as vaccines

Info

Publication number
CA2064194A1
CA2064194A1 CA002064194A CA2064194A CA2064194A1 CA 2064194 A1 CA2064194 A1 CA 2064194A1 CA 002064194 A CA002064194 A CA 002064194A CA 2064194 A CA2064194 A CA 2064194A CA 2064194 A1 CA2064194 A1 CA 2064194A1
Authority
CA
Canada
Prior art keywords
lipid
analog
conjugate
acyl groups
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002064194A
Other languages
French (fr)
Inventor
Nelson Teng
Jerald Sadoff
Apurba K. Bhattacharjee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Univax Biologics Inc
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of CA2064194A1 publication Critical patent/CA2064194A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Saccharide Compounds (AREA)

Abstract

The invention relates to a lipid A analog/immunogenic carrier conjugates and the use thereof as vaccines for the prophylaxis of septic shock in animals.

Description

-~o 91/0l750 ~ PcT/US90/04087 Title of the Inven~ion LlPID A ANALO~/IMMUNOGENIC CARRIER CONJUGATES AND THE USE THEREOF
AS VACCINES

Cross Reference to Related Application This application is a continuation-in part of U.S. Application Serial No. 07/487,898 filed March 6, 1990, which is a continuation-in-part of U.S. Application Serial No. 07/385,860 filed July 27, 1989, the disclosures of which are fully incorporated by reference herein.
FIELD OF THE INVENTION:

This inYention is ;n the f;eld of pharmaceutical compositions and the use thereof as vacc;nes.

BACKGROUND OF JHE INVENTTON: -Each year approximately 1g4,000 patients in U.S. hospitalsdevelop bacteremia; of these, about 75,000 die. Maki, D.G., in ~osocomial Infe_t;ons, R.E. Dixon (ed.), pages 183-196, Yorke Medical Books, New York (1981). This high frequency of mortality occurs despite the aggressive use of potent antibiotics. The shortcomings of antibiotic therapy may be attributed to the relative impermeability of the outer membrane of Gram-negative bacteria to the drugs and to the inability of the drugs to counteract the lethal effects of bacterial endotoxin. ~ Ziegler, E.J., et al., N. Enql. J. Med. 307:1225-1230 (1982); Bogard, W.C.Jr et.al., Infect. Immun. ~5:899-908 (1987).
- One approach toward counteracting the lethal effects of lipopoly-saccharide (LPS) endotoxin is the administration of anti-LPS anti-. . , ~ , ,.

W O 91tO1750 PCT/US90/04 ~ 2-serum. Such antiserum ~ay also facil itate the removal of Gram-negative bacteria by the reticuloendothelial system. SeYeral workers have demonstra~ed successful broad-spectrum protection against challenge by several Gram-negative organisms or endotoxins in dif-ferent animal models by using roush mutants of ~J~ or S. minnesota as i~munizing agents to generate antisera. . Braude, A.I.~ et al./ J.
L 108:505-512 (1972~; Braude, A.I., et_a~ 55~ L
136:S167-S173 (1977); Dunn, D.L.7 et al., Surqerv 92:212-219 ~1982)i McCabe, ~.R., ~. ~mmunol. 1Q~:601-610 (1972); McCabe, ~.R., ~t al., ~.
Infect. Dis. 136:S161-5166 (1977); and Zie~ler, E.J., et .al., J.
Immunol. 111:433^438 (19731. These ~utants lack the O-specific polysaccharide and express on their surface portions of the LP5 core which presumably contains antigenic determinants shared by most Gram-negative organisms. Passive im~un7zation ~f patients with human antisera raised against the rough ~utant E coli J5 has been shown to be an efficacious supplement to antibiotics for the control of nosocomial ~nfections. Z~egler, E.J., et al., N. Enql. J. Med.
307;12~5-1230 (19823.
~ t is now recognized that ~ost of the biological activity of bacterial endotoxins reside in the lipid A ~oiety of the LPS ~olecule.
Luderitz, O., $~ urr. TQP. Membr. TransP. 17:79-151 (1982).
Typical Gram-negative bacterial LPS has three ~ajor structural regions: the O-polysaccharide, the R-core oligosaccharide~ and lipid A. The structure of the 0-pol~saccharide is highly variab1e between organisms, even in the same species. Its antigenicity serves as a basis for serotyp~ng the bacteria. The R region is a bridge between the O-antigen and l~p~d Ai ~ts structure ~s similar ~n ~ost 6ram-ne~ative bacteria. Antibod~es to LPS ~ay promote phagocytosis or the death of the bacteria. 7he 0-antigen is the ~ost antigenic component of the LPS, yet it has little known toxicity. Lipid A, ;n contrast, contains the toxic center of the ~olee~le and is remarkably similar in structure across 2 wide ran~e of bacterial genera.

9 1 /01 750 PCl /US90/04087 2 ~

It is believed that the lipid A region of LPS ;s responsible for a complex array of inflam~atory responses to tissue invasion by Gram^
negative bacteria by directly stimulating host cells such as macro-phages, neutrophils, and endothelial cells which mediate the inflam-~atory changes. Responses are both toxic (hypotension, coagulat;on disturbances, death) and beneficial to the infected host (enhancement of ~ntibody formation, mobilization of pha~ocytes, acute phase protein synthesis~.
~ he structure of l~pid A ~s depicted as Formula ~I) below:
~4--R
~ /~o~O ~o P- ~/ 9 ~ ,P~
V ~, NH H ~ ~.0 C 0 ¦ CLO
< ~ 0 ~.0~.0~ ~0 (1) Lip~d A 1s a glucosam~ne d;saccharide that is phosphorylated at posit~ons l and 4' and has s~x or seven esterified fatty acids. Four ~olec~les of 3-hydro%ytetradecanoate are attached to the glucosamine disacchar~de at posit~ons 2~ ~, 2'~ and 3'; the hydroxyl groups of the 3' 4 H-14:0 residues at positions 2' and 3' (and sometimes 23 are substituted with normal htty acids (dodecanoate, tetradecanoate, hexadecanoate) to form acyloxyacyl groups.
In order to gain insight into the structure-actiYity relationship of lipid A, the biological activity of chemically synthesized lipid A
analogs and biosynthetic precursors of lipid A has been examined. For example, Luderitz, 0., et al., Rev. Infect. Dis. 6:428-431 (19843, -, : : .
.

W o 91/01750 9 ~ PCT/US90/04n `-`

disclose the preparation of lipid A analogs and the relationship thereof between the chemical struc~ure and biologic activity. The authors report that phosphate substitution of the glucosamine disac-charide is not essential for the expression of antigenicity, and tha~
the amine-bound 3-hydroxyacyl residues can be replaced by non-hydroxy-lated fatty acids withou~ reduc~ion of activ~ty. The authors a1so indicat~ that t~e immunodeterminant structure comprises the 1inkaQe region of the amide-linked fatty acids and glucosamine.
6alanos, C., et al., Rev. ~nfect. D~s 6:546-552 (1984), disclose the preparation of semisynthetic lipid A i~munogens comprising glucosamine disaccharide carrying one am~de-linked ~-hydroxymyristic acid conjugated to a protein ~edestin) for immunization of rabbits.
Antibodies were produced in titers comparable to those obtained by immunization under similar condit~ons w~th acid-treated bacteria coated with lipid A.
Behling, U.H., et 21., J. Immunol. 11?:847-8sl ~lg76), disclose the preparation of synthet~c glyco7~p~d ad~uYants cumprising N-pal~itoyl-D-glucosamine, N-oleyl-D-glucosamine, N-~yristoyl-D~glucosa-~ine, N-decanoyl-D-glucosamine, and N~stearoyl-D-glucosa~ine. The authors report that comparable or superior cnhancement of ghe i~une response was obtained with the synthetic glycolipids ~n comparison to the use of LPS endotoxin in assays ~easur~ng anti-sheep red blood cell (SRBC) or gamma-globulin (HGG) ~emagglutinin t~ters. ~he authors also report that the synthetic glycolip~ds have no activity in chick embryo lethality, Shwartzman skin ~ssay, and l~mulus lysate tests which are characterist~c as well as sens~tive assays of endotoxicity. ~here-fore, the authors conclude that the aitogen~c and adjuvant effects by otherwise nonendotoxic glycolip~ts suggest that not all biologic properties of the endotoxin are related to one structural subunit`or to one structural feature of this very complex molecule.
Hodgins, D.S., PCT Application Publication No~ ~087/07297, published December 8, 1987, discloses LPS derivitives of reduced d ~ 91/01750 PCr/US90/~4087 5 2~L~Ll~

toxic~ty obtained by ~reatment of lipid A with an acyloxya hydrolase to g~ve a compou~d of the Fon~ula (II):

.~!,~, ~o~

This altered bacterial LPS is therapeutically useful as a vaccine to pn~vent 6ram-nega~ive bacterial diseases by inducing antibodies to LPS, as 3n antidote to treat or prevent 6ram-negative bacterial sepsis, or as an adjuYant to enhance fon~ation of antibodies to other antigens. The acyloxyacyl hydrolase ~tself ~ay also be prophylacti-cally or therape~t;cally useful to detox~fy endogenous ~PS in patients with Gram-ne~ative bacter~al diseases. ~his enzyme may also be used to remoYe toxic LPS from therapeutic injectants.
Despite the development of lipid A analogs as immunogens for induction of active immunity to LPS, a need continues to exist for new compounds which can be used in the treatment or prevention of sept;c shock in animals.

SUMMARY OF TH I~VENTIQN

The invention relates to a lipid A analog/immunogenic carrier conjugate, wherein said lipid A analog the following Formula (III~:

, .
' WO 91/01750 PCI~/US~0/04~
C~ ~

.
_. _ .

L ~ n (111) wherein n is 1 or 2;
Rl and R3 are the same or different and selected from the group consisting of hyd~ogen, a C2-C18 acyl group~ a 3-hydroxy C3-C18 acyl groups~ a 3-(C2-Cl2-acyloxy)-c3-clg acyl group and a linkage to an immunogenic carrier;
R2 is selected from the group consisting of C~ C1Q asyl groups, 3-hydroxy C3-Clg acyl ~roups, and 3-(C2-Cl~-acyloxy)-c3-clg acyl groups; and . - -R4 is hydrogen, a C2-CI8 acyl group, a phosphate group or a linkage to an immunogenic carrier; - . :
: with ~he proviso that one of Rl, R3 or R4 is a linkage to an im-: munogenic carrier, wherein said linkage does not interfere substan-tially with the ability of the lipid A analog to stimu1ate an i~mune response in an animal.
: ln particular, the invention relates to a lipid A analog/-immunogenic carrier conjugate having the following Formula (IY): -:~ ~

- .

- -~ .. . : : ; . :.
, ) 91/0~750 PC~ i90/040~7 2 ~

/ita3 _g~ .

~0--~A (IV) wherein n, Rl, R~, R3 and R4 are clefined above;
A is an imnunogenic carrier;
is 0 or 1;
p i~ 1 to 200;
L ~s a linker group ~h~ch does not interfere subs~antially with the characterist~c abil~ty of the l~p~d A analog to stimulate an immune response ln an animal;
Rl and R3 are the same or d1fferent and selected from the gru~p consist~ng of hydrogen, C2-Clg acyl groups, 3-hydroxy C3-C~ acyl groups, and 3-~C2-Cl2-acyloxy)-C3-Cl8 acyl groupsi R2 is selected from the group consist~ng of. C2-Clg acyl groups, 3-hydroxy C3-C1g acyl 5roups, and 3-(c2-cl2-acyloxy~-~3-~l8 acyl groups, and R4 is hydrogen, a C2-C1g acyl group or a phosphate group.
The lnvention also relates to a lipid A analog/immunogenic carrier conj~gate ha~ing the ~ollowing Formula (Y):

.

.
:

:

WO 91~1)1750 PCT/US90/040f;~

A7~/la. _~ \ ~V) ~n J
wherein n, m, p, L, Rl, R2, R3 and R4 are defined above.
The inventio~ also relates to a vaccine for the prophylaxis of septic shock comprîsin~ a lipid A analog/immunogenic carrier conjugate of the present invention together with a pharmaceutically acceptable carrier or adjuvant.
The invention also re1ates to a method for treating or preventing septic shock ~n an animal compris~ng admin~stering a pharmaceutical compos~tion to an animal ~ompr~s;ng a lipid A analog/immunogenic carrier conjugate of ~he inYention; and a phanmaceutically acceptable carrier;
~ herein said lipid A analog/~mmunogenic carrier c~njugate is present in an amount effective to induce active i~mun~ty to LPS in an animal.
The invention also relates to intermediates useful for the preparation of the lipid A analog/immunogenic carrier conjugates of the 1nvent~on.
Unexpectedly, the inventors of the present ~pplication have discovered that acyl derivatives of glucose and gentiobiose, when linked to an im0unogenic carrier, induce active immunity to LPS in ani~als. This discovery allows the ready preparation of inexpensive vaccines which may be ~sed for prophylaxis of septic shock.

.

'0 91/01750 PClr/US90/04087 20~d,~ ~
_g DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to lipid A analog/immunogenic carrier conjugates comprising glucose or a polyglucose molecule such as gentiobiose l;nked to an immunogenic carrier, wherein at least C3 posit;on of the glucose or gcnt;ob;ose mo;et;es are subst;tuted w;th an acyl group (R2~. The i~muno~enic carrièr may be linked to any free hydroxyl group on the Lipid A analog molecule, so long as the conj~gate induces active immunity to LPS when administered to an animal. Preferably, the immunogenic carrier is linked to the Cl or c6 positiQn of the glucose or polyglucose moiety.
Equivalents of the lipid A analog/immunogenic carrier conjugates of the present invention include all non-amino saccharides and polysaccharides acylated at least at the C3 position of at least one of the saccharide moieties and linked to an immunogenic carrier, wherein the conjugate induces active immunity to LPS when administered to an animal.
A lipid A analog/immuno~enic carrier conjugate of the invention is considered to induce acti~e immunity to LPS wherein when the conjugate is administered to an animal, anti-LPS antibodies are produced by the animal. The efficacy-of the anti-LPS antibodies can be tested, for example, by admin;stering the sera of an i muni~ed animal to a second animal followed by challenge of the second animal wlth LPS-producing bacteria (see Example 7, below). ~here administra-tion of the anti-LPS sera results in enhanced surviva1 of the second animal, the conjugate is considered to induce immunity to LPS.
Anti-LPS antibodies can also be tested ~y inhibition of the Shwartzman reaction, a well-known biological response to LPS (Lee, L.
et al., In: Zwe~ach, B.W. et al. (eds.) THE INFLAMMATORY PROCESS, Academic Press, NY, 1965, p. 791). Animals, particularly rabbits, actively i~munized with LPS or with one of the compositions of the present invention are given an appropriate LPS challenge to induce a localized Shwart~man reaction. Prevention or amelioration of the erythema, hemorrhage or necrotic reaction is evidence for the presence wo 9l/o175~ P~/US90/04~. ~ ~
,S ~

of anti-LPS antibodies. Alternatively9 serum fnom an animal im~unized wi~h LPS or with one of the compositions of the present invention is transferred to a naive rabbit, which is then tested for a Shwartzman reaction. Inhibition of the reaction is a measure of antibody activity.
The vaccines of the present invention are useful for the prophy-laxis of septic shock in an anima~ which is due to the release of LPS
endotoxin by Gram negative microorganisms. Such Gram negative microorganisms include, but are not limited to Sal~onella, Escherichia, HemoDhilus and Neisseria, Klebsiella, Shiqella, Pseudomonas, E~terobaeter, Acinetobacter, and ~acteroides. See Bergey's Manual of SYstematic M;crob;oloaY.
The conjugates of the invention are useful as vaccines which induce active immunity toward LPS ;n animals. Preferably, such animals are humans, however the invention is not ~ntended to be so limîting. Any animal which ~ay exper;ence the benefic;al effects o~
the ~accines of the .invention are within the scope of animals which may be treated according to the claimed invention.
In particular, the invention relates to lipid A analog/im-munogenic carrier conjugates.having the Fonmula (IV):
.

' ' / \

e~ï ,~

wherein A is an immunogenic carrier;

~ ''O 91/01750 PCT/US90/04087 9~

L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal;
Rl and R3 are selected from the group consisting of hydrogen, C2-Cl8 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy~-C3-clg acyl groups;
is 0 or 1;
n is l or 2;
p is 1 ~o 200;
R2 is selected from the group consisting of C2-Clg acyl groups, 3-hydroxy C3-Clg acyl groups, and 3-(C2-C12-acyloxy)-C3-Clg acyl groups; and R4 is hydrogen, a C2-Clp~ acyl group or a phosphate group.
The invention also relates to a lipid A analog/imTunogenic carrier conjugate having the following Formula (V~:

Af~ Fo~- ~ - (Y~

;;~P
wherein n, m, p, L, Rl, R2, R3 and R4 are defined above.
By the tenm ~immunogenic carrier~ is ~ntended any macromolecule which is capable of inducing an immunogenic reaction in an animal.
Since ~any small ~olecules such as the lipid A analogs of the inven-tion do not induce active immunity by themselves, it is necessary to conjugate the analog to an immunogenic carrier to induce production of antibodies which are specific for the small molecule. Such immunogenic carriers include, but are not 7imited to, proteins such as .

. ~

W O 91/017~0 PCT/US90/04 boYine serum albumin, diphtheria toxoid, tetanus toxo;d, edestin, keyhole-limpet hemocyan;n, Pseudomonal Toxin A, cholerageno;d, cholera toxin, pertussis toxin, viral proteins, and eukaryotic proteins such as interferons, interleukins, or tumor necrosis factor.- Such proteins may be obtained from natural or recombinant sources according to 0ethods known to those skilled in the art. When obtained from recombinant sources, the i~munogenic carrier may comprise a protein .fragment comprising at least the immunogenic portion of the molecule.
Other know~ immunogenic ~acromolecules which 0ay be used in the practice of the invention include, but are not limited to, polysac-charides, tRNA, nonmetabolizable synthetic polymers such as polyvinyl-amine, po~ymethacrylic acid polyvinylpyrrolidone, mixed polyconden-sates (with relatively high molecular weight) of 4'4'-diaminodiphenyl-~ethane-3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid ~See Sela, M., Science 166:136~-1374 (1969)~ or glycolipids, lipids or carbohydrates. Preferably, the immunogenic carrier is a protein.
By the tenm ~linker group~ is intended one or more bifunctional molecules which can be used to link the immunogenic carrier to the lipid A analog and which do not interfere with the production of anti-lipid A antibodies in v;vo. The linker group m~y be attached to any part of the glucose or gentiobiose moiety so long as the point of atta~hment does not interfere with the production of anti-lipid A
antibodies in vivo and thus interfere with the induction of active immun~ty.
Examples of linker groups which can be used to link the lipid A
analog to the in~nunogenic carrier ~ay comprise - (CH2~q-Ntl-, ~YI) wherein q is 2-~0;

-(cH2~q-NH-c-(cH2Jx C- ' (VII) '0 91/017~;0 PCr/U~i90/04087 -13~

wherein q ~ 2-5, x c ~-12; and O
-(cH2~y-c-~ (VIIII) wherein Y e 1-3.
Typica7 acyl groups which can be substituted on the carbohydrate moiety inc~ude, but are not limited to, acetate, propionate, butano-ate, pent~noate, hexanoate, heptanoate, octanoate, nonanoate, decano-ate, palmitoyl~ olelyl, ~yristoyl, stearoyl, 3-hydroxybutanoate, 3-hydroxypentanoate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxy-octanoate, 3-hydraxynonanoate, 3-hydroxydecanoate, 3-hydroxydecanoate, 3-hydroxypalmitoyl, 3-hydr~xyoleyl, 3-hydroxymyristoyl, and 3-hydroxy-stearoyl groups. Also included within the scope of R groups inolude the 3-(C2-C12 aeyloxy)-subst;tuted aforementioned C3-Cl~ acyl groups wherein the C2-C12 acyloxy groups include, but are not ~imited to, acetate, propanoate, butanoate, pentanoate, hexanoate, heptanoate, octanoate, nonanoate, decanoate, and dodecanoate groups.
Preferred lipid A analog/immunogenic carrier conjugates are derive~ from gentiobiose and have the ~ollowin~ Formula (IX):

/ t~ R ~ ~

~/R ~~~ (IX) .:

.. , --,: ~ . .. ~. :

wo 91/017SU ~, P~r/uS9O/04û-3,r~

A preferred lipid A ana~og/immunogenic carrier conjugate is gentiobiose peracetate linked at the Cl-position to an immunogenic carrier having the following Formula (X):

~O~C ~_ ~_0 / 0~~~ H
~ / ~ o~O-CH2-CH2-~-Suc 74 (X) ~ ~Ac Jl~c ~c OAc Another preferred lipid ~ analog/immunogenic carrier conjugate is an acyl glucose molecule linked at the Cl-pos~tion having the Formula (XI): ;

( f / ~ O-CH2-CHz-NH- ~ (XIJ

Another preferred lipid A analog/immunogenic carrier conjugate is an acyl gentiobiose molecule linked at the c6 -position to an in~nunogenic carrier having the Formula (XII):

o gl/0~750 2 ~ 9 ~L PC~/US~0/0~087 , ~Suc--O~_o f~_ ~ (XII) J~c O~/ ~c AC OAc j/
p Ansther preferred lipid A analog/immunogenic carr7er conjugate is an acyl glucose molecule linked at the c6 -position to an immunogenic carrier having the Formula (XIII):
/
A~lSuc--0 ~~ ~ (XIII) ~c A~ ~ /~, The lipid A analog/immunogenic carrier conjugates linked at the Cl-position ~ay be prepared (see Scheme I) by treatment of an appropriately substituted and protected carbohydrate with a reagent capable of linkin~ the carbohydrate to the immunogenic carrier haYing Formula (XIV) either directly or through a linker group. For example, gentiobiose or glucose peracetate (Formula (XV~, n ~ 1 or 2; Rl, R2, R~ and R4 ~ Ac) may be treated with HBr in glacial acetic acid to give the acetobromosaccharide der;vative ~XVI). The ~cetobromo derivative (XVI) may then be reacted with a linker such as 2-aminoethanol (XVII), 3-aminopropanol~ 4-aminobutanol, or 5-aminopentanol, to give the aminoethyl peracetate saccharide derivative (XVIII) which may be :. - ,, ~ .~ : , , .

91/01750 ~ PCI/US90/04~';"'' coupled ~o the immunogenic carrier (XIY) with9 for example, 1-ethyl-3-(3-dimethyl-aminopropy~) carbodiimide (EDAC) to give ~IY). The use of EDAC to form conjugates between amino containing substances and proteins is disclosed in U.S. Patent No. 4,526,714 to Feijen et al.
Alternatively, the immunogenic pept;de may be derivat;zed with a second bifunctional spacer group, such as succ;nic acid or any of the other l;nker groups having two carboxyl groups, to give the derivative (XIX). The derivative having Formula (XIX) may then be condensed with (XYIII) in the presence of EDAC to give (IV~.
~ here R4 is a phosphate group, the hydroxy group may be phosphor-ylated prior to preparation of the conjugate by reacting an appropri-ately protected glucose or gentiobiose compound with $he product of phosphoroùs acid and N-methylimi~a~ole in the presence of HgCl2. Th;s reaction allows the preparation of the phosphate ester at the free hydroxyl group.
The ratio of lipid A analog molecules per immunogenic carrier molecule may var~ considerably according to the molecular weight of the immunogenic carrier, the number of binding sites on the im-~unogenic carrier capable of being coupled to the lipid A molecule, and the antigenic characteristics of the particular lipid A molecule.
ln general, the ratio of lipid A analog molesules to immunogenic carrier molecules may be about 1:1 to about 200:1. Preferably, the ratio may range from about 5:1 to about 100:1. More preferably, where the immunogenic earrier is diphtheria toxoid, the ratio of lipid A
analpg molecules to diphtheria toxoid molecules may range from about 5:1 to about 40:1.

, , ~ - . . . - :
, . .
... . . .

91/01750 PC~/US90/04087 -17- 2~& ~ ~ 9~

Scheme I

8~ _~ ~ R
~;~~ ~ HBr; HOAc ~
R > R ~jJ Br n (XY) / (XVI~

~C ~XVII) ~. .

~ o-cH2cH2-NH2 (XVIII) Imnunogenic Can-ier or / ~nunogenic Carrier-Suc (XIV) ~ XIX) i; \ ~DAC

~ IY) .

W 0 91/01750 ~9 ~ PCT/US90tO40 .
Where the immunogenic carrier is linked to the lipid A molecule at the C6-position, the conjugates may be prepared according to scheme II by the selective tritylation of, for example, the c6 -position of gentiobiose (XX) with trityl chlor;de (XXI) to give the c6 -trityl gentiobiose der;vative (XXII). Acylation of the remaining hydroxyl groups with, for example, acetic anhydride gives 6'-0-tritylgent-iobiose heptaacetate ~XXIII). Removal of the trityl group with, for exa~ple, HBr gives the c6 -hydroxygentio~iose heptaacetate der;vative (XXIV) which may be linked to an i~munogenic carrier such as the succinate derivaLiYe of diphtheria toxoid to give (XXV).

., , . ~ ~ . .

0 91/01750 P~/US90/04087 Scheme I I
_ _ H - ~ Ph~C-Cl (XXI), pyr .
O . -~o~~O~I
L OH ln (XX) _ _ Ph3C- ~ O AC20, pry.

HO~--~OI~
n (XXII) Ph3c--- _ UBr, AcOU
Ac0~ \
A~O ~ Q,~
_ n (XXIII) _ _ DT-SUC; EDAC
~1 () ALO~
ACO~OA~
(XXIV) DT~O 1 ~
(XXVI

.. . : .- .

wo 91/01750 ~ , Pcr/US90~04 The invention also relates to intermediates useful for the preparation of the l;p;d A a~alog~;m~unogen;c carriers of the ;n~en-t;on. In part;c~lar, the invention relates to a l-bromo-saccharide hav i ng the Fo~ul a (XXVI ):

~ -O .

_ _ er (XXVI ) wherei n n-2 .
The inYention also relates to ~he interrnediate having Formula - (XXYII~:

~f~ Br (XXVII) Ac O ~.c Ac ~ he invent~on also relates to lipid A analog/linker conjugates having the Farmula (XXVIII):

~9 _ _ L~~ o CH2CH2 ~H2 n ",. (XXYIII) ~ . . . .

Yj1O 91/017~0 PCI'/US90/04087 -21- 2~ $/7 In particular, the invent;on relates to a lipid A analog~linker conJugate having the Formula (XXIX~:

~t~/O~ O-CH~CH2-NH~ (X%IX~

The invention also relates to a lipid A analog/linker conjugate having the Formula ~XXX):

~c ~_ ~c O~o~o-cH2cH2-RH2 (~

The invention also relates to a lip;d A analog/linker conjugate having the Formula (XXXI): -~ ~ ~O ~ O-CH~H2cH2-NH2 (XXXI) ~c o~ ~ C ~G

The inYention also relates to a lipid A analog/linker conjugate ha~ing the Formula (XXXII):

, :. -.

WO 91/01750 PC'r/US90/04087 s ~ 22-~ ( CH2 ) 5 -NH2 ( XX I I I ) The conjugates of the invention ~ay be purified by any method known to those of ordinary sk;ll ;n the art. For example, the conjugates ~ay be purified by reverse phase chromatoqraphy, ion exchange chromatography, size exclusion chro~atography, or by dialyzing the reaction product against water followed by freeze-drying. Alternatively, the conjugates may be purifi~d by passing a solution of the conjugate through a column having anti-lipid A
antibodies immobilized on a solid phase support. (See the Examples seo~ion of the application for a method of preparing antibodies to lipid A.) Administration of the vaccine comprising the 1 ipid A analog/im-~unogenic carrier conjugate of the present invention may be paren-teral, intravenous, intramuscular, subcutaneous, intranasal, or any other suitable means. Preferably, admin7stration is by subcutaneous ur intramuscular means. The dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered. In general, the conjugate may be ad-~inistered at a dosage of 0.001 ~o 25.0 ~g/kg of animal weight. The initial dose ~ay be followed up with a booster dosage after a period of 4 weeks to enhance the immunogenic response. Further booster doses every six months may be administered for as long as the risk of infection and septic shock exists.
The lipid A analog/i~munogenic carrier conjugates useful in the method of the present invention ~ay be e~ployed in such fo~ms as capsules, liquid solutions, suspensions or elixirs for oral adminis-tration, or sterile liquid forms such as solutions or suspensions.
Any inert carrier is preferably used, such as saline, or phosphate-buffered sal ine~ or any such carrier in which the compounds used in the method of the present invention have suitable solub;lity proper-, . - . . ~ ~ . , .

WO 91/01750 PCI'/US90/04087 Z3 2 Q ~

ties for use in the method of ~he present invention. ~he vaccines may be in the form of single dose preparations or in multi-dose flasks wh;ch can be used for mass vaccination programs. Reference is ~ade to Rem;ngton's Pharmaceutical Sc ences, Mack Publi sh ing Co., Easton~ PA, Osol ~ed.~ (1980~; and New Trends and Develooments in Yacci~nes, Voller et al. (eds.), University Park Press, Baltimore~ MD (1978), ~or methods of preparing and us;ng ~accines.
The vaccines of the invention comprising the lipid A analog/-immunogenic carrier conjugates of the invention may further comprise adjuvants which enhance production of lipid A analog-speeific anti-bodies. Such adiuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant, the dipeptide known as MDP, saponin, aluminum hydroxide, Bordetella ~ertussis, dephosphor-ylated lipid A, or an interferon.
Freund's adjuvant is an emulsion of ~ineral oil and water which is ~ixed with the i~munogenic substance. Although Freund's adjuYant is powerful 9 it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) may be used for administration to a human. The conjugate ~ay be absorbed onto the aluminum hydroxide from which it is slowly released after injection.
The lipid A analog/immunogenic peptide conjugate may also be encapsulated within liposomes according to Fullerton, U.S. Patent No.
4,235,877.
Having now generally described the inventisn, the same ~ay be further understocd by reference to the following examplest which are not intended to be limiting unless so expressly stated.

.

. .
EXAMPLES

EXaMD1e 1: preDaration of a Gentiobiose peracetate-8SA coniuqate Gentiobiose octaacetate, obta;ned from S;gma Chem;cal Co. (St.
Louis, M0), was converted into the acetobromo derivative by dissolu-tion in diehloromethane tl gm ;n S ~1) and treatment with ~3% HBr in glacial ace~ic acid (10 ml) (Fluka~ at 5-C for 45 minutes. A volume of dichloromethane equal to the $otal volume of the reaction mixture was added and the mixture transferred to a separatory funne1. The organic phase was washed with an equal volume of saturated sodium bicarbonate solution (3-4 times), fcllowed by ~wo portions of water.
The organic layer was then dried over MgS0~, filteredS and the solvent eYaporated to gîve a syrup. The acetobromogentiobiose was crystal-lized from dichloromethane and d~ethyl ether, filtered and dried in a vacuum desicca~or. The crystalline compound gave a single spot on T.L.C. and the H-l NMR spectrum was correct.
Acetobromo~entiobiose (5 gm ~n 5 ~1 chloroform) was coupled to 2-aminoethanol (O.S ml, approx. 0.5 gm, in 1 ~1 chlorofo m) in the presence of drierite (1 gm; W.A. Hammond Drierite Co.)~ The mixture was stirred overnight at room temperature then filtered. The result-ing aminoethyl gentiobiose heptaacetate (AGH) was first purified by chromatography on a column of sil~ca gel (1.6X24 cm, 50 ml total volume) using an ethyl acetate-ethanol solvent mixture (19:1) as the eluting solvent. ~he fract~ons were screened by TLC using ethyl acetate-ethanol (19:1) to elute. 7he fractions containing the aminoethyl derivative were combined, the solvent eYaporated in vacuo, and the product crystallized from hot ethanol at 55-C. The crystal-line compound ga~e a single spot on T.L.C. and had a melting point of 176-177~C. Elemental analysis showed C, 49.44%; ~ 6.05%; N, 2.1X
(cakulated for C28H4028 N: C~ 49-56X; H~ 5-94%;

:; ~, ' :

- ~ , .
. ~ ~ , . .

WO 91/01750 PCrtUS90/04087 2~

In order to increase the number of free carboxyl groups and solubility of bovine serum albumin (8SA), the BSA (10-50 mg in PBS at 4-5 mg/ml) was slowly treated with succinic anhydride (5-10 mgtmg protein; Aldrich Gold Label) at pH 8-9. The pH was maintained at B-8.5 by the addition of concentrated NaOH. ~he reaction mixture was then extensiYely dialyzed aqainst water and the resulting succinylated BSA ~Suc-BSA) freeze-dried.
Dioxane was the solvent found capable of dissolving both the protein carriers and the AGH 7igand. Suc-BSA (10 mg) was coupled to A~H jn 20X 1,4-dioxane (2-3 ml; F;sher Scientific) ~n the presence of 1-ethyl-3-(3-dimethylaminopropyl~-carbodiimide (10 mg; EDAC~. The pH
was monitored using pH paper and maintained at 5-6 by the addition of dilute HCl. Three additional portions of EDAC (10 m~) were added after ~0 minute intervals. The resulting conjugate (Suc-BSA-AGH) wa~
dialyzed against water and then frozen. Analysis for reduc;ng sugar by the phenol-sulfur;c acid assay showed that the conjugate consisted of 27.4X AGH. This is about 35 moles of ligand/mole of protein.

Example 2: PreDaration of a Gentiobiose Peracetate-DT Coniuqate ..
Diphtheria Toxoid (DT) from SSVI (Bern, Switzerland) was coupled to AGH (obtained according to Example 1) in 25% 1,4-dioxane and 0.05M
NaCl in the presence of EDAC. The resul~ing conjugate (DT-AGH) was dialyzed against water and freeze-dried. Analysis demonstrated that 16X of the conjugate consisted of AGH.

WO 91/017!iO t;;~ ~ PCI'/US90/04087 ExamDle 3: PreDarat;on of /~in;noDroDYlqentiobiose HeDtaacetate-Succinyl-DT _onjuqate -3-Aminopropanol was l;nked to acetobromogentiob;ose (as described for 2-aminoethanol in Example lJ. Diphther;a toxoid (DT) was succin-ylated (a~ descr;bed for BSA ;n Example 1) to g;ve Suc-DT wh;ch was then coupled to aminopropylgentiobiose heptaacetate (AP~H; according to Example 2) to give Suc-DT-APgH.

Example 4: _ Bindina of Gentiobiose-Protein Carrier Coniuqates to LPS sDeclfic antibodies Althou~h gentiobiose octaacetate is insoluble in water, the protein conjugates dissolYe easily. These soluble conju~ates were tested for their ability to bind anti-lipid A human monoclonal antibody (obtained by fusion of the heteromycloma SHMA6(H4) with Epstein Barr virus (EBV) transformed lymphooytes from the spleen of a patient ;mmunized wi~h the J5 ~utant of E. coli Olll^B4 which is deficient in the enzyme uridine 5'-diphosphategalac~ose 4-epimerase which prevents ~ttachment of the side chains- responsible for the ~arked antigenic diversity among Gram-negative bacteria) by enzyme-linked solid phase immunoassay (ELISA). As shown in Table 1, both BSA-gentiobiose octaacetate and diphtheria toxoid-gentiobiose octaacetate conjugates were able to bind the human monoclonal anti-body. These experiments demonstrate that the important binding epitope remained intact during the conjugation process. Therefore, it would be expected by one of ordinary skill in the art that anti-lipid A ant1bodies would be produced in vivo upon adm;nistration of the lipid A analog/immunogenic carrier conju~ates to an animal.

- ,............ . ................. . . . . .
. ~ , - .............. ........... . ~ ~ - , , .. . - , . . .

WO 91/0~750 PCr/US90/04087 Tabl e Binding of the lipid A Antibody to Gentiobiose Octaacetate/Protein Conjugate Suc-BSA-AGH conjugate 2,560 Suc-BSA-Control 640 DT-AGH eonjugate 48 DT control AGH in ethanol 1,280 E. coli Lipid A 2,560 Suc-DT-APGH 3,200 Suc-DT (control) O
, . . _ . . . , _ _ . _ Exa~ple 5 In Yivo ImmunoqenicitY of the Gentiobio_e Octaacetate/
Protein Coniuqate in ~abbits The gentiobiose octaacetate protein/diphtheria toxoid conjugate was administered to rabb~ts according to the immuni~ation schedule outlined ;n ~able 2. Antibody titers were then determined by ELISA.
Sera from rabbits 1 and 2 was then assayed ~or antibodies specifie for a series:of antigens. Table 3 shows that the rabb;t sera has re-activity with LPS and diphther;a tox;n, but l;ttle react;vity w;th APGH. ~

.

Wo 91/0117~0 ~ PCr/US90/040~7 .--2~-~able 2 B~ g~ Yaccine Dose Adiuvant Date 1 ~ 2 Suc DT-APGH 200 ug Freund's 4/11/88 Lot AKB III-A 200 ug Freund's ~/~0/88 Substitution: 5.2%a 200 ug None 4/29/88 150 ug None 5~25~88 150 ug None 6/30/88 150 ug None 8/15/88 150 ug None 10/28/88 150 ug None 1/4/89 Animals bled out 1/12/89 -3 ~ 4 Suc-DT-APGH
- Lot AKB III-A-171 Subst itution: 2CXa 5 & 6 Sue-DT-APGH
Lot AKB V-33A
Substitution: ~.4Xa All four animals immunized 100 ug Alum 11/2/88 on same schedu~e: 100 ug Alum 11/30/88 100 ug None 12/31/88 100 ug None 1/13/89 . 100 ug None 2/17/89 ~- a -Substitution of succinylated diphtheria toxin by APGH expressed asweight percent.

.. ~ .: :
- : , .. ~ .
.

WO g1/01750 PCl/U~90/04087 -29- 2~

Table 3 Post b7eeds Pre-bleed -----------~------------------Antiq~ Rabb;t 4/11 .4/29 5~13 ~ 7~11 150a 300 400 800 800 2 200 200 300 6~0 1, ~00 Men 6r B

APGH 1 <100 ~100 100 150 150 2 <100 100 100 150 250 DT 1 300 1,600 6,400 12,800 >12,800 2 300 12,80012,800~lZ,800 >1~,800 a All data is expressed as the dilution giving an optical density reading of 0.4 units.

Rabb;t sera was also assayed against a tetanus toxoid-APGH
conjugate ~ APGH), which does not have a spacer ~roup. The results listed in Table 4 show that the antibodies are also specific for this conjugate, confirming that the antibodies are reactive with the APGH
part of the molecule.

WO 91~01750 Pcr/usso/04037 ~,96- 30 Tabl e 4 Post bleeds . _ _ _ _ _ _ _ ~9~ Rabb;t Pre-bleed1/4/89. I~l2~89 TT-APGH
(no spacer~
lOOa 12,800 2 100 >12,~00 3 100 4,800 1,600 4 1 00 800 1, 600 1 001, 200 3, 200 .
~ All data is expressed as the dilution giYing an optical-density reading of 0.4 nits.
Antibody spec;ficity for LPS on boiled J5 was then determined by specific adsorpt~on experiments. In each experiment, the sera of rabbits was diluted 1:500 and adsorbed twice w~th boiled J5 organisms at 10% volume of packed cells. The first adsorption was for 2 hours ~ollowed by a second overnight adsorption (both at 4-C). The J5 oryanisms were analyzed by GLC and found to ~e free of galactose. The sera from the final bleeds of rabbits 1 and 5 were then a~sayed pre-and post-adsorption by ELISA usin~ TT-AP~H. The resuits are shown in Table 5.
Table 5 Rabbit Number X Reduction Exp 1 Exp 2 42 ~1 27%

aPercent reduction at O.D. ~ 0.4.

. ~ . .
: . . . .-- - ~ . - .

-As shown in Table 5, there was significant reduction of antibody titer when the sera was adsorbed to J5 LPS. These data confirm the expectation that the sera of animals immunized with the conJugate is reactive with LPs on boiled J5.

ExamDle 6 PreParation of 6'-Substituted 6entiobiose HeDtaacetate Succinvl-Diphtheria Toxoid (SUC-DT~ con.iuqate A. PreDaration of 6'-0-Tr;t~l-G~ntiobiose Gentiobiose (4 gm, Sigmaj was added slowly with stirring to pyridine (60 ml) and to the resulting suspensio~ was added with stirring triphenylmethylchloride (4 gm, Aldrich)~ The mixture was stirred overnight at room temperature.
The solYent was evaporated off in a rotary eva~orator and the resident pyridine was removed by co-distillation w;th toluene (3 x 15 ml). The solid product was dissolved into dichloromethane 130 ~l) and the solution was loaded onto a washed silica ge~ column (2.0 x 70 cm).
The excess trityl chloride was first eluted with ethyl acetate. The 6'-0-trityl-gentiobiose was then eluted with ~ethanol. The fractions containing the product were combined and evaporated to dryness.

B. PreRaration of 6'-O-TritYl-Gentiobiose Heptaacetate 6'-O-Trityl-gentiobiose was dissolved into pyridine ~35 ml~ and to this solution was added with stirring 17 ~l of acetic anhydride ~Aldrich Chemical Corporation, Milwaukee, ~I). The mixture was stirred oYernight at room temperature and then poured into 400 ml of crushed ice. The resulting precipitate-was filtered, washed with cold water and dried under vacuum tyield 5.5 gms).

~ ' ,... . . .
.. .. .
7~;0 PCr/US90/04087 33`~ ~32-C. Preparation o~ 6'-0-HvdroxY-Gentiobiose HePtaacetate (C-6'-HGH~
1.0 gm of 6'-0-trity7-gentiobiose heptaaceta~e was dissolved into 5.0 ml o~ glacial acetic acid and to this solution was added 33% HBr in glacial acetic acid (Fluka~ to make 2X HBr. The mixture was poured ~int`o ~ ml of iced water and the resulting precipitate (trityl bromide~ was filtered out.
The aqueous so~ution was extracted with dichloromethane ~50 m~) and the organic phase was washed with saturate~ HaHC03 so~ution, cold water, dried with anhydrous sod;um sulfate and evaporated to dryness.
The solid product was crystalli~ed from diethyl ether. Yield 150 mg.

D. PreDaration of 6'-HYdroxv-Gentiobiose HePtaacetate SuccinY
~iphtheria Toxoid (SUC-DT) Con.iuaate.
Succinyl-DT (7.2 mg3 was coupled to 6'-hydroxy-gentiobiose heptaacetate (3.5 mg) in 20% 1,4-dioxane (Fisher Sc;ent;fic Co.) in the presence of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (18 mg, EDAC). The pH was maintained beeween ~.7 and 6.0 with O.lN HCl. ~wo additional portions of EDAC (10 mg) were added at 30 minute intervals.
The resulting conjugate (6'-0-SucDT-~H) was dialyzed against water and then freeze dried. Analysis for reducing sugar by thé phenol-sulfuric acid assay showed that the conjugate consisted of 20X 6'-HGH. This is about 30 moles of ligand/mole of protein.

Example 7 ImmunoqenicitY Studies of 6'-0-SucDT-GH Yaccine The Yaccine was prepared for immunization studies by dissol~ing the lyophilized conjugate in sterile phosphate buffered saline to a final concentration of 200 ug/ml, followed by adsorption to aluminum oxide (Rhesorptar) overnight at 4-C. ~he vaccine was administered intramuscularly to NZW rabbits at 50 ug per dose, and sera were collected periodically during the immunization schedule Vaccine was administered at weeks 0, 3, 6, 9, 12, and 16. Immunogenicity was : .
., . . , .:
.
, WO 91/01750 PCI/U~90/040~7 ~ ~ & ~

determined by ELISA using a heterologous tetanus coniugate vaccine ~ SucDT-GH) as the ~late antigen at a concentration sf 2 ug/ml.
Sera were run in triplicate beg;nning at a 1/50 d;lution, and data reduced to optical density (O.D.) units, the titer at which the O.D.--is equal to 1Ø The results for serial bleeds obtained 1 week after each dose shown in Table 6.

Table 6 Immunogenicity of 6'-0-Suc-DT-GH Vaccine Rabbit #
~k 0 Wk I~k 4 Wk 7 ~k 10Wk 13 ~k 17 R008 22 1112,208 1,844 2,9473,661 1,206 R009 20 27 443 1,053 2,42313,0976,884 ~010 23 82 215 926 3,8903,g25 2,~75 R012 1~ 55 474 663 2,5972,897 2,080 R014 13 432,374 1,232 1,2321,312 S90 The results show that the vacc;ne induced ligand-specific antibodies in each rabbit immunized. To detenmine the protective effieacy of these antibodies, their ability to protect mice in a hog mucin challenge model was assayed. Pooled sera obtained at week 14 from immunized rabbits. or pooled sera obtained from control rabbits not receiving the vaccine, were sterile filtered and administered intraperitoneally to NSA mice at a dose of 1.0 ~l per mouse. Two hours later the ~ice were chal1enged with from 4.8 x 103 to 4.8 x 105 aeruginosa suspended in a 14X preparation o~ hog mucin. Mice were examined hourly for the next 72 hours and the time o~ death recorded.
Table 7 reports the results of these studies.

. . . .
~, ~
.

WO 91/01750 PCI`/US90/04087 L~ _ 3 4 _ Tabl e 7 Hog Muci n Chal 1 enge ~del Protection from Challenge with Pseudomonas aeruginosa Group Challenge 72 hr p* Hours p**
(n=10)(c~u) survival survivinq (X) (Mean + S.E.M.) Immune4.8x103 70 .0049 60.6+6.41 .0001 Normal4.8x103 0 lB.4+0.30 Immune4.8x104 40 .01 47.6+7.01 .0022 Normal4.8x104 0 18.0+0 Immune4.8x105 30 .01 37 8+7.53 .0001 Normal4.8x105 0 18.9+0 .
* x2 analysis of immune group YS all controls receiving < that challenge dose.
** Student's t test, one tailed ~_~,_====,=====_=====__======_=========~================a===========_ These data demonstrate that the vaccine was immunogenic ~nd produced antibodies that protect mice against challen~e with a virulent gram negative bacteria.

Example 8 Active and Passive Protection from a Localized Shwartzman Reaction A. Active Immunization: Three rabbits were immunized twice with Suc-DT-APGH (see Example 3) in aluminum hydroxide gel (20:1, w/w) and subsequently received 4 injections of the ant;gen in saline. To test development of a local;zed Shwartzman reaction, rabbits each received an intracutaneous injection of 50 ~9 E. col; 06 LPS ~ollowed 23 hours later with 25 ~g LPS in 0.1 ml saline, intravenously. Only 1 of 3 immunized rabbits showed a positive erythema, although no hemorrhagic reaction, whereas 3 of 3 non-immune rabbits were positive for erythema and 2 had hemorrhagic reactions. No necrosis was observed.

.. ... ~ . ; ,, , ~

~ .4 ~

B. Passive Immun;~at;on: Rabbits whose sera were to be tPsted were ;mmun;zed once with Suc-DT-APGH (Exa~ple 3) in comp1et~ Freund's adjuvant, followed by 7 injections of the antigen in saline. Serum obtained from these hyper;mmuni~ed rabbits had an antibody t;ter of 1:6400 measured ;n an ELISA w;th TT-APTGH.
Test rabb;ts were injected intracutaneously with 100 ~9 E. coli 06 LPS in 0.2ml saline. After 21 hours, rabb;ts received 15 ml of the hyperimmune SerUm or normal serum intravenously (in the ear ve;n) and 2 hours later were challenged by intraYenous injection of 20 ~9 LPS in 0.2 ml. In the control rabbits receiving normal serum, 4 of 5showed hemorrhage and 1 had tissue necrosis. Only 1 of ~ rabbits receiving hyperimmune serum showed a positive hemorrhagic reaction and none had any signs of necrosis.
Therefore both active and passive immunization against Suc-DT-APGH afforded significant protection against the LPS-induced Shwartz-man reaction, further confirming the capacity of the composition to induce anti-LPS antibodies capable of acting in vivo.

Having now fully descr;bed this invention, it will be understood by those of sk;ll in the art that the same can be performed with any wide equivalent range of compositions, concent.rations, formulation and other parameters w;thout affecting the scope of the invention or any embodiment.

; .
.. . . .

'

Claims (38)

WHAT IS CLAIMED IS:
1. A lipid A analog/immunogenic carrier conjugate comprising a lipid A analog of the formula wherein R1 and R3 are the same or different and selected from the group consisting of hydrogen, C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, a 3-(C2-C12-acyloxy)-C3-C18 acyl group or a linkage to an immunogenic carrier;
R2 is selected from the group consisting of C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups; and R4 is hydrogen, a C2-C18 acyl group, a phosphate group or a linkage to an immunogenic carrier; n = 1 or 2;
with the proviso that one of R1, R3 or R4 is a linkage to an im-munogenic carrier, wherein said linkage does not interfere substan-tially with the ability of the lipid A analog to stimulate an immune response in an animal.
2. A lipid A analog/immunogenic carrier conjugate having the following formula:

wherein A is an immunogenic carrier;
m is O or 1;
n is 1 or 2;
p is 1 to 200;
L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal;
R1 and R3 are the same or different and selected from the group consisting of hydrogen, C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups;
R2 is selected from the group consisting of C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups; and R4 is hydrogen, a C2-C18 acyl group or a phosphate group;
wherein said conjugate induces active immunity to LPS when adminis-tered to an animal.
3. The lipid A analog/immunogenic carrier conjugate of claim 2, wherein said linker group comprises -O-(CH2)q-NH-, wherein q is 2-3.
4. The lipid A analog/immunogenic carrier conjugate of claim 2, wherein L is
5. The lipid A analog/immunogenic carrier conjugate of claim 2, wherein L is , wherein y = 1-3.
6. The lipid A analog/immunogenic carrier conjugate of claim 1, wherein said immunogenic carrier is a protein selected from the group consisting of bovine serum albumin, diphtheria toxoid, edestin, Toxin A and choleragenoid.
7. A lipid A analog/immunogenio carrier conjugate having the formula:

wherein A is an immunogenic carrier;
m is 0 or 1;
p is 1 to 200;
L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal;
R1 and R3 are selected from the group consisting of hydrogen, C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups;

R2 is selected from the group consisting of C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups; and R4 is hydrogen, a C2-C18 acyl group or a phosphate group;
wherein said conjugate induces active immunity to LPS when adminis-tered to an animal.
8. The conjugate of claim 7, wherein the immunogenic carrier is a protein selected from the group consisting of bovine serum albumin, diphtheria toxoid, edestin, Toxin A and choleragenoid.
9. The lipid A analog/immunogenic carrier conjugate of claim 7, wherein said linker group comprises -O-(CH2)q-NH-, wherein q is 2-5.
10. The lipid A analog/immunogenic carrier conjugate of claim 7, wherein said linker group comprises , wherein q = 2-5, and x = 2-12.
11. The lipid A analog/immunogenic carrier conjugate of claim 7, wherein L is , wherein y = 1-3.
12. A lipid A analog/immunogenic carrier conjugate having the formula:

wherein p is 1-200.
13. A lipid A analog/immunogenic carrier conjugate having the formula:

wherein p is 1 to 200.
14. A lipid A analog/immunogenic carrier conjugate having the following formula:

wherein A is an immunogenic carrier;
m is 0 or l;
n is 1 or 2;
p is 1 to 200;
L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal;
R1 and R3 are the same or different and selected from the group consisting of hydrogen, C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups;
R2 is selected from the group consisting of C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups; and R4 is hydrogen, a C2-C18 acyl group or a phosphate group;
wherein said conjugate induces active immunity to LPS when adminis-tered to an animal.
15. The lipid A analog/immunogenic carrier conjugate of claim 14, wherein L is
16. The lipid A analog/immunogenic carrier conjugate of claim 14, wherein said immunogenic carrier is a protein selected from the group consisting of bovine serum albumin, diphtheria toxoid, edestin, Toxin A and choleragenoid.
17. A lipid A analog/immunogenic carrier conjugate having the formula:

wherein A is an immunogenic carrier;
m is 0 or 1;
p is 1 to 200;
L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal;
R1 and R3 are selected from the group consisting of hydrogen, C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups;
R2 is selected from the group consisting of C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups; and R4 is hydrogen, a C2-C18 acyl group or a phosphate group;
wherein said conjugate induces active immunity to LPS when adminis-tered to an animal.
18. The conjugate of claim 17, wherein the immunogenic carrier is a protein selected from the group consisting of bovine serum albumin, diphtheria toxoid, edestin, Toxin A and choleragenoid.
19. The lipid A analog/immunogenic carrier conjugate of claim 17, wherein said linker group comprises
20. A lipid A analog/immunogenic carrier conjugate having the formula:

wherein p is 1-200.
21. A lipid A analog/immunogenic carrier conjugate having the formula:
wherein p is 1 to 200.
22. A vaccine for the prophylaxis of septic shock in an animal comprising:
(a) the lipid A analog/immunogenic carrier conjugate of claim 1;
and (b) a pharmaceutically acceptable carrier;
wherein said conjugate is present in an amount effective to induce active immunity to LPS in an animal.
23. The vaccine of claim 22, further comprising an adjuvant.
24. The vaccine of claim 22, wherein said conjugate has the formula:

wherein p is 1 to 200.
25. The vaccine of claim 22, wherein said conjugate has the formula:

wherein p is 1 to 200.
26. The vaccine of claim 22, wherein said conjugate has the formula:

wherein p is 1 to 200.
27. The vaccine of claim 22, wherein said conjugate has the formula:

wherein p is 1 to 200.
28. A method for treating or preventing septic shock in an animal comprising administering a pharmaceutical composition to an animal comprising:
(a) the lipid A analog/immunogenic carrier conjugate of claim 1;
and (b) a pharmaceutically acceptable carrier;
wherein said lipid A analog/immunogenic carrier conjugate is present in an amount effective to induce active immunity to LPS in an animal.
29. The method of claim 28, wherein said amount effective is 0.001 to 25.0 µg/kg body weight of said animal.
30. The method of claim 28, wherein said conjugate has the formula:

wherein p is 1 to 200.
31. The method of claim 28, wherein said conjugate has the formula:

wherein p is 1 to 200.
32. The method of claim 28, wherein said conjugate has the formula:

wherein p is 1 to 200.
33. The method of claim 28, wherein said conjugate has the formula:

wherein p is 1 to 200.
34. A compound having the formula:

wherein n=2, R1 and R3 are the same or different and selected from the group consisting of hydrogen, C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups;
R2 is selected from the group consisting of C2-C18 acyl groups, 3-hydroxy C3-C18 acyl groups, and 3-(C2-C12-acyloxy)-C3-C18 acyl groups; and R4 is hydrogen, a C2-C18 acyl group or a phosphate group.
35. A compound having the formula:

36. A compound having the formula:

37. A compound having the formula:

38. A method of prepaparing a hyperimmune serum comprising administering the lipid A analog/immunogenic carrier conjugate of claim 1 to an animal and collecting the hyperimmunized serum.
CA002064194A 1989-07-27 1990-07-20 Lipid a analog/immunogenic carrier conjugates and the use thereof as vaccines Abandoned CA2064194A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US38586089A 1989-07-27 1989-07-27
US385,860 1989-07-27
US48789890A 1990-03-06 1990-03-06
US487,898 1990-03-06
US50255590A 1990-04-02 1990-04-02
US502,555 1990-04-02

Publications (1)

Publication Number Publication Date
CA2064194A1 true CA2064194A1 (en) 1991-01-28

Family

ID=27409738

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002064194A Abandoned CA2064194A1 (en) 1989-07-27 1990-07-20 Lipid a analog/immunogenic carrier conjugates and the use thereof as vaccines

Country Status (5)

Country Link
EP (1) EP0484403A4 (en)
JP (1) JPH04507244A (en)
AU (1) AU651319B2 (en)
CA (1) CA2064194A1 (en)
WO (1) WO1991001750A1 (en)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5583112A (en) * 1987-05-29 1996-12-10 Cambridge Biotech Corporation Saponin-antigen conjugates and the use thereof
US5827819A (en) * 1990-11-01 1998-10-27 Oregon Health Sciences University Covalent polar lipid conjugates with neurologically active compounds for targeting
US5256641A (en) * 1990-11-01 1993-10-26 State Of Oregon Covalent polar lipid-peptide conjugates for immunological targeting
US5543389A (en) * 1990-11-01 1996-08-06 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University, A Non Profit Organization Covalent polar lipid-peptide conjugates for use in salves
US5543390A (en) 1990-11-01 1996-08-06 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University Covalent microparticle-drug conjugates for biological targeting
US5824310A (en) * 1991-10-22 1998-10-20 The United States Of America As Represented By The Department Of Health And Human Services Lipopplysaccharide conjugate vaccines
ES2104984T3 (en) * 1992-05-07 1997-10-16 American Cyanamid Co GLIPOCONJUGATED ANTIGENS WITH LIPID TO SYNTHETICS FOR USE IN VACCINES.
NZ507791A (en) 1998-05-19 2003-12-19 Res Dev Foundation Triterpene compositions from Acacia victoriae and methods for use thereof
JP2004528321A (en) 2001-04-04 2004-09-16 ノルディック ワクチン テクノロジー アクティーゼルスカブ Polynucleotide binding complexes containing sterols and saponins
WO2004058788A1 (en) * 2002-12-24 2004-07-15 Sankyo Company, Limited Lipid a analogues having glucose as the right sugar
ES2926662T3 (en) 2012-08-21 2022-10-27 Janssen Pharmaceutica Nv Antibodies against haptens of risperidone and use thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3224788A1 (en) * 1981-07-17 1983-02-03 South African Inventions Development Corp., Scientia, Pretoria,Transvaal CARRIER-TIED IMMUNOGENIC MATERIAL
US4902506A (en) * 1983-07-05 1990-02-20 The University Of Rochester Immunogenic conjugates
US4695624A (en) * 1984-05-10 1987-09-22 Merck & Co., Inc. Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and of confirming covalency
US4844894A (en) * 1984-07-12 1989-07-04 Ribi Immunochem Research Inc. Method of inhibiting the onset of septicemia and endotoxemia

Also Published As

Publication number Publication date
EP0484403A4 (en) 1993-03-17
AU6079590A (en) 1991-03-11
JPH04507244A (en) 1992-12-17
WO1991001750A1 (en) 1991-02-21
AU651319B2 (en) 1994-07-21
EP0484403A1 (en) 1992-05-13

Similar Documents

Publication Publication Date Title
US5977081A (en) Triterpene saponin analogs having adjuvant and immunostimulatory activity
JP3717511B2 (en) Saponin-antigen complex and its use
JPH05222099A (en) Process for producing synthetic n-bonded glyco complex
CA2064194A1 (en) Lipid a analog/immunogenic carrier conjugates and the use thereof as vaccines
US11420993B2 (en) Semi-synthetic meningococcal conjugate vaccine
JP6622804B2 (en) Vaccine against Streptococcus pneumoniae serotype 4
EP1212334B1 (en) Oligosaccharides derived from ribose-ribitol-phosphate, and vaccines containing them
US20240024489A1 (en) Protected disaccharides, their process of preparation and their use in the synthesis of zwitterionic oligosaccharides, and conjugates thereof
EP0575517A1 (en) Lipid a analog/immunogenic carrier conjugates, the use thereof as vaccines and polyvalent hyperimmune gammaglobulins
EP1000076A1 (en) HEXADECASACCHARIDE-PROTEIN CONJUGATE VACCINE FOR $i(SHIGELLA DYSENTERIAE) TYPE 1
US8716469B2 (en) Synthetic oligosaccharides for Moraxella vaccine
JPH0641200A (en) Synthetic lipid a sugar complex antigen
KR830002059B1 (en) Process for the preparation of novel compounds in the form of mul-amyl-pentide
EP3000820A1 (en) Synthetic vaccines against Streptococcus pneumoniae serotype 8
MXPA99010675A (en) Triterpene saponin analogs having adjuvant and immunostimulatory activity

Legal Events

Date Code Title Description
FZDE Dead