CA2128212A1 - Detoxified lps-cholera toxin conjugate vaccine for prevention of cholera - Google Patents
Detoxified lps-cholera toxin conjugate vaccine for prevention of choleraInfo
- Publication number
- CA2128212A1 CA2128212A1 CA002128212A CA2128212A CA2128212A1 CA 2128212 A1 CA2128212 A1 CA 2128212A1 CA 002128212 A CA002128212 A CA 002128212A CA 2128212 A CA2128212 A CA 2128212A CA 2128212 A1 CA2128212 A1 CA 2128212A1
- Authority
- CA
- Canada
- Prior art keywords
- lps
- vaccine
- cholera
- lipopolysaccharide
- detoxified
- 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
Links
Classifications
-
- 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
- A61K39/107—Vibrio
-
- 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
- A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- 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
-
- 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
- A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Communicable Diseases (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
A vaccine formulation comprising conjugates of detoxified LPS
with proteins including cholera toxin (CT) is disclosed. Treatment with hydrazine (DeA-LPS) reduces the endotoxic properties of the LPS to clinically acceptable levels and results in a larger and more antigenic molecule than the saccharide produced by acid hydrolysis. The conjugates utilizing the cholera toxin of V.
cholerae) are disclosed which have low levels of pyrogen, no toxic activity upon Chinese hamster overay cells and elicit booster responses of vibriocidal and CT antibodies when injected subcutaneously as saline solutions into mice. The conjugates produced as a cholera vaccine induce the same antibodies as parenterally injected cellular vaccines but have improved safety and immunologic properties.
with proteins including cholera toxin (CT) is disclosed. Treatment with hydrazine (DeA-LPS) reduces the endotoxic properties of the LPS to clinically acceptable levels and results in a larger and more antigenic molecule than the saccharide produced by acid hydrolysis. The conjugates utilizing the cholera toxin of V.
cholerae) are disclosed which have low levels of pyrogen, no toxic activity upon Chinese hamster overay cells and elicit booster responses of vibriocidal and CT antibodies when injected subcutaneously as saline solutions into mice. The conjugates produced as a cholera vaccine induce the same antibodies as parenterally injected cellular vaccines but have improved safety and immunologic properties.
Description
~093/13797 2 ~ 2 ~ 2 1 ~ PCT/US93/002~3 Deto~ifi~d L~B-Cholera To~in Co~jugate Vaccino for Pr~enkion of Ch~ler~
The invention disclosed herein relates broadly to the production of vaccines for the amelioration of bacterial infections. More specifically, the invention : describe~ the production of an antibacterial vaccine by conjugation o~ detoxified lipopolysaccharide derived from ~: ; the bacterial target strain to proteins also produced by ~: the bacteria target.
BACKGROUND ART
~ Cholera~persists as a cause of illness and death in : at least 40 countries on 3 three continents: ~340,000 cases have been reported in the Western hemisphere since an epidemic~startéd in Peru, January 1~91 ~16,33~.
Worldwide preventi~on ~f cholera by immunization has not bean~achieved because of the limltations of available vaccines~ Research into new vaccines is difficult because there ~is no;~consensus as~to the moieties which best eliclt a protective~:immune~response. The absence of
The invention disclosed herein relates broadly to the production of vaccines for the amelioration of bacterial infections. More specifically, the invention : describe~ the production of an antibacterial vaccine by conjugation o~ detoxified lipopolysaccharide derived from ~: ; the bacterial target strain to proteins also produced by ~: the bacteria target.
BACKGROUND ART
~ Cholera~persists as a cause of illness and death in : at least 40 countries on 3 three continents: ~340,000 cases have been reported in the Western hemisphere since an epidemic~startéd in Peru, January 1~91 ~16,33~.
Worldwide preventi~on ~f cholera by immunization has not bean~achieved because of the limltations of available vaccines~ Research into new vaccines is difficult because there ~is no;~consensus as~to the moieties which best eliclt a protective~:immune~response. The absence of
2~0~ ~bacte:rial invasion, the systemic sy~ptoms and the lack of intestina~l inflammation characterizing the disease haYe led~to the understanding~that~cholera is a toxin-mediated disease of the~lumlnal~surface of the jejeunum, and to ; :the notion that a local intestinal respon~e is re~uired :~for~protective immunity ~4,10-12,21,22,24,26,32,36,44].
The~lipopolysaccharide (LPS) of Vibrio cholerae is considered to be a~protective antigen [3,18,22,36,38,43,51,60]~but the structures, pathogenic role and:~host~moieties involved in protective immunity to 30 ~cholera are incompletely understood. V. cholerae 01 LPS
contains lipld:~A~and a core oliqosaccharide composed of : 4-amino-4-deoxy~ arabinose, quinovosamine, D-glucose, :: :
D-fructose and heptose [23,30,47]. 3-deoxy-D-manno octulosonic acid :(KDO) has been identified recently and presumed to ~e in the core adjacent to the lipid A [5].
:
WO93/13797 PCT/US93/00~5;~
212821~ -2-The O-specific polysaccharide (O~SP) of V. cholerae o, serotype Inaba, contains a saccharide of -12 residues composed of l~2-linked D-pero~amine whose amino groups are acylated by 3-deoxy-~-glycero-tetronic acid ~23,30,47]. The relationship between the sequence of the genes that encode the enzymes which synthesize the V.
: cholerae hPS and ~he serological specific~ty of the serotypes (LPS types~ Inaba and Ogawa ~23,36~ has not been c~arified.
~Parenterally administered cellular vaccines or '::l0 partially purified LPS~induce a statistically significant pr~tection against~cholera in adults (-60%) for -6 months [3,7,18,22,38,45]. Cellular vaccines are less effective for infants and young children and ineffective for contrsl of outbreaks of cholera [38~5l~ The protective immu~e moiety induced by these vaccines is proposed to be serum LPS anti~odies with vibriocidal activity
The~lipopolysaccharide (LPS) of Vibrio cholerae is considered to be a~protective antigen [3,18,22,36,38,43,51,60]~but the structures, pathogenic role and:~host~moieties involved in protective immunity to 30 ~cholera are incompletely understood. V. cholerae 01 LPS
contains lipld:~A~and a core oliqosaccharide composed of : 4-amino-4-deoxy~ arabinose, quinovosamine, D-glucose, :: :
D-fructose and heptose [23,30,47]. 3-deoxy-D-manno octulosonic acid :(KDO) has been identified recently and presumed to ~e in the core adjacent to the lipid A [5].
:
WO93/13797 PCT/US93/00~5;~
212821~ -2-The O-specific polysaccharide (O~SP) of V. cholerae o, serotype Inaba, contains a saccharide of -12 residues composed of l~2-linked D-pero~amine whose amino groups are acylated by 3-deoxy-~-glycero-tetronic acid ~23,30,47]. The relationship between the sequence of the genes that encode the enzymes which synthesize the V.
: cholerae hPS and ~he serological specific~ty of the serotypes (LPS types~ Inaba and Ogawa ~23,36~ has not been c~arified.
~Parenterally administered cellular vaccines or '::l0 partially purified LPS~induce a statistically significant pr~tection against~cholera in adults (-60%) for -6 months [3,7,18,22,38,45]. Cellular vaccines are less effective for infants and young children and ineffective for contrsl of outbreaks of cholera [38~5l~ The protective immu~e moiety induced by these vaccines is proposed to be serum LPS anti~odies with vibriocidal activity
3 ~18 ~ 22 r 38 ~ 40] . ~ The cellular vacci~es do not elicit serum antit~xin~[37] nor, ~y analogy with simiIar products, secreto~y antibodies ~563. Similar effects are 2~ al~o ob~ained with orally administered inactivated V.
cholerae [7,10-lZ~]~ ~ddition of the B subunit of CT to : the f:ormulat;lon~o~f~this vaccine does no~ recruit :additional protection ~12].~
Although considered by many workers as a "marker"
and~not as a~protective moiaty, vibriocidal antibody : :levels~are~a~reliable method for predicting resistanGe to cholera. Serum vibriocidal activity is correlated with resistance ag,ainst dis~ase following convales~ence from cholera, by administration of live attenuated strains, or by inacti~a~ed V. cholerae alone or with the ~ subunit of . ~ .
CT. Also,~the age-rela~ed acquisition of vibriocidal antibodies in endemic areas parallels the increasing ~: resistance to ~holera observed in older children and adults [l,3,4,7,lO,Il,18,22,38,50]. Our interpretation : 35 of these data is that cellular cholera vaccines, as ~ 093/13797 212 g 212 PCTtUS93/00253 v observed with similar products and polysaccharides, are poor immunogens and have T-cell independent prGperties :~ ~37,49~. , ,: ,Based on similar reasoning, Kabir synthesized a bivalent con~ugate composed of NaOH-treated LPS from s~rotypes Inaba and Ogawa bound to a protein extract of V. cholerae 395 (ogawa) [27]. One mg of this conjugate, , ~, in complete Freund's; adjuvant, elicited antibodies in rabbits with vibriocidal activity against the two ::
:serotypes. The route of immunization, using CFA, and the dosage used ~are clinically unacceptable.
; ~ ~
Although~the~ use;of another component from V.
cholerae~may obscure the nature of protection elicited by our conjugates,~we chose CT because it served as an immunogenic carrler for both the H. influenzae type b ;and the Vi:polysa~ harides:[49,54]. Fuxther, there remains~the poss~ibility that:serum antitoxin, specific for the CT~o~f~the infecting strain,:may be protective or exert:synergistic;~:protective activity with LPS antibodies ;[~28,~:43~,;52~
,Con3~ugate~vacci~nes have many~advantages compared to ;cel~lular~vaccines~ no`serious adverse reactions are anticipated;~because the LPS~:levels arç low; 2) conjugated :saccharides`~can;~be~expected to~have greater ;immunogenic~ity~and T- ell dependent properties compared to~cellular~vaccines~[9,l4,3l,48], thus the conjugate may represent:a~sa~fer and:more immunogeni~ ~and thereby more e~ffective)~vaccine~ 3~::conjugates may be administer~d ;concurrently with~Diptheria and Tetanus toxoid, Pertussis (DTP~: and~H.~infIuenzae type~b~o~jugates to infants ~48], thus~the~con~ugate might; be incorporated into routine immunlzat,ion~of in~fants and children, that age :group~with th~e hlghest attack~rate in areas endemic for cholera ~38~]~and:4):the composition of our conjugate can be standardlzed:so~that the potency of new lots can be controlled by~ abaratory assays.
W093/137g7 P~T/US93/~025^ , 2~2~ 4-S ~ Y OF TH INVENTION
To sol~e the problems of adverse reactions, the lescer immunogenicity in infants and young children and the T-cell independent properties of t~e LPS in cellular vaccines, we synth sized conjuga~es, composed of detoxified LPS of V. c~olerae serotype Inaba, to several proteins including CT ~48]. The synthesis a~d immunologic properties of these vaccines, using CT as a carrier, are described infra.
The preparation ~f conjugates to elicit LPS
: 10 antibodies is di~ficult because; 1) the complete structures of the LPS of the two serotypes are not known : and; 2) the size of the Inaba O-SP is relatively small (approximately 6,000 daltons molecular weight (d)) (The : immunogenicity of saccharides alone or in conjugates is directly related to their siz~) ~2,17,54~. Tre2tment of LPS from strain 569B~(Inaba) with 1% acetic acid, lOO~C, for 90 minutes, results in a product of about 5,900 d, which has clinlca~lly acceptable levels of endotoxin but :: d~es not precipitate~w;ith hyperimmune sera. Treatment 20~ ~with hydrazine detoxifies the LPS to acceptable levels, resulting~i~:produ~ts having molecular weights of approxlmately 13,000 and 6,000-d and retaining their ; ` antigenicity. A~cordinglyt tne hydrazine treated LPS is use~ to prepare the conjugates. As demonstrated for O-SP
25 ~ of Shigella dysenteriae type 1 ~9], conjugates prepared ;by multipoint~a:tta~hment (DeA-LPS-CTII) elicit higher levels of LPS antlbodi~es than ~hose prepared by single point attachment ~DeA-LPS CTI). Our conjugates, injected subcutaneously in saline at 1/lOth the proposed 30 ~ human ~ose, elicit LPS antibodies with vibriocidal activity in young~:outbred mice. This immunization scheme was predictive of immunogenicity of H. influenzae type b-tetanus toxoid c3njugates in infants injected concurrently with DTP [49J. The low levels of "endotoxic" activity, as measured by the L~L and rabbit ' '.WO93/13797 PCT/US93/00253 21~21~
pyrogen a~says, provide assurance that our conjugates will elicit little or no adverse reactions encountered with cellular vaccines for cholera [25].
The most ~eneral description of the inventio~ is an anti-bacterial vaccine formulation which comprises a conjugate betwe~n lipopolysaccharide moietie~ derived from the target bacterial strain and proteins derived from the same s~rain. A vaccine is formulated using this conjuga~e and any of the pharmaceutically accepta~le : carriers, stabilizers, adjuvants and the like that are known in the art of vaccine preparation. Such a carrier : :; may be sterile saline for the preparation of an injectable vaccine. The conjugates may alss be incorporated into formulations currently in use in ~; : childhood immunization~protocols, i~ particular, the diptheria and tetanus toxoid, pertussis (DTP~ vaccine commonly a~ministered~to children in the United States.
A large adv ntage in terms of clinical usefulness of the conjugate v~c~ine is obtained by detaxifying the LPS
: ; component of the vaccine.: Such detoxificatio~ can be ~c~:~eved by~ removal of the esterified fatty acids from th~ lipid A component of the LPS using hydrazine or by acid ~ydrolysis ~f~ the LPS. Acfu.ordingly, one objçct of the pre:sent inYention is to provide LPS-protein conjugate :; vac:cines using~su~h a:detoxified: LPS componentO
25 ~ : ~ FurthermQre, it might ~be expected that c:onjugation of the detoxified; LPS to a~ protein synthesized by the bacterial target would provide a useful vac ::ine, particularly if a protein localized on the surface of th2 bacterium is~usedu A vaccine:which produc~s a ; 30 : neutralizing antibody response to toxins secreted by bacteria would~also be a useful vaccine. Accordingly, it is a second objec:t of the present invention to provide a vaccine wherein a detoxified LPS is conjugated to such a secreted toxin.
A preferred embodiment of the invention is the ~' ~
W093/13797 PCT/US93/002.
conjugation of detoxified LPS to a toxin protein produced by ~he bacterial target.
The conjugation reaction can be ~arried out using a variety of reagents. The conjugation can be directly ~; between the LPS and the protein or carried out using a cross-linking agent. Such a cross-linking agent can be a bifunctional linker. Examples of hifunctional linkers which can be employed in the present invention include, but are not limited to, adipic acid dihydrazide, diaminohexane, amino-~-caproic acid, and an N-hydrosuccinimide acid anhydride-ba-ed heterobifunctional linker.
As preferred~embodiments, two methods of conjugation are described for the LPS detoxified by treatment with hydrazine; reaction with N-succinimidyl 3-(2-~pyridyldithlo)~propionate (SPDP) or reaction with adipicacld~ dihydrazide (ADH) followed~by reaction with l-ethyl-3(3-dimethyl~aminopropy) carbodiimide (EDAC). The latter method results in~the formation of a covalently bonded aggregatc~(lattice)~ of the LPS-protein conjugate. A
'2~ preferred~ embodiment~of~the~invention utilizes one of these~methods o~f~conJugation.~
The~polyclonal or monoclonal antibodies raised by administrati~n~of~the conjugate~vaccine to a laboratory animal~may~f~ind~use~as components;o~ a diagnostic kit or ~as~companents~of~ a; method of treatment of infection by the~ targeted~organ~ism.~ Accordingly, it is another object of the invention~to provide for~diagnostic kits for the detecbion of~or~anisms bearing either the LPS or protein portlon,~or both;~, o~f the conjugate. A final object of the~invention is~to provide antibodies which might be used to treat infections caused by an organism ~earing either the~LPS or protein portion, or both, of the conjugate or~whlch~neutralize a toxin secreted by such an ; organism.
35 ~ ;
:
.
-~: :
~:~VO93/13797 2 ~ 2 PCT/USg3/00253 BRIEF DESCRIPTION OF THE DRA~INGS
Figure l shows a silver-stained SDS-PAGE gel (14%3 of 2.5 mg ~PS from Vibrio cholerae serotype Inaba (lane l) and Escherichia coli Olll (lane 2).
Figure 2 shows ~he characterization of LPS and LPS-CT conjugates by double immunodiffusion. Left: A.
Hyperimmune Vibrio cholerae serotype Inaba serum, outer wells: l - Inaba LPS, 250 mlml, 2 - Inaba DeA;LPS, 250 : mlml, 3 - Inaba 0 SP, 250 mlml. Right: A. Hyperimmune V. cholerae serotype Inaba serum, B. hyperimmune cholera toxin an iserum, 3 Inaba DeA-LPS, 250 mlml, 4 -Con}ugate DeA-LPS-CTII.
Figure 3 shows13C nuclear magnetic resonance spectrum of the hydrazine-treated lipopolysac~haride (DeA-LPS) from Vibrio cholerae, serotype Inaba. The l9 15; major signals are identical o t~ose reported by Xenne et al., ~30]. The 13C N.M.R. spectrum of the acid~reated }ipop~ly~accharide (O-SP) was almost identical to this spectrum.
Figure 4 shows HPLC profiles of l00 mL samples (l.0 mg/ml) thrvugh a l0x300 mm column of Superose 12 in 0.2 M
Na~l, 0.0l M TRIS, 0.00l M EDTA, 0~25% deoxycholic acid, pH~8. a. LPS serotype Inaba; b. DeA-LPS Inaba; c. 0-SP
Inaba.
DIscLnsuRE OF THE INVENTION
~: 25 : ~Scientific~papers and other literature cited in this application are incorporated herein by r~ference in their entirety.
~, ~ Preferred embodiments o the invention are described in detail below by means of representative examples.
~:: 30 Th~se examples are meant only as illustrations of the invention and are~not to be taken as limiting of the scope of the in~ention.
Chemical reagents for executing the procedures described in the examples can be obtained from the sources noted below:
.
W093/13797 PCT/US93/0025~s`
Anhydrous hydrazine (Lot 104F-3523), adipic acid dihyrazide (ADH, Lot 77F-5016), dithiothreitol (DTT, Lot 49F-0138), l-ethyl-3(3-dimethylaminopropyl~ carbodiimide (EDAC, ~ot 105F-0308), disodium EDTA (Lot ll9F-0275), ; XDO, RNase (Lot 12~8F-0462), DNase (Lot 89F-9605) and pronase (Lot ~9F-0391) can be purchased from Si~ma ;
Chemical Co., St. Louis, MO. HEPES (Lot 051790) and : deoxycholi~ acid (Lot 264I0l) can be purchased from ; Calbiochem, La Jolla, CA. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Lot 900707084), alum (Lot 891120103:) and 8CA reagent for protein determination;can be:~obtained from Pierce Chemical Co, ~ :: Rockford, TL.~ Cyanog~en bromide (CNBr, Lot 014783A) can be ~ purchased from Eastman Chemical, Rochester, NY. Sephadex G-25 (Lot Pl0036),~Sephacryl S-300, l0x300 mm Superose 12 co1umn~and dextrans~for~molecular weight assay can be purchased from~Pharmac1a-LKB,~ Piscataway, NJ. LPS from V.: d olerae~:strain~569B~(Inaba) can~be purchased from List Biologicals~,~Campbell,~CA.~ Limulus amoebocyte lysate ~L~L)~can be purchased~from Associates of Cape Cod,~Woods~Ho1e,;:MA;.~; p-nitrophenyl phosphate can be obtained~from`Fluka,~:Ronkonkoma,~ NY. The US Standard for endotoxin~oan be~obtainet from Donald Hochs~e~int U.nited States~:~Food~and~Drug~Admin1stration ~253. Cholera toxin, variant~ Lot~58~2~can~be obtained from Pasteur Merieux 5erums~&~Vaccins~ Lyon~ France) and cholera toxin variant lot rst is~ purified from V. c holerae Inaba strain 569B
2lr28~].~ Anti-mouse~IgG:and IgM~alkaline phosphatase conjugates:can be:~purchased~from Kirkegaard & Perry Laboratories,~ nc,~ Gaithersburg,~MD.
The bacteri~al~:;stra1ns used:1n the examples are: V.
cholerae, biotype~c1asslca1, serotype Inaba, strain 569B
:and V.: cho1~erae, b1otype class1cal, serotype Ogawa strain NIH 41:are~used~for vibriocida1 assay. V. cholerae, :: : classical Inaba:strain~2524 (Katherine Greene, C.D.C., : 35 `Atlanta, GA1 is ~used for raising antiserum in mice 2 1 ~ 8 2 1 2 PCr/US93/0~253 against the LPS. V. chol erae, serotype Inaba, biotype El Tor, cholera toxin (C~) variant 2, strain 075, is a recent isolate from South America (Richard Haberberger, Naval Research Medical Institute, Bethesda, MD~. All of these strains may be obtained by contacting the laboratory of Dr. Shousun Szu, Laboratory of Developmen~al and Molecular Immunity, National Institute of Child Health and Human Developmentl National ~: Institutes of Health, Bethesda, MD 20892 or the laboratory of Dr. John:B. Robbins at the same address.
Equivalent strains may be~obtained from the American Type Culture Collection, Rockville, MD. These strains are catalogued as ~Vibrio cholerae ~Inaba), ATCC 9459 and Vibrio cholerae (Inaba, biotype El Tor) ATCC 14033.
15;~ Exa~le l: Production~and characterization of detoxified i~opolysaccharides ::~ ~
LPS is detoxif:ied;by two methods. For acid-hydrolysis, LPS~ lO mg/ml in :1% acetic acid is heated~at lOO~'C~for;90 min~59]~. The r~action mixture is ultracentrifuged~at~60,000 x:g, lOnC, for 5 hr. and the supernatant~passed~through a sterile 0.22 micron filter (Nalge,~Roche~ster,:NY): and~ freeze-dried (d~signated as 0-SP)~ For~detoxi:fication by~base-hydrolysis:, LPS at 10 25 :: mg/ml,~is treated~wit~hydrazine at 37~C for 2 hr.
Hydrazine treatment has been reported to remove esterified~fatty~acids:~from the lipid A, accordingly this product is designated as DeA-LPS. This material is mixed with acetone in~an~lce bath until a precipitate formed 30 : (approx;imately~90%:a:cetone) and the reac~ion mixture : centrlfuged~at~15,000::x g, lO C, for 30 min. The : precipitate is~dissolYed in 0.15 M NaCl, pH 7.0 to about 3 mg/ml. The~reaction mixture is centrifuged at 60,000 x g for 5 hr. at~10-C, the supernatant dialyzed against H20 exhaustively, passed:~through a 0.22 micron filter, and WO 93/13797 PCI`/US93/002~
~.?, ?. ~'Z~ h freeze dried. The protein and nucleic acid concentration of the 0-SP and the DeA-LPS are ~1%. LPS, extracted from acetone-dried V. holerae cel}s of El Tor biotype Ogawa serotype strain 3083-13, is used for inhibition of vibriocidal activity.
The~LPS~is sub~ected to.various preliminary characterizations,~using~both in vitro and, in vivo techniques. ~SDS-PAG~E~is used for detection of LPS [56].
LPS concentration,~assàyed by LAL, is expressed in endotoxin units ~(EU)~ related to the US standard [25].
10~ The molecular sizes~of~LPS, 0-SP and~deacylated LPS
tD'eA-1PS~) are~ estimated~by gel;filtration through Superose~12~in 0.2 M NaCl, l~mM EDTA, 10 mM Tris, 0.25%
deoxycholic acid,;~pH~8~.0,~using the dextran standards to calibrate t~e column.~KDO is measured by the thiobarblturic~acid~assay,~using KDO~ as a standard [5].
Double immunod~i~ffusi'on is performed in 1% agarose in ph~ ~ ate- ~ Pe d~sal~ine~(PBS)~ Data for` ~ spectra a're~-~recorded~on a~JEO~GSX-500 spectrometer. Each s ~ ~ is~;a ~ ired`~with'~broad-band~H~decoupling at 90-,20~ 0~msec~ca`rb,on~observed~pulse~: 32~, aoo data points whichar`e;~zero-fil~le~d~to~ 64~,~000~points~prior to Fourier transormation'~ 30'~KHz~spectral~window~ (0.54 sec ;ac ~ isition~;t~ e~ 3.0~s c~d lay~be ~ een pulse cycles.
Prior~to'FQuri~r~transformation;~each free-induction-decay a~ is expon~ent~ial~1y~,multiplied so as to result in an additional 4Mz'~ ne-broadenin~in the frequency domain ctrum.~ App~xima~tely~10 mg~o~each samplè are dissolved in;0.5'~mL of D O and recorded at ambient probe temperature;~,(21-~C)~
,~ S~i1vér-sta~ined~SDS~PAGE~of~2.~5 mg of LPS from Inaba shows~2~faint~ban~ds with a "~la~dder" in the middle and two dense bands~nea'r~ th~e~bottom of~-~the gel (Fig. 1). Typical "ladders" of hi,gher~mo~lecular~weight 0-SP are ~formed by the~LPS~from~;E~ coll 0111.;~ No~bands are observed with 10 ~mg~samples of;either the 0-SP~or the DeA-LPS. The LPS of ~093/13797 21 ~ ~ 2 1 ~ PCT/US93/00253 v serotype Inaba has 3-6x103 EU/mg and the DeA-LPS had 3 EU/mg by LAL assay: this level represents >1000-fold reduction~ Immu~odiffusion showc a single band of precipitation between the LPS and the hyperinmune LPS
sera (Fig~ 2). A less intense and more di~fuse band is observed with the DeA-LPS which yields a partial identity reaction with the LPS. Neither the 0-SP nor the CT
pr~cipitates with this hyperimmune serum. The molecular : sizes of the LPS, 0-SP and DeA-LPS are estimated by HPLC
on Superose 12 (Fig. 3~. The LPS and DeA-LPS show two peaks: the LPS has Rd val~es of 0.~0 (16tOOO d) and 0.46 (8,700 d) and the DeA-LPS has Kd values of 0.38 (13,000 d) and 0.50: (6,000 d). The 0-SP exhi~ited only one peak corresponding to the second peak of the DeA-LPS (Kd 0.51, ,900 d~. Because of its greater antigenlcity and high molecular weight~, DeA-LPS is preferably used as the ; saccharide ~or the conjugates. We cannot detect KD0 in ; e:ither the:0-SP:~or:the DeA-LPS by the ~hiobar~ituric acid assay~ tS~57~. ~The~3C NMR spectra of the DeA-LPS and 0-SP are in a~reement with pr vious report~ ~30,47].
;20 ~ Each spectrum sh w s 10 major signals with identical, or nearly identical,~chemi~al shifts ~o those reported (Fig.
Example~2:~ Production~and characterization of DeA-2~ LPS-Cholera Toxin~conluqates : Conjugation of~DeA-LPS with proteins is performed using either.of two methods. In method 1, the covalent attachment :o. the LPS to the protein is accomplished by using~ SPDP;to~thi:olate both the: protein and the DeA-LPS
as described:for:the cell wall polysaccharide of pneumococci [52~].:: DeA-LPS ~3 mg/ml) or protein (lo : mg/ml) are dissolved in 0.15 M HEPES, 2 mM EDTA, pH 7.5.
SP~P (20 mM in ethanol) is added dropwise at weight ratios of 0.5 for DeA-LPS and 0.2 for protein. The :~
~:
:~:
~,~?~..;.~;,~.3;'f~ r '~
W093/13?9~2~ 2 PCr/USg3/002,.`'"' reaction mixture is stirred at ambient temperature for 1 hrO and passed through a 5x35 cm Sephadex G-25 column in H2O for DeA-LPS--SPDP and in PBS for pro~ein. The D~A LPS-SP~P is freeæe-dried and the protein is concentrated by membrane filtration (Amicon, YM10). The extent of derivatization with SPDP in aliqunts of DeA-LPS
or he proteins is determined spectrophotometrically following reduction of the N-pyridyl disulfide bond with 40 mM DTT and ~ssuming a molar extinction coefficient at 340 nm of 8.08x104 [53]. The N-pyridyl disulfide on the DeA LPS-SPDP is reduced with 40 mM DTT, passed through a 2.5x50 cm column of G-25 S~phadex in 0.2 M NaCl and the void volume fractions mixed with the SPDP derivative of the protein. This r action mixture is stirred at room tPmperature~for 2 hrs., passed through a 5x100 cm column of S-300 Sephacryl:in 0.2 M NaCl and the void volume fractions pooIed:.: Th~ conjugate synthesized by this method using Cholera Toxin as the protein component is designa ed as DeA-LPSCTIO An aliquot of DeA-LP5-CTI in sa:line i treated with 0.05 M ED~C at room temperature for 1 hr~ at pH 6.0 to cross-link the conjuga~e. The non-reacted ED~ is removed~by exhaustive dialysis agalnst:water. ~ ~:
In method~2~ DeA-LPS is derivatized with ~DH as described for Naemophilus influenz~e type b ~9,48].
DeA-LPS, 10:mg/ml::in ~aline, is~brought to pH 10.5 with 1 NaO8~and an equal weight of~CNBr (1 g/ml in ace`tonitrile) is added. The pH is maintained be~ween 10.0 and 11.0 with 1 N NaOH for 3 minutes. An equal volume of 0.5 M ADH in 0.5 M NaHCO3 is added and the pH adjusted to 8.5. The reaction mixture is stirred at room temperature for 1 hr. and then at 3-8 C ov~rnight and passed through a 5x35 cm Sephadex G-2~ column in H2O. Fractions from the void ~olume are~ pooled and freeze dri~d. The DeA-LPS-AH
derivative is dissolved in 0.I5 M NaCl to 10 mg/ml. An equal volume of protein (~10 mg/ml) is added and the pH
~V 93/13797 21~ ~ 212 PCT/US93/00253 adjusted to 5.S with O.l M HCl. EDAC is added to a final concentration of 0.05 M and the pH is maintained at 5.5-6.0 for l hr.. The reaction mix~ure is passed through 2.5x90 cm column of S-300 Sephacryl in 0.2 M NaCl and the fractions in the void volume are pooled.
Conjugates synthesized using CT (Lot 5~2) and CT tLot rst) as the prbtein component are d~signated as DeA-LPS-CTII and DeA-LPS-CTIII.
.
As in example 1, the conjugates are characterized by : several in vi~ro and in vivo methods. The extent of derivatization of DeA-LPS with adipic acid hydrazide is ~ ~ ~ measured by reactlon with trinitrobenzene sulfonic acid : (TNBS) using ADH as a standard [9]. Protein is measured by the BCA reagent using bovine serum albumin as a : standard ~17~. Hexose is measured by the ~nthrone ; 15 react~ion using:the:0-SP as the stardard [55]. Endotoxin concentration~is~estimated as in Example l. Double immunodiffusion~is also performed as in Example l. In : vitro cy*otoxioity of CT is measured by obsexYation of élongati~n of CHO cells [ 15 ] o Pyrogenicity of the 2;0~ conjugate is assayed in rabbits using the meth~d of Hochstein et al.~ C2~].
Table l hows~some~of the results of characterization of the conjugakes.
:~
i, j i .
30::
; 35 .
: :`
~ .
WO ~3/13797 Pcr/US93/OO~ ~
~,~?.~ 14-Tab . Chara~terization of Vibrio cholera~ hydrazine-treated lipopolysaccharide-protein :~: conJugates.
: DeA-LPS/Composition DeA -LPS protein Yield protein mglml Conjugate ~ Linker (wt/wt) ~(wt/wtj 9~ wt/wt protem CHO
. _ ~ _ . .
DeA-LPS-CT~ SPDP 1.18 :~ 8.8 88 ~ 0.72 2.Q 1.44 ~ : ~ . ;
DeA-LPS-CT~* SPDP ~l.~I8 ~ 8.8 ~ NA 0.65 1.15 0.25 : ~~ ~ : , ~ , DeA-LPS-CTD ADH~ 1.76 ; : : NA 79 0.80 0.48 0.38 : : ~ : ~ , :
DeA-LPS-cTm: ADH :: : 1.76 ~: ~: ~ NA ~ 88~ 1.50 1.0 1.50 : ~ ~ ~ ~ :
*Fùrther; -~atecl ~with :O.OSM: w~ EDAC ~(MATERJ.ALS AND METHODS) NA:`not~icable ~
Polys~tccha~ide~measured~by anthrone~ reaction~with DeA-LPS~ a standard [ ]. The yield alculat~d ~based upon;~e~ weight of the sacchaJide~:in :the con~ugate compared to the wçight:of t~e:adipic~acld hydrazide derivati~e. ~: ~
:,, ~ ~D93/13797 2 1 2 8 2 1 2 P~T/US93/00253 v The extent of derivatization of the DeA-LPS with SPDP or with ADH, is similar. The De~-LPS/protein (wt/wt) ratios are slightly lower for the conjugat~s of ;~ : CT prepared with SPDP than with ADH, ranging from 0.72 for DeA-LPS-CTI to 1.5 for DeA-LPS-CTII. The yields for all the conjugates are ~80~ as calculated by the re~overy of saccharide in the conjugates compared to the derivatized polysaccharide. Similar results are obtained by Method~II: with tetanus toxoid as the protein component of the conjugate. ~ representative double im~unodiffusion experiment shows that the serotype Inaba hyperimmune~antiserum~yields an identical line of precipItation~with the::DeA-LPS and DeA-LPS-CTII (Fig. 2).
SimilarIy the CT;~and LPS antisera yields a line of identity~with~the-~DeA-LPS-CTII and~the CT. A faint spur fr~m the CT antiserum~extends over the LPS antiserum and :the conjugate~,~:suggesting:that:there is a slight amount of unbound CT in~:this prepara~ion. The residual ~oxicity :of:the CT and the~DeA-LPS in the conjugateC as estimated by~ he in vitro~and~}~_y}~Q~:~assays described abovç is ~ery~low. ~In~the~thermal~inductlon~test, the DeA-LPS was not pyro~enic when~ injected~at 1 mg/Kg rabbit body weight.~The endotoxin~content of the conjugates was ~2 EU/mg: by thé: LAL:~assay. CT induced elongation in CHO
cel:ls:at 0.4 ng/m~. The:amount of CT, as a conju~ated 5~ form~required~to~elicit~the sa~e~degree of elongation was lO3:~to~:101~g~reater~ DeA-LPS-CTIII, a preparation : intended for ~lini;cal~use,~ad no detectable toxicity in CH0 cell~assay~;at~l~.0 mq/ml and passed the general safety :te~st in guinea~plgs~at lO human doses (25 m~;~DeA-LPS per ~:dose) as described~in~the~Code of Federal Regulations C.
Example 3: ComDarison of efflcac~_of cellular and LPS-CT
con~uqate~vaccines~
: Hyperimmune LPS antiserum is prepared by injecting ~: ~
":: :~ :
WO 93/137g7 PCI`/US93/002',~
female, adult BAL~/c mice with heat-killed V. chol~rae strain 2524 ~41]. Burro CT antiserum is prepared as described [13]. For evaluation of immunogenicity, 6-weeks-old B~LB/~ or general purpose m~ce (NIH) are injected subcutaneously with 2.5 mg or 10 mg of DeA-LPS
alone or as a conjugate in saline. Mice are injected at 2 week intervals and`bled 7 days after each immunization.
The fourth dose is given 4 weeks after the third injection and mlce are~bled 7 days and 6 months later.
Groups of mice:are immunized similarly with conjugates adsorbed with 0.:125 or 1.2S mg of aluminum hydroxide per , ~
dose. Cellular cholera~vaccine (purchasable from Wyeth Ayerst Laboratories, Marietta, PAj containing 4x109 :~ ~ each of Inaba~and Ogawa serotypes, is used as a control.
Mice are immunized with:0.1 or 0.2 ml of the vaccine 5 ~ Complement-mediated~vibr~iocida} antibody is measured :against Inaba~:and~Ogawa s~rains tl9,20~. Ten-fold serum dilutions are:~mlxed;~with equal~volumes of -lO00 cells/ml c~ :diluted guinea~pig~senum;and:~incubated at 37'C for 1 hr.
A~hyperimmune~s~erum~is~used as~standard in each assay.
;20~ ~The~serum~titer~is~expressed~as~the~recip~ocal sf the highest diluti~on~ of::serum that~yielded 50%;vibriocidal activity. ~Some~;sera were~assayed for vibriocidal .
antibodies~agai~nst strains 569B and 075 of serotype Inaba~ the~:titers:~of~these~sera were identical against both strains.~ There:~ore, vibrlocidal activities of the sera~àre~ass;ayed~w;ith~s~rain 569B. Inhibition of vibrlocidal ~activity: is assayed by mixing 100 mg/ml of LPS,: DeA-LPS,~:O-SP:or CT with various dilutions of antisera at~:~37~C~for~I:hr. prlor to the addition of the bacte~ia ~20~
LPS~and;proteln~antibody levels are determined by enzyme-linked lmmunosorbent assay (ELISA) using Immunolon
cholerae [7,10-lZ~]~ ~ddition of the B subunit of CT to : the f:ormulat;lon~o~f~this vaccine does no~ recruit :additional protection ~12].~
Although considered by many workers as a "marker"
and~not as a~protective moiaty, vibriocidal antibody : :levels~are~a~reliable method for predicting resistanGe to cholera. Serum vibriocidal activity is correlated with resistance ag,ainst dis~ase following convales~ence from cholera, by administration of live attenuated strains, or by inacti~a~ed V. cholerae alone or with the ~ subunit of . ~ .
CT. Also,~the age-rela~ed acquisition of vibriocidal antibodies in endemic areas parallels the increasing ~: resistance to ~holera observed in older children and adults [l,3,4,7,lO,Il,18,22,38,50]. Our interpretation : 35 of these data is that cellular cholera vaccines, as ~ 093/13797 212 g 212 PCTtUS93/00253 v observed with similar products and polysaccharides, are poor immunogens and have T-cell independent prGperties :~ ~37,49~. , ,: ,Based on similar reasoning, Kabir synthesized a bivalent con~ugate composed of NaOH-treated LPS from s~rotypes Inaba and Ogawa bound to a protein extract of V. cholerae 395 (ogawa) [27]. One mg of this conjugate, , ~, in complete Freund's; adjuvant, elicited antibodies in rabbits with vibriocidal activity against the two ::
:serotypes. The route of immunization, using CFA, and the dosage used ~are clinically unacceptable.
; ~ ~
Although~the~ use;of another component from V.
cholerae~may obscure the nature of protection elicited by our conjugates,~we chose CT because it served as an immunogenic carrler for both the H. influenzae type b ;and the Vi:polysa~ harides:[49,54]. Fuxther, there remains~the poss~ibility that:serum antitoxin, specific for the CT~o~f~the infecting strain,:may be protective or exert:synergistic;~:protective activity with LPS antibodies ;[~28,~:43~,;52~
,Con3~ugate~vacci~nes have many~advantages compared to ;cel~lular~vaccines~ no`serious adverse reactions are anticipated;~because the LPS~:levels arç low; 2) conjugated :saccharides`~can;~be~expected to~have greater ;immunogenic~ity~and T- ell dependent properties compared to~cellular~vaccines~[9,l4,3l,48], thus the conjugate may represent:a~sa~fer and:more immunogeni~ ~and thereby more e~ffective)~vaccine~ 3~::conjugates may be administer~d ;concurrently with~Diptheria and Tetanus toxoid, Pertussis (DTP~: and~H.~infIuenzae type~b~o~jugates to infants ~48], thus~the~con~ugate might; be incorporated into routine immunlzat,ion~of in~fants and children, that age :group~with th~e hlghest attack~rate in areas endemic for cholera ~38~]~and:4):the composition of our conjugate can be standardlzed:so~that the potency of new lots can be controlled by~ abaratory assays.
W093/137g7 P~T/US93/~025^ , 2~2~ 4-S ~ Y OF TH INVENTION
To sol~e the problems of adverse reactions, the lescer immunogenicity in infants and young children and the T-cell independent properties of t~e LPS in cellular vaccines, we synth sized conjuga~es, composed of detoxified LPS of V. c~olerae serotype Inaba, to several proteins including CT ~48]. The synthesis a~d immunologic properties of these vaccines, using CT as a carrier, are described infra.
The preparation ~f conjugates to elicit LPS
: 10 antibodies is di~ficult because; 1) the complete structures of the LPS of the two serotypes are not known : and; 2) the size of the Inaba O-SP is relatively small (approximately 6,000 daltons molecular weight (d)) (The : immunogenicity of saccharides alone or in conjugates is directly related to their siz~) ~2,17,54~. Tre2tment of LPS from strain 569B~(Inaba) with 1% acetic acid, lOO~C, for 90 minutes, results in a product of about 5,900 d, which has clinlca~lly acceptable levels of endotoxin but :: d~es not precipitate~w;ith hyperimmune sera. Treatment 20~ ~with hydrazine detoxifies the LPS to acceptable levels, resulting~i~:produ~ts having molecular weights of approxlmately 13,000 and 6,000-d and retaining their ; ` antigenicity. A~cordinglyt tne hydrazine treated LPS is use~ to prepare the conjugates. As demonstrated for O-SP
25 ~ of Shigella dysenteriae type 1 ~9], conjugates prepared ;by multipoint~a:tta~hment (DeA-LPS-CTII) elicit higher levels of LPS antlbodi~es than ~hose prepared by single point attachment ~DeA-LPS CTI). Our conjugates, injected subcutaneously in saline at 1/lOth the proposed 30 ~ human ~ose, elicit LPS antibodies with vibriocidal activity in young~:outbred mice. This immunization scheme was predictive of immunogenicity of H. influenzae type b-tetanus toxoid c3njugates in infants injected concurrently with DTP [49J. The low levels of "endotoxic" activity, as measured by the L~L and rabbit ' '.WO93/13797 PCT/US93/00253 21~21~
pyrogen a~says, provide assurance that our conjugates will elicit little or no adverse reactions encountered with cellular vaccines for cholera [25].
The most ~eneral description of the inventio~ is an anti-bacterial vaccine formulation which comprises a conjugate betwe~n lipopolysaccharide moietie~ derived from the target bacterial strain and proteins derived from the same s~rain. A vaccine is formulated using this conjuga~e and any of the pharmaceutically accepta~le : carriers, stabilizers, adjuvants and the like that are known in the art of vaccine preparation. Such a carrier : :; may be sterile saline for the preparation of an injectable vaccine. The conjugates may alss be incorporated into formulations currently in use in ~; : childhood immunization~protocols, i~ particular, the diptheria and tetanus toxoid, pertussis (DTP~ vaccine commonly a~ministered~to children in the United States.
A large adv ntage in terms of clinical usefulness of the conjugate v~c~ine is obtained by detaxifying the LPS
: ; component of the vaccine.: Such detoxificatio~ can be ~c~:~eved by~ removal of the esterified fatty acids from th~ lipid A component of the LPS using hydrazine or by acid ~ydrolysis ~f~ the LPS. Acfu.ordingly, one objçct of the pre:sent inYention is to provide LPS-protein conjugate :; vac:cines using~su~h a:detoxified: LPS componentO
25 ~ : ~ FurthermQre, it might ~be expected that c:onjugation of the detoxified; LPS to a~ protein synthesized by the bacterial target would provide a useful vac ::ine, particularly if a protein localized on the surface of th2 bacterium is~usedu A vaccine:which produc~s a ; 30 : neutralizing antibody response to toxins secreted by bacteria would~also be a useful vaccine. Accordingly, it is a second objec:t of the present invention to provide a vaccine wherein a detoxified LPS is conjugated to such a secreted toxin.
A preferred embodiment of the invention is the ~' ~
W093/13797 PCT/US93/002.
conjugation of detoxified LPS to a toxin protein produced by ~he bacterial target.
The conjugation reaction can be ~arried out using a variety of reagents. The conjugation can be directly ~; between the LPS and the protein or carried out using a cross-linking agent. Such a cross-linking agent can be a bifunctional linker. Examples of hifunctional linkers which can be employed in the present invention include, but are not limited to, adipic acid dihydrazide, diaminohexane, amino-~-caproic acid, and an N-hydrosuccinimide acid anhydride-ba-ed heterobifunctional linker.
As preferred~embodiments, two methods of conjugation are described for the LPS detoxified by treatment with hydrazine; reaction with N-succinimidyl 3-(2-~pyridyldithlo)~propionate (SPDP) or reaction with adipicacld~ dihydrazide (ADH) followed~by reaction with l-ethyl-3(3-dimethyl~aminopropy) carbodiimide (EDAC). The latter method results in~the formation of a covalently bonded aggregatc~(lattice)~ of the LPS-protein conjugate. A
'2~ preferred~ embodiment~of~the~invention utilizes one of these~methods o~f~conJugation.~
The~polyclonal or monoclonal antibodies raised by administrati~n~of~the conjugate~vaccine to a laboratory animal~may~f~ind~use~as components;o~ a diagnostic kit or ~as~companents~of~ a; method of treatment of infection by the~ targeted~organ~ism.~ Accordingly, it is another object of the invention~to provide for~diagnostic kits for the detecbion of~or~anisms bearing either the LPS or protein portlon,~or both;~, o~f the conjugate. A final object of the~invention is~to provide antibodies which might be used to treat infections caused by an organism ~earing either the~LPS or protein portion, or both, of the conjugate or~whlch~neutralize a toxin secreted by such an ; organism.
35 ~ ;
:
.
-~: :
~:~VO93/13797 2 ~ 2 PCT/USg3/00253 BRIEF DESCRIPTION OF THE DRA~INGS
Figure l shows a silver-stained SDS-PAGE gel (14%3 of 2.5 mg ~PS from Vibrio cholerae serotype Inaba (lane l) and Escherichia coli Olll (lane 2).
Figure 2 shows ~he characterization of LPS and LPS-CT conjugates by double immunodiffusion. Left: A.
Hyperimmune Vibrio cholerae serotype Inaba serum, outer wells: l - Inaba LPS, 250 mlml, 2 - Inaba DeA;LPS, 250 : mlml, 3 - Inaba 0 SP, 250 mlml. Right: A. Hyperimmune V. cholerae serotype Inaba serum, B. hyperimmune cholera toxin an iserum, 3 Inaba DeA-LPS, 250 mlml, 4 -Con}ugate DeA-LPS-CTII.
Figure 3 shows13C nuclear magnetic resonance spectrum of the hydrazine-treated lipopolysac~haride (DeA-LPS) from Vibrio cholerae, serotype Inaba. The l9 15; major signals are identical o t~ose reported by Xenne et al., ~30]. The 13C N.M.R. spectrum of the acid~reated }ipop~ly~accharide (O-SP) was almost identical to this spectrum.
Figure 4 shows HPLC profiles of l00 mL samples (l.0 mg/ml) thrvugh a l0x300 mm column of Superose 12 in 0.2 M
Na~l, 0.0l M TRIS, 0.00l M EDTA, 0~25% deoxycholic acid, pH~8. a. LPS serotype Inaba; b. DeA-LPS Inaba; c. 0-SP
Inaba.
DIscLnsuRE OF THE INVENTION
~: 25 : ~Scientific~papers and other literature cited in this application are incorporated herein by r~ference in their entirety.
~, ~ Preferred embodiments o the invention are described in detail below by means of representative examples.
~:: 30 Th~se examples are meant only as illustrations of the invention and are~not to be taken as limiting of the scope of the in~ention.
Chemical reagents for executing the procedures described in the examples can be obtained from the sources noted below:
.
W093/13797 PCT/US93/0025~s`
Anhydrous hydrazine (Lot 104F-3523), adipic acid dihyrazide (ADH, Lot 77F-5016), dithiothreitol (DTT, Lot 49F-0138), l-ethyl-3(3-dimethylaminopropyl~ carbodiimide (EDAC, ~ot 105F-0308), disodium EDTA (Lot ll9F-0275), ; XDO, RNase (Lot 12~8F-0462), DNase (Lot 89F-9605) and pronase (Lot ~9F-0391) can be purchased from Si~ma ;
Chemical Co., St. Louis, MO. HEPES (Lot 051790) and : deoxycholi~ acid (Lot 264I0l) can be purchased from ; Calbiochem, La Jolla, CA. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Lot 900707084), alum (Lot 891120103:) and 8CA reagent for protein determination;can be:~obtained from Pierce Chemical Co, ~ :: Rockford, TL.~ Cyanog~en bromide (CNBr, Lot 014783A) can be ~ purchased from Eastman Chemical, Rochester, NY. Sephadex G-25 (Lot Pl0036),~Sephacryl S-300, l0x300 mm Superose 12 co1umn~and dextrans~for~molecular weight assay can be purchased from~Pharmac1a-LKB,~ Piscataway, NJ. LPS from V.: d olerae~:strain~569B~(Inaba) can~be purchased from List Biologicals~,~Campbell,~CA.~ Limulus amoebocyte lysate ~L~L)~can be purchased~from Associates of Cape Cod,~Woods~Ho1e,;:MA;.~; p-nitrophenyl phosphate can be obtained~from`Fluka,~:Ronkonkoma,~ NY. The US Standard for endotoxin~oan be~obtainet from Donald Hochs~e~int U.nited States~:~Food~and~Drug~Admin1stration ~253. Cholera toxin, variant~ Lot~58~2~can~be obtained from Pasteur Merieux 5erums~&~Vaccins~ Lyon~ France) and cholera toxin variant lot rst is~ purified from V. c holerae Inaba strain 569B
2lr28~].~ Anti-mouse~IgG:and IgM~alkaline phosphatase conjugates:can be:~purchased~from Kirkegaard & Perry Laboratories,~ nc,~ Gaithersburg,~MD.
The bacteri~al~:;stra1ns used:1n the examples are: V.
cholerae, biotype~c1asslca1, serotype Inaba, strain 569B
:and V.: cho1~erae, b1otype class1cal, serotype Ogawa strain NIH 41:are~used~for vibriocida1 assay. V. cholerae, :: : classical Inaba:strain~2524 (Katherine Greene, C.D.C., : 35 `Atlanta, GA1 is ~used for raising antiserum in mice 2 1 ~ 8 2 1 2 PCr/US93/0~253 against the LPS. V. chol erae, serotype Inaba, biotype El Tor, cholera toxin (C~) variant 2, strain 075, is a recent isolate from South America (Richard Haberberger, Naval Research Medical Institute, Bethesda, MD~. All of these strains may be obtained by contacting the laboratory of Dr. Shousun Szu, Laboratory of Developmen~al and Molecular Immunity, National Institute of Child Health and Human Developmentl National ~: Institutes of Health, Bethesda, MD 20892 or the laboratory of Dr. John:B. Robbins at the same address.
Equivalent strains may be~obtained from the American Type Culture Collection, Rockville, MD. These strains are catalogued as ~Vibrio cholerae ~Inaba), ATCC 9459 and Vibrio cholerae (Inaba, biotype El Tor) ATCC 14033.
15;~ Exa~le l: Production~and characterization of detoxified i~opolysaccharides ::~ ~
LPS is detoxif:ied;by two methods. For acid-hydrolysis, LPS~ lO mg/ml in :1% acetic acid is heated~at lOO~'C~for;90 min~59]~. The r~action mixture is ultracentrifuged~at~60,000 x:g, lOnC, for 5 hr. and the supernatant~passed~through a sterile 0.22 micron filter (Nalge,~Roche~ster,:NY): and~ freeze-dried (d~signated as 0-SP)~ For~detoxi:fication by~base-hydrolysis:, LPS at 10 25 :: mg/ml,~is treated~wit~hydrazine at 37~C for 2 hr.
Hydrazine treatment has been reported to remove esterified~fatty~acids:~from the lipid A, accordingly this product is designated as DeA-LPS. This material is mixed with acetone in~an~lce bath until a precipitate formed 30 : (approx;imately~90%:a:cetone) and the reac~ion mixture : centrlfuged~at~15,000::x g, lO C, for 30 min. The : precipitate is~dissolYed in 0.15 M NaCl, pH 7.0 to about 3 mg/ml. The~reaction mixture is centrifuged at 60,000 x g for 5 hr. at~10-C, the supernatant dialyzed against H20 exhaustively, passed:~through a 0.22 micron filter, and WO 93/13797 PCI`/US93/002~
~.?, ?. ~'Z~ h freeze dried. The protein and nucleic acid concentration of the 0-SP and the DeA-LPS are ~1%. LPS, extracted from acetone-dried V. holerae cel}s of El Tor biotype Ogawa serotype strain 3083-13, is used for inhibition of vibriocidal activity.
The~LPS~is sub~ected to.various preliminary characterizations,~using~both in vitro and, in vivo techniques. ~SDS-PAG~E~is used for detection of LPS [56].
LPS concentration,~assàyed by LAL, is expressed in endotoxin units ~(EU)~ related to the US standard [25].
10~ The molecular sizes~of~LPS, 0-SP and~deacylated LPS
tD'eA-1PS~) are~ estimated~by gel;filtration through Superose~12~in 0.2 M NaCl, l~mM EDTA, 10 mM Tris, 0.25%
deoxycholic acid,;~pH~8~.0,~using the dextran standards to calibrate t~e column.~KDO is measured by the thiobarblturic~acid~assay,~using KDO~ as a standard [5].
Double immunod~i~ffusi'on is performed in 1% agarose in ph~ ~ ate- ~ Pe d~sal~ine~(PBS)~ Data for` ~ spectra a're~-~recorded~on a~JEO~GSX-500 spectrometer. Each s ~ ~ is~;a ~ ired`~with'~broad-band~H~decoupling at 90-,20~ 0~msec~ca`rb,on~observed~pulse~: 32~, aoo data points whichar`e;~zero-fil~le~d~to~ 64~,~000~points~prior to Fourier transormation'~ 30'~KHz~spectral~window~ (0.54 sec ;ac ~ isition~;t~ e~ 3.0~s c~d lay~be ~ een pulse cycles.
Prior~to'FQuri~r~transformation;~each free-induction-decay a~ is expon~ent~ial~1y~,multiplied so as to result in an additional 4Mz'~ ne-broadenin~in the frequency domain ctrum.~ App~xima~tely~10 mg~o~each samplè are dissolved in;0.5'~mL of D O and recorded at ambient probe temperature;~,(21-~C)~
,~ S~i1vér-sta~ined~SDS~PAGE~of~2.~5 mg of LPS from Inaba shows~2~faint~ban~ds with a "~la~dder" in the middle and two dense bands~nea'r~ th~e~bottom of~-~the gel (Fig. 1). Typical "ladders" of hi,gher~mo~lecular~weight 0-SP are ~formed by the~LPS~from~;E~ coll 0111.;~ No~bands are observed with 10 ~mg~samples of;either the 0-SP~or the DeA-LPS. The LPS of ~093/13797 21 ~ ~ 2 1 ~ PCT/US93/00253 v serotype Inaba has 3-6x103 EU/mg and the DeA-LPS had 3 EU/mg by LAL assay: this level represents >1000-fold reduction~ Immu~odiffusion showc a single band of precipitation between the LPS and the hyperinmune LPS
sera (Fig~ 2). A less intense and more di~fuse band is observed with the DeA-LPS which yields a partial identity reaction with the LPS. Neither the 0-SP nor the CT
pr~cipitates with this hyperimmune serum. The molecular : sizes of the LPS, 0-SP and DeA-LPS are estimated by HPLC
on Superose 12 (Fig. 3~. The LPS and DeA-LPS show two peaks: the LPS has Rd val~es of 0.~0 (16tOOO d) and 0.46 (8,700 d) and the DeA-LPS has Kd values of 0.38 (13,000 d) and 0.50: (6,000 d). The 0-SP exhi~ited only one peak corresponding to the second peak of the DeA-LPS (Kd 0.51, ,900 d~. Because of its greater antigenlcity and high molecular weight~, DeA-LPS is preferably used as the ; saccharide ~or the conjugates. We cannot detect KD0 in ; e:ither the:0-SP:~or:the DeA-LPS by the ~hiobar~ituric acid assay~ tS~57~. ~The~3C NMR spectra of the DeA-LPS and 0-SP are in a~reement with pr vious report~ ~30,47].
;20 ~ Each spectrum sh w s 10 major signals with identical, or nearly identical,~chemi~al shifts ~o those reported (Fig.
Example~2:~ Production~and characterization of DeA-2~ LPS-Cholera Toxin~conluqates : Conjugation of~DeA-LPS with proteins is performed using either.of two methods. In method 1, the covalent attachment :o. the LPS to the protein is accomplished by using~ SPDP;to~thi:olate both the: protein and the DeA-LPS
as described:for:the cell wall polysaccharide of pneumococci [52~].:: DeA-LPS ~3 mg/ml) or protein (lo : mg/ml) are dissolved in 0.15 M HEPES, 2 mM EDTA, pH 7.5.
SP~P (20 mM in ethanol) is added dropwise at weight ratios of 0.5 for DeA-LPS and 0.2 for protein. The :~
~:
:~:
~,~?~..;.~;,~.3;'f~ r '~
W093/13?9~2~ 2 PCr/USg3/002,.`'"' reaction mixture is stirred at ambient temperature for 1 hrO and passed through a 5x35 cm Sephadex G-25 column in H2O for DeA-LPS--SPDP and in PBS for pro~ein. The D~A LPS-SP~P is freeæe-dried and the protein is concentrated by membrane filtration (Amicon, YM10). The extent of derivatization with SPDP in aliqunts of DeA-LPS
or he proteins is determined spectrophotometrically following reduction of the N-pyridyl disulfide bond with 40 mM DTT and ~ssuming a molar extinction coefficient at 340 nm of 8.08x104 [53]. The N-pyridyl disulfide on the DeA LPS-SPDP is reduced with 40 mM DTT, passed through a 2.5x50 cm column of G-25 S~phadex in 0.2 M NaCl and the void volume fractions mixed with the SPDP derivative of the protein. This r action mixture is stirred at room tPmperature~for 2 hrs., passed through a 5x100 cm column of S-300 Sephacryl:in 0.2 M NaCl and the void volume fractions pooIed:.: Th~ conjugate synthesized by this method using Cholera Toxin as the protein component is designa ed as DeA-LPSCTIO An aliquot of DeA-LP5-CTI in sa:line i treated with 0.05 M ED~C at room temperature for 1 hr~ at pH 6.0 to cross-link the conjuga~e. The non-reacted ED~ is removed~by exhaustive dialysis agalnst:water. ~ ~:
In method~2~ DeA-LPS is derivatized with ~DH as described for Naemophilus influenz~e type b ~9,48].
DeA-LPS, 10:mg/ml::in ~aline, is~brought to pH 10.5 with 1 NaO8~and an equal weight of~CNBr (1 g/ml in ace`tonitrile) is added. The pH is maintained be~ween 10.0 and 11.0 with 1 N NaOH for 3 minutes. An equal volume of 0.5 M ADH in 0.5 M NaHCO3 is added and the pH adjusted to 8.5. The reaction mixture is stirred at room temperature for 1 hr. and then at 3-8 C ov~rnight and passed through a 5x35 cm Sephadex G-2~ column in H2O. Fractions from the void ~olume are~ pooled and freeze dri~d. The DeA-LPS-AH
derivative is dissolved in 0.I5 M NaCl to 10 mg/ml. An equal volume of protein (~10 mg/ml) is added and the pH
~V 93/13797 21~ ~ 212 PCT/US93/00253 adjusted to 5.S with O.l M HCl. EDAC is added to a final concentration of 0.05 M and the pH is maintained at 5.5-6.0 for l hr.. The reaction mix~ure is passed through 2.5x90 cm column of S-300 Sephacryl in 0.2 M NaCl and the fractions in the void volume are pooled.
Conjugates synthesized using CT (Lot 5~2) and CT tLot rst) as the prbtein component are d~signated as DeA-LPS-CTII and DeA-LPS-CTIII.
.
As in example 1, the conjugates are characterized by : several in vi~ro and in vivo methods. The extent of derivatization of DeA-LPS with adipic acid hydrazide is ~ ~ ~ measured by reactlon with trinitrobenzene sulfonic acid : (TNBS) using ADH as a standard [9]. Protein is measured by the BCA reagent using bovine serum albumin as a : standard ~17~. Hexose is measured by the ~nthrone ; 15 react~ion using:the:0-SP as the stardard [55]. Endotoxin concentration~is~estimated as in Example l. Double immunodiffusion~is also performed as in Example l. In : vitro cy*otoxioity of CT is measured by obsexYation of élongati~n of CHO cells [ 15 ] o Pyrogenicity of the 2;0~ conjugate is assayed in rabbits using the meth~d of Hochstein et al.~ C2~].
Table l hows~some~of the results of characterization of the conjugakes.
:~
i, j i .
30::
; 35 .
: :`
~ .
WO ~3/13797 Pcr/US93/OO~ ~
~,~?.~ 14-Tab . Chara~terization of Vibrio cholera~ hydrazine-treated lipopolysaccharide-protein :~: conJugates.
: DeA-LPS/Composition DeA -LPS protein Yield protein mglml Conjugate ~ Linker (wt/wt) ~(wt/wtj 9~ wt/wt protem CHO
. _ ~ _ . .
DeA-LPS-CT~ SPDP 1.18 :~ 8.8 88 ~ 0.72 2.Q 1.44 ~ : ~ . ;
DeA-LPS-CT~* SPDP ~l.~I8 ~ 8.8 ~ NA 0.65 1.15 0.25 : ~~ ~ : , ~ , DeA-LPS-CTD ADH~ 1.76 ; : : NA 79 0.80 0.48 0.38 : : ~ : ~ , :
DeA-LPS-cTm: ADH :: : 1.76 ~: ~: ~ NA ~ 88~ 1.50 1.0 1.50 : ~ ~ ~ ~ :
*Fùrther; -~atecl ~with :O.OSM: w~ EDAC ~(MATERJ.ALS AND METHODS) NA:`not~icable ~
Polys~tccha~ide~measured~by anthrone~ reaction~with DeA-LPS~ a standard [ ]. The yield alculat~d ~based upon;~e~ weight of the sacchaJide~:in :the con~ugate compared to the wçight:of t~e:adipic~acld hydrazide derivati~e. ~: ~
:,, ~ ~D93/13797 2 1 2 8 2 1 2 P~T/US93/00253 v The extent of derivatization of the DeA-LPS with SPDP or with ADH, is similar. The De~-LPS/protein (wt/wt) ratios are slightly lower for the conjugat~s of ;~ : CT prepared with SPDP than with ADH, ranging from 0.72 for DeA-LPS-CTI to 1.5 for DeA-LPS-CTII. The yields for all the conjugates are ~80~ as calculated by the re~overy of saccharide in the conjugates compared to the derivatized polysaccharide. Similar results are obtained by Method~II: with tetanus toxoid as the protein component of the conjugate. ~ representative double im~unodiffusion experiment shows that the serotype Inaba hyperimmune~antiserum~yields an identical line of precipItation~with the::DeA-LPS and DeA-LPS-CTII (Fig. 2).
SimilarIy the CT;~and LPS antisera yields a line of identity~with~the-~DeA-LPS-CTII and~the CT. A faint spur fr~m the CT antiserum~extends over the LPS antiserum and :the conjugate~,~:suggesting:that:there is a slight amount of unbound CT in~:this prepara~ion. The residual ~oxicity :of:the CT and the~DeA-LPS in the conjugateC as estimated by~ he in vitro~and~}~_y}~Q~:~assays described abovç is ~ery~low. ~In~the~thermal~inductlon~test, the DeA-LPS was not pyro~enic when~ injected~at 1 mg/Kg rabbit body weight.~The endotoxin~content of the conjugates was ~2 EU/mg: by thé: LAL:~assay. CT induced elongation in CHO
cel:ls:at 0.4 ng/m~. The:amount of CT, as a conju~ated 5~ form~required~to~elicit~the sa~e~degree of elongation was lO3:~to~:101~g~reater~ DeA-LPS-CTIII, a preparation : intended for ~lini;cal~use,~ad no detectable toxicity in CH0 cell~assay~;at~l~.0 mq/ml and passed the general safety :te~st in guinea~plgs~at lO human doses (25 m~;~DeA-LPS per ~:dose) as described~in~the~Code of Federal Regulations C.
Example 3: ComDarison of efflcac~_of cellular and LPS-CT
con~uqate~vaccines~
: Hyperimmune LPS antiserum is prepared by injecting ~: ~
":: :~ :
WO 93/137g7 PCI`/US93/002',~
female, adult BAL~/c mice with heat-killed V. chol~rae strain 2524 ~41]. Burro CT antiserum is prepared as described [13]. For evaluation of immunogenicity, 6-weeks-old B~LB/~ or general purpose m~ce (NIH) are injected subcutaneously with 2.5 mg or 10 mg of DeA-LPS
alone or as a conjugate in saline. Mice are injected at 2 week intervals and`bled 7 days after each immunization.
The fourth dose is given 4 weeks after the third injection and mlce are~bled 7 days and 6 months later.
Groups of mice:are immunized similarly with conjugates adsorbed with 0.:125 or 1.2S mg of aluminum hydroxide per , ~
dose. Cellular cholera~vaccine (purchasable from Wyeth Ayerst Laboratories, Marietta, PAj containing 4x109 :~ ~ each of Inaba~and Ogawa serotypes, is used as a control.
Mice are immunized with:0.1 or 0.2 ml of the vaccine 5 ~ Complement-mediated~vibr~iocida} antibody is measured :against Inaba~:and~Ogawa s~rains tl9,20~. Ten-fold serum dilutions are:~mlxed;~with equal~volumes of -lO00 cells/ml c~ :diluted guinea~pig~senum;and:~incubated at 37'C for 1 hr.
A~hyperimmune~s~erum~is~used as~standard in each assay.
;20~ ~The~serum~titer~is~expressed~as~the~recip~ocal sf the highest diluti~on~ of::serum that~yielded 50%;vibriocidal activity. ~Some~;sera were~assayed for vibriocidal .
antibodies~agai~nst strains 569B and 075 of serotype Inaba~ the~:titers:~of~these~sera were identical against both strains.~ There:~ore, vibrlocidal activities of the sera~àre~ass;ayed~w;ith~s~rain 569B. Inhibition of vibrlocidal ~activity: is assayed by mixing 100 mg/ml of LPS,: DeA-LPS,~:O-SP:or CT with various dilutions of antisera at~:~37~C~for~I:hr. prlor to the addition of the bacte~ia ~20~
LPS~and;proteln~antibody levels are determined by enzyme-linked lmmunosorbent assay (ELISA) using Immunolon
4~ plates (Dynate~ch, Chantilly, VA). The plates are coated with l00~ml~:per well of either LPS, 10 mg/ml, or : CT,:5 mg/ml, in~phosphate:buffered saline (PBS). LPS
.~ ~
~: : `
~0 93/137g7 2 1 2 ~ 2 1 2 P~T/US93/00253 antibody levels are ~xpressed in ELISA units using hyperi~mune sera as a reference. CT antibody levels are expressed in EL~SA units with a hyperimmune mouse pooled standard sera prepared using methods typically known in the art by repeated immunization of mice with CT.
Antibody levels are expressed as the geometric mean.
Antibody concentrations below the sensitivity of ~he ELISA are assigned values of one-half of that level.
Comparison of geometric means is perfonmed with the two--sided t test and the Wilcoxon test.
There are no detectable LPS antibodies in either general purpose or BALB/c mice immunized with 2.5 or lO
mg of DeA-LPS alone after any injection. Table 2 shows the leYels of antibodies to LPS in general purpose mice immunized with DeA-LPS-CTI and DeA-LPS~CTII.
~ , ~ :
: ~ ~ l. J
~: .; : :
~ 20 . ~
~ 25 : , :
, ~: ': .
~ :
~ 30 ~ ~:
:::: ::
~ ' ~:
.
WO 93/13797 Pcr/us93/oo2 Table 2. Serum lgM and IgG LPS antibodies (ELISA) elicited in general pu~ose mi~e immunized with DeA-LPS alone or as conjugatesa Dose Inject. G~me~ic mean (25-7 jth ~ntiles) Vaccine (~g) Number n-- IgG IgM
_ ~ . . . __ LPS 2.S 2 10 < lO 320 : ~ : 3 9 320 ~40 LPS lO.0 2 3 ~ 10 50 (1~160) 3: ~ 10 149C(5-~153) 260(4~522) ~: ~ : 4 10 1085d: (8~12800) 1525 (269-5 120) eA-LPS~ 2.5 2 : 5 < 10 < 10 . :3 8 ~ 22e (5 95) 1 1 (5-24) : ~ :
~ ~ 4 ~ 3 I g (7 202391 1 52 ( 1 ~2263) :DeA-LPS~ 2.5 ~ ~ 2; ~ ~5 ~ 35~: (14^gO~ 23 (14~0) : ~ ~ ~ . . --: ~~ ~: 3~ : ~ 11 80g (20~3~0) 150 (2~40) : ~ 4:~ ~ I 154~ (32~12800) ~19 (4~6~0) , ~ ~ 4b ; :5 640' (453-9~5) 80 ~4~226) : aFemale-;gene~l~pur~se~m~ 6~wks~ld, were in~ d s.c. with saline solu~ions of the:anûgen every ~week:for Ihroe ~imes and then were given a four~h inJee~ion 4 ~ weeks ~1ater.
~he~mice were bl~ 7 days:after each lnjec~ion and then again 6 months ~ter the four~h lnieeoon.~
h~ vs f, h vs d, p=NS; h vs~:g, p=O.OQ2, d vs c, p=0.08; f vs e, p~0.06, :: h ~s i, NS
~ ~ .
:: :
., : ... , .. ,.. ... . ,.. . . . ., .. ,, .. , . , ~, .. ..... , ,,,. ,, ., ,~ .. ... ..
` 'Y~ 93/13797 2 ~ 2 ~ 2 1 2 P~r/US93/00253 Neither conjugate elicits LPS antibodies after the first immunization. DeA-LPS-CTII elicits IgG and IgM
antibodies after the second injection. Both conjugates elicit a si~nificant rise of IgG antibodies after the ~: third and fourth injections (P~O.Ol). The IgG levels : ~ after fourth înjection are simi}ar in mice injected with ;~ ~ either the LPS or DeA-LPS-CTII. LPS doses of 2.5 or 10.0 mg elicit IgG antibodies only after the third injection.
: The IgG levels~are similar in the sera taken 7 days or 6 : months after the fourth injection of DeA-LPS-CTII.
: 10 Similar levels~of:antibodies are elici~ed by 10 mg doses of:the conjugates, by:EDAC treated DeA-LPS-CTI and by conjugates~:adsorbed onto~alum.
:: Table 3 shows that the LPS antibody levels elicited by con~ugates in BALB/c mice are lower than those of the general~ purpose;mice.
: 30 ; 3 . .
WO 93/137g7 Pcr/us93/
Table 3. Serum lgM and lgG LPS anh-LPS antibodies elicited in BALB/c mice immunized with DeA-LPS-CT c~njugates or cellular cholera va~cine Dose Inj~ct. Geometric mean ~25-75th centiles) Vaccine (~g) Number n = IgC IgM
__ . ~ . _ ,, DeA-LPS-CTI 2.5 /ug 1 5 < 10 40 (4040) 2 5 ~ lO 40 (40 40) 3 10 ~ 10 53 (~40) 3* 10 13 (5-24)a 65 (40-160) DeA-LPS-CTLI 2.~ ~g I S < ln 30 (20~0) 2 ~ < 10 70 ~4 3 10 46 (5-7~1) 130 (4~
: 3- 6 32 (1~95)b 50 (4~80) Whole~ell 0.1 ml 1 5 ~ C10 106 (57-226) :; ~ 2 5 139 ~4~640)C 1114 (453-~560) : ~ ~3 ~ 9 1742 (905-2560)d 741 (400 1600 ~~ 3- 6 90 (67-160)e 101 ~67-190) * mic~ ;bled 5 motlths after the~ third immunization e Y3 a, P=0.0004, e vs b7 P-NS,~e vs d, P=0.0001, d vs c, P=O.û2 :
.,^~093/13797 2 1 2 ~ 2 1 2 PCT/US93/00253 After the first and second dose, there is low IgM
and no detectab~e IgG antibodies in BALB/c mice injected with the conjugates. Low levels are detected after the third injection. The antibody levels remain similar 5 months after the las~ injection. The cellular vaccine induces high levels of both IgG and IgM after the second injeGtion and a booster effect upon the IgG antibody ;: levels following the third injection. Dasages of 0.l ml and 0.2 ml elicit simiIar levels. The IgG levels of the mice injected with the cellular vaccine decline to ~l~20 I0 of thelr optimal values~5 months after the last injection (P--O.O00l) and~are~similar to those elicited by : : DeA-LPS-CTI~
~ ~ , ` Neither 2.5 mg nor l0 mg of DeA-LPS or PBS
(controls~)~elicits vibriocidal antibodies to V. cholerae serotypes Inaba or Ogawa. As shown in Table 4, ;DeA-LPS-CTII elicits~low levels of vibriocid~l antibodies to the:Inaba~strain in:general purpose mice after the first injection ~ , ~ 35 ~ -';~: :
Table 4. Vibriocidal an~ibody titers of pooled sera from NIH general pu~ose mice immunized with conjuga~es or LPS
Dose Challenge Immuniza~ion number Immunogen (~g) serotype 1 2 __ ~_= , , _ . , . .
LPS 10.0 Inaba ND50,000 500,000 eA-LPS-CTI 2.5 ~ Inaba ND500 50,000 2.5 Ogawa ~ 100 50,000 DeA-LPS~ 2.5 Inaba l0025t000 50,000 2.5 Ogawa < 101,000 25,000 : 10.0 ~ Inaba 5~25,000 l00,~0 : 10.0 Ogawa 50l,000 50,0 * ~I:) - Not done : ~ ~
Geneial purpo~e m~ from the: NIH were inJected s.c. wi~h 2.5 ,L~g of .
DeA-LPS~and their sera~were pooled in equal arnount fo~ each group ~ , :: :
~ i :: ~: : : : :
: ~
. -~093/l3797 ` 2 1 ~ ~ 2 1 2 PCT/US93/00253 Both DeA-LPS-C~I and DeA-LPS-CTII elicit booster ~: reæponses after the nsxt two injections. LPS elicits the : : highest level of vibriocidal antibodies. In BALB/c mice, both conjugates elicit vibriocidal antibodies after the first injection; only DeA-LPS-CTII elicits booster responses following the second and third injections ; ~ (Table 5).
:
~:
2~0 35`
::~'; ~ : :
:: ::~:: :
-: ~
WO 93/1379~ Pc~r/u~93/002.~
2~
Table 5. Vibriocidal ac~ivities of sera f~om BALB/c mice immunized with DeA^LPS alone, conjugated ~o cholera toxin (CT) or with whole cell cholera vaccine Recipro~ vibriocidal titer Vaccine Dose Targetls~ inj 2nd inj 3rd inj --. _ __ .
DeA-LPS~ 2.5,~g lnaba 100 100 5,000 ~: ~ ~ Ogawa 25 100 l,000 DeA-LPs-CTn 2.5 ~h Inaba 250 5,000 l00,000 Ogawa 100 500 50,000 ~ol~ cell: 0. 1 ml Inaba 2,~00 50,000 100,000 ~ : Ogawa25,000 500,000 1,0007000 +: ;Vibnocidal antibody ~iter of ~led sera a~er each dose Who~ olera~ Yaccine ~ ~093/~3797 2 ~ 2 8 2 1 2 PCr/~S93/00253 None of our conjugates elicits IgG antibodies fo11Owing the first injection. Thi~ apparent lesser immunogenicity ~f DeA-LPS-CT conjugates, compared to those with capsular polysaccharides [48], could be due to ~wo factors: 1) the lesser immunogenicity of the O~-SP of V. cho7er 01 is due t~ its simp1icity ~linear homopo1ymer of perosamine acy1ated by 4-amino-4,6-dideoxy-L-glycero-tetronic acid) [30,47J; 2) the : relatively low molecular weight of the O-SP of V.
; ~ cholerae O1 [36,46].
We propose the following mechanism by which serum vibriocidal~ antibodies prevent cholera, a disease which is caused by:a non-invasive organism, whose s~mptoms are mediated by an exotoxin and which is not accompanied ~y inflammation.
~ First,~serum~antlbod:ies, especially those of the IgG
: class, penetrate :into:t~e lumen of the intestine ~28,59~.
It is:likely~complement prQteins are also present.
Second, the~walIs~of~the intestine are in contact due to peristalsis.~: Third, ~the inoculum~that survives the acid 20~ conditio~s;of~ the~:~stomach~is probably approximately 103 : V.~ ~holerae~: [22~,31j. ~Fourth, V. ~cholerae hav~ short polysaccharldes:on~their~LPS, this trait is associated with a high ~su c;eptibility to the comp1ement-dependent action of serum antibodies [42]. These factors, name1y ~low inocula~ serum vibriocidal~antabodies and complement ;at~the mu~osàl;surface whose su~faces are pressed upon ; ea~h~ other and;:churnin~ a susceptib1e organism, provide an explanation for how serum vibriocida1 antibodies onfer protectlon~against cholera; ingested V. cholerae are Iysed o~ the~ intestinal muccsal surface.
The~conjugates ellclt higher vibriocida1 activity to the homologou~s~serotype (Inaba~ than to the hetero1Oyous serotype (Ogawa3. The ce11u1ar vaccine, which contains both serotypes, induces higher leve1s of vibriocida1 : : 35 antibodies against Ogawa than Inaba. The vibriocidal ::
~:
::: ::
WQ93/13797 PCT/US93/0025- .
levels to serotype Inaba are elicited earlier and ir.
higher titer by the cellular vaccine compared to the conjugates. After the third injection, the vibriocidal : levels to Inaba elicited by the whole cell and conjugate vaccines are similar. All vibriocidal activity i5 ~:: 5 removed from the conjugate-induced antibodies following adsorbtion with either the LPS, DeA-LPS or the O-SP of the Inaba serotype.~ Adsorption with the Inaba LPS also removes all of the vibriocidal activity from the sera of mice injected with the cellular vaccine. The DeA-LPS and : 10 O-SP, in contrast, removes approxima~ly 90% of the vibriocidal acti~ity from these sera. Absorption with ~ the Ogawa LPS~removes about 90% of the vibriocidal :~ activity against strain Inaba. Absorption with CT does-not change the vibriocidal titers from the sera of mice 15 ~ ~injected with~either the con~ugates or the cellular vaccines. ~ Adsorption of DeA-~PS-CTI, DeA-LPS-CTII or other conjugates~onto alum has no e~fect upon their immunogenicity.~
Table 6 shows~the cholera toxin antibodies that are induced by immunization with the conjugates. Significant : rises of CT;antibodies are elicited in all mice of both strains by both::conjugates after each injection.
:25 : ~ ~
, - :::: : ~ :
~ 30 ~:
~ 35 ~F'"`'~ ~3/13797 2 1 2 8 2 1 2 Pcr/US93/00253 Ta~le 6. Serum IgG cholera toxin antibodies in mice immunized with DeA-LPS conjugates (Geome~ic mean (25-75th cen~iles) Injection Strain of mice Vaccme number BALBIc Gener~l purpose . .. . . .. .:.. .
DeA-LPS-CTl 1 0.1~(0.1~.2) 0. lf(0. 1-0.2) . 2 ~56.9b (44-76) ~ 49.0g (37-69) 3 217.4b5152-270) 157.1h(134-198 :
DeA-LPS~ ~ ~ ~0.1C(~. 1~.2) o. }i~o.03-0.3) ~ , : ; ~2 ~ ; ~30.6d (11-80) 17.~ (11-31) : 3 ~: ~ 136 7R(102- 198)156.0k(105~201) * Mice Immun~ wi~:whole cell;cholera vaccine, DeA-LPS or PBS
had ~O.Ol E~LlSA~antibody 'evels. ~ ~ ~
b vs a, P= <O.OOl:;: d :vs c~, P~=O.OO~ Y5 d, P=0.04; h,g YS f, P<0 01;
YS~, P--0.01;~ vs~, P<0.01.
: ~:: ~: : ::
, ~ ~
~ .
WO 93/13797 PCr/US93/002~ .3 xample 4 Diaqnostic and Thera~eutic Appl ications The LPS-protein conjugate of the present invention may be administered to human or animal subj ects in the form of a vaccine for the purpose o~ treating or preventing infections caused by organisms containing the LPS and/or protein antigen. Such vaccines c:an contain approximately 5 to 100 ~g of the LPS-protein conjugate.
These vaccines can be administered subcutaneously or intramuscularly. The antibodies raised to the conjugate 10 can be introduced into sterile filtered or radiation sterilized milk~(bovine, ovine or caprine) and ~: administered orally. The conjugates can be suspended in alum, saline, buffered saline, or oil-water emulsions, , :and subjects can be vaccinated with a series of injections, preferably one to five injections over a twelYe ~onth:period.
MonoclonaI or polyclonal antibodies of human ~r animal origin can be produced via the use of the above-described vaccines. These antibodies can be administered 2~0 :~to: animals and~humans, alone:or in combination with the LPS-protein~conjugate vaccines of t~e present invention, f:or the:prevention~:or treat~ent of infec~ions caused by th~ organism(~s)::from which the LPS and protein components of:the ~conjugate~are~obtained.~ These antibodies can be :: 25 adminis~ered~:to:a~subject in need the~eo~, either alone, for the purpose of passive immunization, or in combinatlon with~the LPS-protein conjugate vaccines of the~present invention, as an adjunct ~h~rapy. Such anti~odies can take the form of serum or gamma globulin containing the::antibodies of interest.
Of particu1ar~interest are antibodies to the LPS and ~`~: : CT an~igens of V. cholerae. These antibodies demonstrate neutralizing activ~ity against relat~d bacterial toxin ant1gens, particuIar1y the toxins secreted hy Eschericia 35 coli, Campylo~acter jejeuni and Aeromonas hydrophilia.
~:
` ~093/13797 2 ~ 2 ~ 212 PCT/US93/00253 --2g--v Monoclonal or polyclonal antibodies prepared via the use of the conjugate vaccines of the present invention can also be used for diagnostic purposes, or for the investigation of the developmental processes, pathogenesis, prevention, immunopathology, of LPS alone, as a component of a complex molecule, such as the LPS-protein conjugate, or of organisms expressing this polysaccharide~ fragment or derivatives thereof. The antibodies can~also be used to investigate the immunologic responses to the~above antigens.
Such antibodies can be derivatized or reacted with ; other substances to;produce kits~for disease diagnosis, or for the identification of organisms containing the LPS
or protein~used in the conjugate.
~ ; The invention being thus described,~ it will be J~ 5~ ;obvious that~the~same may be varied in many ways. Such variations~are~not~ to be~regarded as a departure from the spirit;and~scope of~the~invention,~and all such modifications~as~ would be~obvious to;one skilled in the art are~int~nded~to be included~within the scope of the 20~ ~f~llowing~claims.
3~5~
~: ~
LITER~TURE CITED
1. Ahmed, A. et al., J. Immunol. 105:431-441 (1970) 2. Anderson, P.W. et aI., J. Immuol.137.1181~ 6 (1986~
3. Benenson, ~. S., Proceedings of the 12th Joint Conference of US Japan CooperatiYe Medical Sciences Program, Cholera Panel, Sapporo, pp.
~ 228-242 (1976) : ~ : 4. 81ack, R.E.et al., Infect. Immun. 55:1166-1120 ~ 87) : 10 5. Brade, H., J. Bacteriol. 161:795_798 (1985) 7. Cash, R.A.~ et al., J. Infect. Dis. 130:325~333 1974j 9. Chu, C.Y. ~et al~,~ Infec. Immun. 59:4450-4458 (1991): ~ ;
10.~ Clemens,~J.D. et al.:, J. Infect~ Dis.
63:1235_1242 (1991)~
Clemens, J.D~ et~al., Vàccine 8:469-472 (1990) 12. Clemens, J.D. et al., Lancet 335:270-273 (1990) :13.~ Dafni,~Z::. and~J.B:. Robbins, J.~ Infect. Dis.
13 :S138-S141~1976) ~
14. DeMa:ria,~::A.~;et~al., J. Infect. Dis. 158:301-311 15. Elson,:C.~ and~W. Ealding,:J. Immunol.
32:2~36 274~ 1984 25 ~ 16~.~Enteric~Diseases~Branch~, MVWR 40:860 (I991~
17.~ Fatt~ml~A~ et al.~, Infect. Immun. 58:2309-2312 : : 18. Feeley, JoC~ and E.J. Gangarosa, 43rd Nobel ymposlum,~Stockholm 1978, pp. 204-2}0 (1980) : 30 :l9. Finkelstein:,:R.A. et:al~ Bull. Johns Hopkins Hosp. 116:~152-160 (1965)~ ~
~ ~ , 20. Finkelste1n,~R.A., J. Immunol. 89:264-271 (1962) 21. Finkelstein,:R.A.~,:CRC Crit. Rev. Microbiol.
: 2:553-623 (1973) ~: :
~ ;093/13797 2 1 2 ~ 2 1 2 P~T/US93/00253 v 22. Finkelstein, R.A., pp. 107-136 in R. Germanier .(ed.) "Bacterial Vaccines", c. 1984 by Academic Press Inc, ~ew York, NY
: 23. ~isatsune, K~ et al., Adv. in ~xp. Med. Biol.
: 256:189-197 (1~90) 24, Holmgren, J. and A~o Svennerholm, J. Infect.
Dis. 136:S105-108 (1977) 25. Hochstein, H.D., pp. 38-49 in "Clinical : Applications of the Liumulus amoeboc~te lysate test", R.B. Prior (ed.), c. 1990 by CRC Press, Boca Raton : 26. Jertborn, ~.: et al., Infect. ~mmun. 24:203-209 , (13~) 27. Kabir, S., J. Med. Microbiol. 23:9-18 tl987) : 28.:Kaur, J. et al., J. Infect. Dis. 12~:359-366 I5~ 971) ~
30. Kenne, L.~ et al., CarbohydrO Res. ~QQ:341-349 (1982):
31. Landy~, M. et al~., Amer. J. Publ. Hlth.
44:1572-1~579 (~1954)~
20 :: 32.~ Levine, ~:.M.:et al., Infect. Immun. 43:~15-522 33.~ evine~ N.M.~Lancet i:45-46 (1991) 3~5~.~ McGroa~y, E~.J. and M. Rivera,:Infect. Immun.
58~:1030-1037~:~(1990) ~
2~ :36.~ Mann~ing,~P~.A~ et al., Infect. Immun. 53:272-277 37~ Me~ritt, C.B.;: and R.B. Sack., J. Infect~. Dis.
21:52~5 S30~1970) ; 38.;Reviewed~1n~ Mos1ey, W.H.,;;Texas Rep. Biol. Med.
: 3~0 ~ :~ 27;:5upp~1ement 1:~27-241 (1969) 40.~Neoh,:S~.~H.~and D. Rowley, J. Infect. Dis.
26:41_~7 (~ 972) 41. Orskov,~F~. and I. Orskov, Methods in Microbiology 11:1-77 (1978) :,; ~ ~ , '~ ' :
42. Parsot, C. e~ a}., Proc. Natl. Acad. Sci. (~SA) 88:1641-1645 ~1991) : 43. Peterson, J.W.,:InfectO Immun. 26:528-533 (1979) 44. Pierce, N.F. et al., Infec~. Immun. 56:142-148 (lg8~) 45. Philippines Cholera Committee, Bull. W.H.O.
4~:38~-3~4 (1973) 46. Raziuddin, S., Immunochem. 15:611_61~ ~1978) ~ 47~ Redmond, J.W., Biochem. Biophys. Acta.
`:: ~ 5a4:346-352 ~1979) 10 ~ 48. Robbins, J.B. and R. Schneerson, J. Infect. Dis, : I61:8~1-832 (1990):
4g. Sack, D.A.~ et al~., J. Infect. 164:407-411 (1991) ~: ~ 50. Sommer, A. and W.H. Mosley, Lancet i:l232-1235 : (1973) :~ ~51. Svenn~rholm, A.-M.:;and J.~ Holmgren~ Infect. Immun.
735-740 tl~76) 52.:~Szu, S.C. et al.,;Infe~t~.~ Immun. ~:448-455 (19~6) 53:.~S~zu, S~C~et al:., In~ect. I~mun. 57:3823-3827 :~ :54.~:Trevelyant:W.E. and J.S. Harrison, Biochem. J~
50::298-:303~(19~52)::
55.: Tsai,~C-M.~ J. Biol. Sta`nd. 14 25~33 tl986) 56~.~Waldman~ R~H.:~et;al.~ Infect. Dis.
26:~401-4~07~(1972)~
.~ ~
~: : `
~0 93/137g7 2 1 2 ~ 2 1 2 P~T/US93/00253 antibody levels are ~xpressed in ELISA units using hyperi~mune sera as a reference. CT antibody levels are expressed in EL~SA units with a hyperimmune mouse pooled standard sera prepared using methods typically known in the art by repeated immunization of mice with CT.
Antibody levels are expressed as the geometric mean.
Antibody concentrations below the sensitivity of ~he ELISA are assigned values of one-half of that level.
Comparison of geometric means is perfonmed with the two--sided t test and the Wilcoxon test.
There are no detectable LPS antibodies in either general purpose or BALB/c mice immunized with 2.5 or lO
mg of DeA-LPS alone after any injection. Table 2 shows the leYels of antibodies to LPS in general purpose mice immunized with DeA-LPS-CTI and DeA-LPS~CTII.
~ , ~ :
: ~ ~ l. J
~: .; : :
~ 20 . ~
~ 25 : , :
, ~: ': .
~ :
~ 30 ~ ~:
:::: ::
~ ' ~:
.
WO 93/13797 Pcr/us93/oo2 Table 2. Serum lgM and IgG LPS antibodies (ELISA) elicited in general pu~ose mi~e immunized with DeA-LPS alone or as conjugatesa Dose Inject. G~me~ic mean (25-7 jth ~ntiles) Vaccine (~g) Number n-- IgG IgM
_ ~ . . . __ LPS 2.S 2 10 < lO 320 : ~ : 3 9 320 ~40 LPS lO.0 2 3 ~ 10 50 (1~160) 3: ~ 10 149C(5-~153) 260(4~522) ~: ~ : 4 10 1085d: (8~12800) 1525 (269-5 120) eA-LPS~ 2.5 2 : 5 < 10 < 10 . :3 8 ~ 22e (5 95) 1 1 (5-24) : ~ :
~ ~ 4 ~ 3 I g (7 202391 1 52 ( 1 ~2263) :DeA-LPS~ 2.5 ~ ~ 2; ~ ~5 ~ 35~: (14^gO~ 23 (14~0) : ~ ~ ~ . . --: ~~ ~: 3~ : ~ 11 80g (20~3~0) 150 (2~40) : ~ 4:~ ~ I 154~ (32~12800) ~19 (4~6~0) , ~ ~ 4b ; :5 640' (453-9~5) 80 ~4~226) : aFemale-;gene~l~pur~se~m~ 6~wks~ld, were in~ d s.c. with saline solu~ions of the:anûgen every ~week:for Ihroe ~imes and then were given a four~h inJee~ion 4 ~ weeks ~1ater.
~he~mice were bl~ 7 days:after each lnjec~ion and then again 6 months ~ter the four~h lnieeoon.~
h~ vs f, h vs d, p=NS; h vs~:g, p=O.OQ2, d vs c, p=0.08; f vs e, p~0.06, :: h ~s i, NS
~ ~ .
:: :
., : ... , .. ,.. ... . ,.. . . . ., .. ,, .. , . , ~, .. ..... , ,,,. ,, ., ,~ .. ... ..
` 'Y~ 93/13797 2 ~ 2 ~ 2 1 2 P~r/US93/00253 Neither conjugate elicits LPS antibodies after the first immunization. DeA-LPS-CTII elicits IgG and IgM
antibodies after the second injection. Both conjugates elicit a si~nificant rise of IgG antibodies after the ~: third and fourth injections (P~O.Ol). The IgG levels : ~ after fourth înjection are simi}ar in mice injected with ;~ ~ either the LPS or DeA-LPS-CTII. LPS doses of 2.5 or 10.0 mg elicit IgG antibodies only after the third injection.
: The IgG levels~are similar in the sera taken 7 days or 6 : months after the fourth injection of DeA-LPS-CTII.
: 10 Similar levels~of:antibodies are elici~ed by 10 mg doses of:the conjugates, by:EDAC treated DeA-LPS-CTI and by conjugates~:adsorbed onto~alum.
:: Table 3 shows that the LPS antibody levels elicited by con~ugates in BALB/c mice are lower than those of the general~ purpose;mice.
: 30 ; 3 . .
WO 93/137g7 Pcr/us93/
Table 3. Serum lgM and lgG LPS anh-LPS antibodies elicited in BALB/c mice immunized with DeA-LPS-CT c~njugates or cellular cholera va~cine Dose Inj~ct. Geometric mean ~25-75th centiles) Vaccine (~g) Number n = IgC IgM
__ . ~ . _ ,, DeA-LPS-CTI 2.5 /ug 1 5 < 10 40 (4040) 2 5 ~ lO 40 (40 40) 3 10 ~ 10 53 (~40) 3* 10 13 (5-24)a 65 (40-160) DeA-LPS-CTLI 2.~ ~g I S < ln 30 (20~0) 2 ~ < 10 70 ~4 3 10 46 (5-7~1) 130 (4~
: 3- 6 32 (1~95)b 50 (4~80) Whole~ell 0.1 ml 1 5 ~ C10 106 (57-226) :; ~ 2 5 139 ~4~640)C 1114 (453-~560) : ~ ~3 ~ 9 1742 (905-2560)d 741 (400 1600 ~~ 3- 6 90 (67-160)e 101 ~67-190) * mic~ ;bled 5 motlths after the~ third immunization e Y3 a, P=0.0004, e vs b7 P-NS,~e vs d, P=0.0001, d vs c, P=O.û2 :
.,^~093/13797 2 1 2 ~ 2 1 2 PCT/US93/00253 After the first and second dose, there is low IgM
and no detectab~e IgG antibodies in BALB/c mice injected with the conjugates. Low levels are detected after the third injection. The antibody levels remain similar 5 months after the las~ injection. The cellular vaccine induces high levels of both IgG and IgM after the second injeGtion and a booster effect upon the IgG antibody ;: levels following the third injection. Dasages of 0.l ml and 0.2 ml elicit simiIar levels. The IgG levels of the mice injected with the cellular vaccine decline to ~l~20 I0 of thelr optimal values~5 months after the last injection (P--O.O00l) and~are~similar to those elicited by : : DeA-LPS-CTI~
~ ~ , ` Neither 2.5 mg nor l0 mg of DeA-LPS or PBS
(controls~)~elicits vibriocidal antibodies to V. cholerae serotypes Inaba or Ogawa. As shown in Table 4, ;DeA-LPS-CTII elicits~low levels of vibriocid~l antibodies to the:Inaba~strain in:general purpose mice after the first injection ~ , ~ 35 ~ -';~: :
Table 4. Vibriocidal an~ibody titers of pooled sera from NIH general pu~ose mice immunized with conjuga~es or LPS
Dose Challenge Immuniza~ion number Immunogen (~g) serotype 1 2 __ ~_= , , _ . , . .
LPS 10.0 Inaba ND50,000 500,000 eA-LPS-CTI 2.5 ~ Inaba ND500 50,000 2.5 Ogawa ~ 100 50,000 DeA-LPS~ 2.5 Inaba l0025t000 50,000 2.5 Ogawa < 101,000 25,000 : 10.0 ~ Inaba 5~25,000 l00,~0 : 10.0 Ogawa 50l,000 50,0 * ~I:) - Not done : ~ ~
Geneial purpo~e m~ from the: NIH were inJected s.c. wi~h 2.5 ,L~g of .
DeA-LPS~and their sera~were pooled in equal arnount fo~ each group ~ , :: :
~ i :: ~: : : : :
: ~
. -~093/l3797 ` 2 1 ~ ~ 2 1 2 PCT/US93/00253 Both DeA-LPS-C~I and DeA-LPS-CTII elicit booster ~: reæponses after the nsxt two injections. LPS elicits the : : highest level of vibriocidal antibodies. In BALB/c mice, both conjugates elicit vibriocidal antibodies after the first injection; only DeA-LPS-CTII elicits booster responses following the second and third injections ; ~ (Table 5).
:
~:
2~0 35`
::~'; ~ : :
:: ::~:: :
-: ~
WO 93/1379~ Pc~r/u~93/002.~
2~
Table 5. Vibriocidal ac~ivities of sera f~om BALB/c mice immunized with DeA^LPS alone, conjugated ~o cholera toxin (CT) or with whole cell cholera vaccine Recipro~ vibriocidal titer Vaccine Dose Targetls~ inj 2nd inj 3rd inj --. _ __ .
DeA-LPS~ 2.5,~g lnaba 100 100 5,000 ~: ~ ~ Ogawa 25 100 l,000 DeA-LPs-CTn 2.5 ~h Inaba 250 5,000 l00,000 Ogawa 100 500 50,000 ~ol~ cell: 0. 1 ml Inaba 2,~00 50,000 100,000 ~ : Ogawa25,000 500,000 1,0007000 +: ;Vibnocidal antibody ~iter of ~led sera a~er each dose Who~ olera~ Yaccine ~ ~093/~3797 2 ~ 2 8 2 1 2 PCr/~S93/00253 None of our conjugates elicits IgG antibodies fo11Owing the first injection. Thi~ apparent lesser immunogenicity ~f DeA-LPS-CT conjugates, compared to those with capsular polysaccharides [48], could be due to ~wo factors: 1) the lesser immunogenicity of the O~-SP of V. cho7er 01 is due t~ its simp1icity ~linear homopo1ymer of perosamine acy1ated by 4-amino-4,6-dideoxy-L-glycero-tetronic acid) [30,47J; 2) the : relatively low molecular weight of the O-SP of V.
; ~ cholerae O1 [36,46].
We propose the following mechanism by which serum vibriocidal~ antibodies prevent cholera, a disease which is caused by:a non-invasive organism, whose s~mptoms are mediated by an exotoxin and which is not accompanied ~y inflammation.
~ First,~serum~antlbod:ies, especially those of the IgG
: class, penetrate :into:t~e lumen of the intestine ~28,59~.
It is:likely~complement prQteins are also present.
Second, the~walIs~of~the intestine are in contact due to peristalsis.~: Third, ~the inoculum~that survives the acid 20~ conditio~s;of~ the~:~stomach~is probably approximately 103 : V.~ ~holerae~: [22~,31j. ~Fourth, V. ~cholerae hav~ short polysaccharldes:on~their~LPS, this trait is associated with a high ~su c;eptibility to the comp1ement-dependent action of serum antibodies [42]. These factors, name1y ~low inocula~ serum vibriocidal~antabodies and complement ;at~the mu~osàl;surface whose su~faces are pressed upon ; ea~h~ other and;:churnin~ a susceptib1e organism, provide an explanation for how serum vibriocida1 antibodies onfer protectlon~against cholera; ingested V. cholerae are Iysed o~ the~ intestinal muccsal surface.
The~conjugates ellclt higher vibriocida1 activity to the homologou~s~serotype (Inaba~ than to the hetero1Oyous serotype (Ogawa3. The ce11u1ar vaccine, which contains both serotypes, induces higher leve1s of vibriocida1 : : 35 antibodies against Ogawa than Inaba. The vibriocidal ::
~:
::: ::
WQ93/13797 PCT/US93/0025- .
levels to serotype Inaba are elicited earlier and ir.
higher titer by the cellular vaccine compared to the conjugates. After the third injection, the vibriocidal : levels to Inaba elicited by the whole cell and conjugate vaccines are similar. All vibriocidal activity i5 ~:: 5 removed from the conjugate-induced antibodies following adsorbtion with either the LPS, DeA-LPS or the O-SP of the Inaba serotype.~ Adsorption with the Inaba LPS also removes all of the vibriocidal activity from the sera of mice injected with the cellular vaccine. The DeA-LPS and : 10 O-SP, in contrast, removes approxima~ly 90% of the vibriocidal acti~ity from these sera. Absorption with ~ the Ogawa LPS~removes about 90% of the vibriocidal :~ activity against strain Inaba. Absorption with CT does-not change the vibriocidal titers from the sera of mice 15 ~ ~injected with~either the con~ugates or the cellular vaccines. ~ Adsorption of DeA-~PS-CTI, DeA-LPS-CTII or other conjugates~onto alum has no e~fect upon their immunogenicity.~
Table 6 shows~the cholera toxin antibodies that are induced by immunization with the conjugates. Significant : rises of CT;antibodies are elicited in all mice of both strains by both::conjugates after each injection.
:25 : ~ ~
, - :::: : ~ :
~ 30 ~:
~ 35 ~F'"`'~ ~3/13797 2 1 2 8 2 1 2 Pcr/US93/00253 Ta~le 6. Serum IgG cholera toxin antibodies in mice immunized with DeA-LPS conjugates (Geome~ic mean (25-75th cen~iles) Injection Strain of mice Vaccme number BALBIc Gener~l purpose . .. . . .. .:.. .
DeA-LPS-CTl 1 0.1~(0.1~.2) 0. lf(0. 1-0.2) . 2 ~56.9b (44-76) ~ 49.0g (37-69) 3 217.4b5152-270) 157.1h(134-198 :
DeA-LPS~ ~ ~ ~0.1C(~. 1~.2) o. }i~o.03-0.3) ~ , : ; ~2 ~ ; ~30.6d (11-80) 17.~ (11-31) : 3 ~: ~ 136 7R(102- 198)156.0k(105~201) * Mice Immun~ wi~:whole cell;cholera vaccine, DeA-LPS or PBS
had ~O.Ol E~LlSA~antibody 'evels. ~ ~ ~
b vs a, P= <O.OOl:;: d :vs c~, P~=O.OO~ Y5 d, P=0.04; h,g YS f, P<0 01;
YS~, P--0.01;~ vs~, P<0.01.
: ~:: ~: : ::
, ~ ~
~ .
WO 93/13797 PCr/US93/002~ .3 xample 4 Diaqnostic and Thera~eutic Appl ications The LPS-protein conjugate of the present invention may be administered to human or animal subj ects in the form of a vaccine for the purpose o~ treating or preventing infections caused by organisms containing the LPS and/or protein antigen. Such vaccines c:an contain approximately 5 to 100 ~g of the LPS-protein conjugate.
These vaccines can be administered subcutaneously or intramuscularly. The antibodies raised to the conjugate 10 can be introduced into sterile filtered or radiation sterilized milk~(bovine, ovine or caprine) and ~: administered orally. The conjugates can be suspended in alum, saline, buffered saline, or oil-water emulsions, , :and subjects can be vaccinated with a series of injections, preferably one to five injections over a twelYe ~onth:period.
MonoclonaI or polyclonal antibodies of human ~r animal origin can be produced via the use of the above-described vaccines. These antibodies can be administered 2~0 :~to: animals and~humans, alone:or in combination with the LPS-protein~conjugate vaccines of t~e present invention, f:or the:prevention~:or treat~ent of infec~ions caused by th~ organism(~s)::from which the LPS and protein components of:the ~conjugate~are~obtained.~ These antibodies can be :: 25 adminis~ered~:to:a~subject in need the~eo~, either alone, for the purpose of passive immunization, or in combinatlon with~the LPS-protein conjugate vaccines of the~present invention, as an adjunct ~h~rapy. Such anti~odies can take the form of serum or gamma globulin containing the::antibodies of interest.
Of particu1ar~interest are antibodies to the LPS and ~`~: : CT an~igens of V. cholerae. These antibodies demonstrate neutralizing activ~ity against relat~d bacterial toxin ant1gens, particuIar1y the toxins secreted hy Eschericia 35 coli, Campylo~acter jejeuni and Aeromonas hydrophilia.
~:
` ~093/13797 2 ~ 2 ~ 212 PCT/US93/00253 --2g--v Monoclonal or polyclonal antibodies prepared via the use of the conjugate vaccines of the present invention can also be used for diagnostic purposes, or for the investigation of the developmental processes, pathogenesis, prevention, immunopathology, of LPS alone, as a component of a complex molecule, such as the LPS-protein conjugate, or of organisms expressing this polysaccharide~ fragment or derivatives thereof. The antibodies can~also be used to investigate the immunologic responses to the~above antigens.
Such antibodies can be derivatized or reacted with ; other substances to;produce kits~for disease diagnosis, or for the identification of organisms containing the LPS
or protein~used in the conjugate.
~ ; The invention being thus described,~ it will be J~ 5~ ;obvious that~the~same may be varied in many ways. Such variations~are~not~ to be~regarded as a departure from the spirit;and~scope of~the~invention,~and all such modifications~as~ would be~obvious to;one skilled in the art are~int~nded~to be included~within the scope of the 20~ ~f~llowing~claims.
3~5~
~: ~
LITER~TURE CITED
1. Ahmed, A. et al., J. Immunol. 105:431-441 (1970) 2. Anderson, P.W. et aI., J. Immuol.137.1181~ 6 (1986~
3. Benenson, ~. S., Proceedings of the 12th Joint Conference of US Japan CooperatiYe Medical Sciences Program, Cholera Panel, Sapporo, pp.
~ 228-242 (1976) : ~ : 4. 81ack, R.E.et al., Infect. Immun. 55:1166-1120 ~ 87) : 10 5. Brade, H., J. Bacteriol. 161:795_798 (1985) 7. Cash, R.A.~ et al., J. Infect. Dis. 130:325~333 1974j 9. Chu, C.Y. ~et al~,~ Infec. Immun. 59:4450-4458 (1991): ~ ;
10.~ Clemens,~J.D. et al.:, J. Infect~ Dis.
63:1235_1242 (1991)~
Clemens, J.D~ et~al., Vàccine 8:469-472 (1990) 12. Clemens, J.D. et al., Lancet 335:270-273 (1990) :13.~ Dafni,~Z::. and~J.B:. Robbins, J.~ Infect. Dis.
13 :S138-S141~1976) ~
14. DeMa:ria,~::A.~;et~al., J. Infect. Dis. 158:301-311 15. Elson,:C.~ and~W. Ealding,:J. Immunol.
32:2~36 274~ 1984 25 ~ 16~.~Enteric~Diseases~Branch~, MVWR 40:860 (I991~
17.~ Fatt~ml~A~ et al.~, Infect. Immun. 58:2309-2312 : : 18. Feeley, JoC~ and E.J. Gangarosa, 43rd Nobel ymposlum,~Stockholm 1978, pp. 204-2}0 (1980) : 30 :l9. Finkelstein:,:R.A. et:al~ Bull. Johns Hopkins Hosp. 116:~152-160 (1965)~ ~
~ ~ , 20. Finkelste1n,~R.A., J. Immunol. 89:264-271 (1962) 21. Finkelstein,:R.A.~,:CRC Crit. Rev. Microbiol.
: 2:553-623 (1973) ~: :
~ ;093/13797 2 1 2 ~ 2 1 2 P~T/US93/00253 v 22. Finkelstein, R.A., pp. 107-136 in R. Germanier .(ed.) "Bacterial Vaccines", c. 1984 by Academic Press Inc, ~ew York, NY
: 23. ~isatsune, K~ et al., Adv. in ~xp. Med. Biol.
: 256:189-197 (1~90) 24, Holmgren, J. and A~o Svennerholm, J. Infect.
Dis. 136:S105-108 (1977) 25. Hochstein, H.D., pp. 38-49 in "Clinical : Applications of the Liumulus amoeboc~te lysate test", R.B. Prior (ed.), c. 1990 by CRC Press, Boca Raton : 26. Jertborn, ~.: et al., Infect. ~mmun. 24:203-209 , (13~) 27. Kabir, S., J. Med. Microbiol. 23:9-18 tl987) : 28.:Kaur, J. et al., J. Infect. Dis. 12~:359-366 I5~ 971) ~
30. Kenne, L.~ et al., CarbohydrO Res. ~QQ:341-349 (1982):
31. Landy~, M. et al~., Amer. J. Publ. Hlth.
44:1572-1~579 (~1954)~
20 :: 32.~ Levine, ~:.M.:et al., Infect. Immun. 43:~15-522 33.~ evine~ N.M.~Lancet i:45-46 (1991) 3~5~.~ McGroa~y, E~.J. and M. Rivera,:Infect. Immun.
58~:1030-1037~:~(1990) ~
2~ :36.~ Mann~ing,~P~.A~ et al., Infect. Immun. 53:272-277 37~ Me~ritt, C.B.;: and R.B. Sack., J. Infect~. Dis.
21:52~5 S30~1970) ; 38.;Reviewed~1n~ Mos1ey, W.H.,;;Texas Rep. Biol. Med.
: 3~0 ~ :~ 27;:5upp~1ement 1:~27-241 (1969) 40.~Neoh,:S~.~H.~and D. Rowley, J. Infect. Dis.
26:41_~7 (~ 972) 41. Orskov,~F~. and I. Orskov, Methods in Microbiology 11:1-77 (1978) :,; ~ ~ , '~ ' :
42. Parsot, C. e~ a}., Proc. Natl. Acad. Sci. (~SA) 88:1641-1645 ~1991) : 43. Peterson, J.W.,:InfectO Immun. 26:528-533 (1979) 44. Pierce, N.F. et al., Infec~. Immun. 56:142-148 (lg8~) 45. Philippines Cholera Committee, Bull. W.H.O.
4~:38~-3~4 (1973) 46. Raziuddin, S., Immunochem. 15:611_61~ ~1978) ~ 47~ Redmond, J.W., Biochem. Biophys. Acta.
`:: ~ 5a4:346-352 ~1979) 10 ~ 48. Robbins, J.B. and R. Schneerson, J. Infect. Dis, : I61:8~1-832 (1990):
4g. Sack, D.A.~ et al~., J. Infect. 164:407-411 (1991) ~: ~ 50. Sommer, A. and W.H. Mosley, Lancet i:l232-1235 : (1973) :~ ~51. Svenn~rholm, A.-M.:;and J.~ Holmgren~ Infect. Immun.
735-740 tl~76) 52.:~Szu, S.C. et al.,;Infe~t~.~ Immun. ~:448-455 (19~6) 53:.~S~zu, S~C~et al:., In~ect. I~mun. 57:3823-3827 :~ :54.~:Trevelyant:W.E. and J.S. Harrison, Biochem. J~
50::298-:303~(19~52)::
55.: Tsai,~C-M.~ J. Biol. Sta`nd. 14 25~33 tl986) 56~.~Waldman~ R~H.:~et;al.~ Infect. Dis.
26:~401-4~07~(1972)~
5:7.~Ung~r,~F.~ M.~ Adv. Carb. Chem. Biochem~
38:~3~3-357:~ 1981:) sg. Westphal, O.~:and::~. Jann,:Meth. Carbohydr. Chem.
:: 5:83-gl (~96:S)~
:: 60. Winner,~ L. I~ et al., Infect. Immun. 59:977-982 3~0 ;~ 199~
~: ~
~ ~ :
~:, 3~
: , : :
38:~3~3-357:~ 1981:) sg. Westphal, O.~:and::~. Jann,:Meth. Carbohydr. Chem.
:: 5:83-gl (~96:S)~
:: 60. Winner,~ L. I~ et al., Infect. Immun. 59:977-982 3~0 ;~ 199~
~: ~
~ ~ :
~:, 3~
: , : :
Claims (29)
[received by the International Bureau on 22 November 1993 (22.11.93);
original claims 1-21 replaced by amended claims 1-29 (3 pages)]
1. A detoxified Vibrio cholera lipopolysaccharide-protein carrier conjugate comprising the detoxified lipopolysaccharide of Vibrio cholera, said lipopolysaccharide detoxified using anhydrous hydrazine, covalently attached to a protein carrier by means of a bifunctional linker to form a detoxified Vibrio cholera lipopolysaccharide-protein carrier conjugate.
2. A cholera vaccine comprising: the detoxified Vibrio cholera lipopolysaccharide-protein carrier conjugate according to claim 1.
3. A cholera vaccine comprising: the detoxified Vibrio cholera lipopolysaccharide-protein carrier conjugate according to claim 1, wherein the anhydrous hydrazine selectively removes acyl linked fatty acids from a lipid A
component of lipopolysaccharide.
component of lipopolysaccharide.
4. A vaccine according to claim 2 wherein the protein carrier is isolated from or secreted by a bacterial strain.
5. A vaccine according to claim 4, wherein said protein carrier is a secreted protein.
6. A vaccine according to claim 4, wherein said protein carrier is a bacterial toxin.
7. A vaccine according claim 6, wherein said toxin is the cholera toxin of Vibrio cholera.
8. A vaccine according to claim 2, wherein said covalent attachment is accomplished by reaction with a bifunctional linker selected from the group consisting of adipic acid dihydrazide, diaminohexane, amino-.epsilon.-caproic acid, and an N-hydrosuccinimide acid anhydride-based heterobifunctional linker.
9. A vaccine according to claim 8, wherein said N-hydrosuccinimide acid anhydride-based heterobifunctional linker is N-succinimidyl-3-(2-pyridylidithio) propionate.
10. A vaccine of claim 2, wherein the conjugate if formed between a detoxified derivatized LPS and a protein carrier by reaction with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide.
11. A method for preparing a covalently linked detoxified Vibrio cholera lipopolysaccharide-protein carrier conjugate comprising:
1) detoxifying Vibrio cholera lipopolysaccharide using anhydrous hydrazine under conditions that selectively remove acyl linked fatty acids from the lipid A component, 2) reacting the detoxified Vibrio cholera lipopolysaccharide of step 1 with a bifunctional linker, 3) mixing the derivatized, detoxified Vibrio cholera lipopolysaccharide from step (2) with a protein carrier, 4) adding to the mixture of step (3) a reagent, said reagent causing covalent linkage to occur to form the conjugate.
1) detoxifying Vibrio cholera lipopolysaccharide using anhydrous hydrazine under conditions that selectively remove acyl linked fatty acids from the lipid A component, 2) reacting the detoxified Vibrio cholera lipopolysaccharide of step 1 with a bifunctional linker, 3) mixing the derivatized, detoxified Vibrio cholera lipopolysaccharide from step (2) with a protein carrier, 4) adding to the mixture of step (3) a reagent, said reagent causing covalent linkage to occur to form the conjugate.
12. A vaccine according to claim 7, which further comprises a second vaccine directed against a second microorganism.
13. A vaccine according to claim 12, wherein said second vaccine is selected from the group consisting of diphtheria (D), tetanus toxoid (T), pertussis (P), DPT, and Hepatitis B vaccine.
14. A vaccine comprising the conjugate of claim 4, and a second vaccine directed against a second microorganism.
15. The vaccine of claim 14, wherein said second vaccine is selected from the group consisting of diphtheria (D), tetanus toxoid (T), pertussis (P), DPT, and Hepatitis B vaccine.
16. Use of a vaccine according to claim 2 for the manufacture of a medicament for use in a method for immunizing a human against cholera which comprises administering an amount of the vaccine of claim 2, sufficient to provide protection against cholera to the human.
17. Use of a vaccine according to claim 4 for the manufacture of a medicament for use in a method for immunizing a human against cholera which comprises administering an amount of the vaccine of claim 4, sufficient to provide protection against cholera to the human.
18. Use of a vaccine according to claim 7 for the manufacture of a medicament for use in a method for immunizing a human against cholera which comprises administering an amount of the conjugate of claim 7, sufficient to provide protection against cholera, to the human.
19. A method for detoxifying Vibrio cholera lipopolysaccharide and retaining protective antigenicity comprising the steps:
A) treating the lipopolysaccharide with an effective amount of anhydrous hydrazine to selectively remove acyl linked fatty acids fromthe lipid A component.
A) treating the lipopolysaccharide with an effective amount of anhydrous hydrazine to selectively remove acyl linked fatty acids fromthe lipid A component.
20. A method according to claim 19 wherein step (A) is performed at a temperature of about 37°C.
21. A method according to claim 19 wherein step (A) is performed for about 120 minutes.
22. A method according to claim 19 further comprising step (B) purifying the lipopolysaccharide.
23. A method according to claim 22 step (B) wherein the purification is performed by precipitation of the detoxified lipopolysaccharide using about 90% acetone at about 4°C.
24. A purified and isolated antibody produced by a mammal in response to immunization with the vaccine according to claim 2, 4, or 7 said antibody characterized in that it reacts with lipopolysaccharide on Vibrio cholera and has vibriocidal activity.
25. A purified and isolated antibody of claim 24 wherein the antibody is polyclonal.
26. A pharmaceutical composition comprising the antibody according to claim 24 and a pharmaceutically acceptable carrier.
27. Use of an antibody of claim 24 for the manufacture of a medicament for use in a method for prevention or treatment of cholera in a human, comprising: administration of the antibodies in an amount effective for prevention or treatment of cholera.
28. A method of claim 11 further comprising: Step (5) isolating the detoxified Vibrio cholera lipopolysaccharide-protein conjugate.
29. A pharmaceutical composition comprising the vaccine according to claims 2, 4, or 7 and a pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US82145392A | 1992-01-16 | 1992-01-16 | |
| US07/821,453 | 1992-01-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2128212A1 true CA2128212A1 (en) | 1993-07-22 |
Family
ID=25233450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002128212A Abandoned CA2128212A1 (en) | 1992-01-16 | 1993-01-14 | Detoxified lps-cholera toxin conjugate vaccine for prevention of cholera |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0623026A1 (en) |
| JP (1) | JPH07503238A (en) |
| AU (1) | AU678549B2 (en) |
| CA (1) | CA2128212A1 (en) |
| WO (1) | WO1993013797A2 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU1420897A (en) * | 1996-12-18 | 1998-07-15 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Conjugate vaccine for (salmonella paratyphi) a |
| US6531131B1 (en) | 1999-08-10 | 2003-03-11 | The United States Of America As Represented By The Department Of Health And Human Services | Conjugate vaccine for Neisseria meningitidis |
| AU2001253568A1 (en) * | 2000-04-18 | 2001-10-30 | Dan C. Deborde | Lipopolysaccharide-conjugate vaccine for sepsis treatment |
| US7749511B2 (en) | 2000-04-18 | 2010-07-06 | Endobiologics, Incorporated | Anti-sepsis conjugate vaccine |
| US7527797B1 (en) | 2000-09-01 | 2009-05-05 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Vibrio cholerae 0139 conjugate vaccines |
| US20030068324A1 (en) * | 2001-04-06 | 2003-04-10 | Jean-Michel Fournier | Conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid |
| WO2003094961A1 (en) | 2002-05-09 | 2003-11-20 | Massimo Porro | Improved polysaccharide and glycoconjugate vaccines_____________ |
| BR0316271A (en) * | 2002-11-14 | 2005-10-11 | Inst Finlay Ct De Investigacio | Method for obtaining multivalent conjugate vaccine preparations, multivalent vaccine composition and use thereof |
| US8048432B2 (en) | 2003-08-06 | 2011-11-01 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Polysaccharide-protein conjugate vaccines |
| CN102824632A (en) * | 2012-09-12 | 2012-12-19 | 北京民海生物科技有限公司 | Polysaccharide conjugate vaccine of vibrio cholera group O1, preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2532850B1 (en) * | 1982-09-15 | 1985-12-20 | Pasteur Institut | IMMUNOGENIC CONJUGATES BETWEEN A HAPTENA AND A CARRIER MOLECULE DERIVED FROM A TOXIN, THE VACCINES CONTAINING THEM AND PROCESS FOR OBTAINING THEM |
| US4663160A (en) * | 1983-03-14 | 1987-05-05 | Miles Laboratories, Inc. | Vaccines for gram-negative bacteria |
-
1993
- 1993-01-14 EP EP93903428A patent/EP0623026A1/en not_active Withdrawn
- 1993-01-14 CA CA002128212A patent/CA2128212A1/en not_active Abandoned
- 1993-01-14 AU AU34696/93A patent/AU678549B2/en not_active Ceased
- 1993-01-14 WO PCT/US1993/000253 patent/WO1993013797A2/en not_active Application Discontinuation
- 1993-01-14 JP JP5512624A patent/JPH07503238A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU3469693A (en) | 1993-08-03 |
| EP0623026A1 (en) | 1994-11-09 |
| WO1993013797A3 (en) | 1993-10-28 |
| AU678549B2 (en) | 1997-06-05 |
| JPH07503238A (en) | 1995-04-06 |
| WO1993013797A2 (en) | 1993-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0549617B1 (en) | Improved vaccine compositions | |
| Gupta et al. | Synthesis, characterization, and some immunological properties of conjugates composed of the detoxified lipopolysaccharide of Vibrio cholerae O1 serotype Inaba bound to cholera toxin | |
| ES2659050T3 (en) | Conjugation procedure for PNAG and a transporter protein | |
| EP0598818B1 (en) | Escherichia coli o-polysaccharide-protein conjugate vaccine | |
| AU2005316864B2 (en) | Glycoconjugate vaccines containing peptidoglycan | |
| US6531131B1 (en) | Conjugate vaccine for Neisseria meningitidis | |
| CA2128212A1 (en) | Detoxified lps-cholera toxin conjugate vaccine for prevention of cholera | |
| Tsay et al. | Preparation and characterization of a nontoxic polysaccharide-protein conjugate that induces active immunity and passively protective antibody against Pseudomonas aeruginosa immunotype 1 in mice | |
| JPH04507244A (en) | Lipid A analog/immunogenic carrier conjugate and its use as a vaccine | |
| EP0471954A2 (en) | Immunogenic conjugates of nontoxic oligosaccharide derived from bordetella pertussis lipooligosaccharide | |
| JP6091214B2 (en) | Anti-gram positive bacterial vaccine based on poly-glycerol phosphate | |
| US10772946B2 (en) | Immunogenic compositions against S. aureus | |
| US20030068324A1 (en) | Conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid | |
| KR20010034124A (en) | Lipooligosaccharide-based vaccine for prevention of Moraxella(Branhamella) catarrhalis infections in mammals | |
| WO2005044861A1 (en) | Polysaccharides of helicobacter pylori | |
| EP0575517A1 (en) | Lipid a analog/immunogenic carrier conjugates, the use thereof as vaccines and polyvalent hyperimmune gammaglobulins | |
| SVENSON et al. | Protection against Mouse Typhoid by Artificial Sa/mone//a Vaccines |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |