WO1993013797A2 - Vaccin conjugue a base de toxine du cholera et de lypopolysaccharide (lps) detoxique, utilise pour la prevention du cholera - Google Patents

Vaccin conjugue a base de toxine du cholera et de lypopolysaccharide (lps) detoxique, utilise pour la prevention du cholera Download PDF

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WO1993013797A2
WO1993013797A2 PCT/US1993/000253 US9300253W WO9313797A2 WO 1993013797 A2 WO1993013797 A2 WO 1993013797A2 US 9300253 W US9300253 W US 9300253W WO 9313797 A2 WO9313797 A2 WO 9313797A2
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lps
conjugate
vaccine
protein
cholera
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PCT/US1993/000253
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WO1993013797B1 (fr
WO1993013797A3 (fr
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Shousun C. Szu
John B. Robbins
Rajesh K. Gupta
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The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services
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Priority to JP5512624A priority Critical patent/JPH07503238A/ja
Priority to AU34696/93A priority patent/AU678549B2/en
Priority to EP93903428A priority patent/EP0623026A1/fr
Publication of WO1993013797A2 publication Critical patent/WO1993013797A2/fr
Publication of WO1993013797A3 publication Critical patent/WO1993013797A3/fr
Publication of WO1993013797B1 publication Critical patent/WO1993013797B1/fr

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    • 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

Definitions

  • the invention disclosed herein relates broadly to the production of vaccines for the amelioration of 5 bacterial infections. More specifically, the invention describes the production of an antibacterial vaccine by conjugation of detoxified lipopolysaccharide derived from the bacterial target strain to proteins also produced by the bacteria target. 0 BACKGROUND ART
  • cholera is a toxin-mediated disease of the luminal surface of the jejeunum, and to the notion that a local intestinal response is required 5 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
  • V. cholerae 01 LPS contains lipid A and a core oligosaccharide composed of 4-amino-4-deoxy-L-arabinose, quinovosamine, D-glucose, D-fructose and heptose [23,30,47], 3-deoxy-D-manno octulosonic acid (KDO) has been identified recently and 5 presumed to be in the core adjacent to the lipid A [5].
  • serotype Inaba contains a saccharide of -12 residues composed of l-»2-linked D-perosamine whose amino groups are acylated by 3-deoxy-L-glycero-tetronic acid [23,30,47].
  • the relationship between the sequence of the genes that encode the enzymes which synthesize the V. cholerae LPS and the serological specificity of the serotypes (LPS types) Inaba and Ogawa [23,36] has not been clarified.
  • cellular vaccines or partially purified LPS induce a statistically significant protection 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 control of outbreaks of cholera [38,51].
  • the protective immune moiety induced by these vaccines is proposed to be serum LPS antibodies with vibriocidal activity [1,3,18,22,38,40].
  • the cellular vaccines do not elicit serum antitoxin [37] nor, by analogy with similar products, secretory antibodies [56]. Similar effects are also obtained with orally administered inactivated V. cholerae [7,10-12]. Addition of the B subunit of CT to the formulation of this vaccine does not recruit additional protection [12].
  • vibriocidal antibody levels are a reliable method for predicting resistance to cholera. Serum vibriocidal activity is correlated with resistance against disease following convalescence from cholera, by administration of live attenuated strains, or by inactivated V. cholerae alone or with the B subunit of CT. Also, the age-related acquisition of vibriocidal antibodies in endemic areas parallels the increasing resistance to cholera observed in older children and adults [1,3,4,7,10,11,18,22,38,50]. Our interpretation of these data is that cellular cholera vaccines, as observed with similar products and polysaccharides, are poor immunogens and have T-cell independent properties [37,49].
  • the route of immunization, using CFA, and the dosage used are clinically unacceptable.
  • Conjugate vaccines have many advantages compared to cellular vaccines; 1) no serious adverse reactions are anticipated because the LPS levels are low; 2) conjugated saccharides can be expected to have greater immunogenicity and T-cell dependent properties compared to cellular vaccines [9,14,31,48], thus the conjugate may represent a safer and more immunogenic (and thereby more effective) vaccine; 3) conjugates may be administered concurrently with Diptheria and Tetanus toxoid, Pertussis (DTP) and H.
  • DTP Pertussis
  • influenzae type b conjugates to infants [48] thus the conjugate might be incorporated into routine immunization of infants and children, that age group with the highest attack rate in areas endemic for cholera [38] and 4) the composition of our conjugate can be standardized so that the potency of new lots can be controlled by laboratory assays.
  • influenzae type b-tetanus toxoid conjugates in infants injected concurrently with DTP [49].
  • the low levels of "endotoxic" activity as measured by the LAL and rabbit pyrogen assays, provide assurance that our conjugates will elicit little or no adverse reactions encountered with cellular vaccines for cholera [25].
  • the most general description of the invention is an anti-bacterial vaccine formulation which comprises a conjugate between lipopolysaccharide moieties derived from the target bacterial strain and proteins derived from the same strain.
  • a vaccine is formulated using this conjugate and any of the pharmaceutically acceptable 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 also be incorporated into formulations currently in use in childhood immunization protocols, in particular, the diptheria and tetanus toxoid, pertussis (DTP) vaccine commonly administered to children in the United States.
  • DTP diptheria and tetanus toxoid, pertussis
  • a large advantage in terms of clinical usefulness of the conjugate vaccine is obtained by detoxifying the LPS component of the vaccine.
  • Such detoxification can be achieved by removal of the esterified fatty acids from the lipid A component of the LPS using hydrazine or by acid hydrolysis of the LPS.
  • one object of the present invention is to provide LPS-protein conjugate vaccines using such a detoxified LPS component.
  • conjugation of the detoxified LPS to a protein synthesized by the bacterial target would provide a useful vaccine, particularly if a protein localized on the surface of the bacterium is used.
  • a vaccine which produces a neutralizing antibody response to toxins secreted by bacteria would also be a useful vaccine.
  • a second object 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 conjugation of detoxified LPS to a toxin protein produced by the bacterial target.
  • the conjugation reaction can be carried 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.
  • a cross-linking agent can be a bifunctional linker.
  • bifunctional linkers which can be employed in the present invention include, but are not limited to, adipic acid dihydrazide, diaminohexane, amino-e-caproic acid, and an N- hydrosuccinimide acid anhydride-based heterobifunctional linker.
  • two methods of conjugation are described for the LPS detoxified by treatment with hydrazine; reaction with N-succinimidyl 3-(2- pyridyldithio) propionate (SPDP) or reaction with adipic acid dihydrazide (ADH) followed by reaction with 1-ethyl- 3(3-dimethylaminopropy) carbodiimide (EDAC) .
  • SPDP N-succinimidyl 3-(2- pyridyldithio) propionate
  • ADH adipic acid dihydrazide
  • EDAC 1-ethyl- 3(3-dimethylaminopropy carbodiimide
  • a preferred embodiment of the invention utilizes one of these methods of conjugation.
  • the polyclonal or monoclonal antibodies raised by administration of the conjugate vaccine to a laboratory animal may find use as components of a diagnostic kit or as components of a method of treatment of infection by the targeted organism. Accordingly, it is another object of the invention to provide for diagnostic kits for the detection of organisms bearing either the LPS or protein portion, or both, of the conjugate.
  • a final object of the invention is to provide antibodies which might be used to treat infections caused by an organism bearing either the LPS or protein portion, or both, of the conjugate or which neutralize a toxin secreted by such an organism.
  • Figure 1 shows a silver-stained SDS-PAGE gel (14%) of 2.5 mg LPS from Vibrio cholerae serotype Inaba (lane 1) and Escherichia coli 0111 (lane 2) .
  • Figure 2 shows the characterization of LPS and LPS- CT conjugates by double immunodiffusion. Left: A.
  • Figure 3 shows 13 C nuclear magnetic resonance spectrum of the hydrazine-treated lipopolysaccharide (DeA-LPS) from Vibrio cholerae , serotype Inaba.
  • the 10 major signals are identical to those reported by Kenne et al., [30].
  • the 13 C N.M.R. spectrum of the acid-treated lipopolysaccharide (0-SP) was almost identical to this spectrum.
  • Figure 4 shows HPLC profiles of 100 mL samples (1.0 mg/ml) through a 10x300 mm column of Superose 12 in 0.2 M NaCl, 0.01 M TRIS, 0.001 M EDTA, 0.25% deoxycholic acid, pH 8.
  • Chemical reagents for executing the procedures described in the examples can be obtained from the sources noted below: Anhydrous hydrazine (Lot 104F-3523) , adipic acid dihyrazide (ADH, Lot 77F-5016) , dithiothreitol (DTT, Lot 49F-0138) , l-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDAC, Lot 105F-0308) , disodium EDTA (Lot 119F-0275) , KDO, RNase (Lot 128F-0462) , DNase (Lot 89F-9605) and pronase (Lot 99F-0391) can be purchased from Sigma Chemical Co., St.
  • HEPES Lit 051790
  • deoxycholic acid Lit 264101
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • SPDP Lot 900707084
  • alum Lit 891120103
  • BCA reagent for protein determination can be obtained from Pierce Chemical Co, Rockford, IL.
  • Cyanogen bromide CBr, Lot 014783A
  • Sephadex G-25 (Lot P10036) , Sephacryl S-300, 10x300 mm Superose 12 column and dextrans for molecular weight assay can be purchased from Pharmacia-LKB, Piscataway, NJ.
  • LPS from V. cholerae strain 569B (Inaba) can be purchased from List Biologicals, Campbell, CA.
  • Limulus amoebocyte lysate (LAL) can be purchased from Associates of Cape Cod, Woods Hole, MA.
  • p-nitrophenyl phosphate can be obtained from Fluka, Ronkonkoma, NY.
  • the US Standard for endotoxin can be obtained from Donald Hochstein, United States Food and Drug Administration [25].
  • Cholera toxin, variant 1, Lot 582 can be obtained from Pasteur Merieux Serums & Vaccins, Lyon, France) and cholera toxin variant 1, lot rst is purified from V. cholerae Inaba strain 569B [21,28].
  • Anti-mouse IgG and IgM alkaline phosphatase conjugates can be purchased from Kirkegaard & Perry Laboratories, Inc, Gaithersburg, MD.
  • the bacterial strains used in the examples are: V. cholerae , biotype classical, serotype Inaba, strain 569B and V. cholerae , biotype classical, serotype Ogawa strain NIH 41 are used for vibriocidal assay.
  • V. cholerae biotype classical, serotype Inaba
  • strain 569B and V. cholerae , biotype classical, serotype Ogawa strain NIH 41 are used for vibriocidal assay.
  • cholerae classical Inaba strain 2524 (Katherine Greene, C.D.C. , Atlanta, GA) is used for raising antiserum in mice ⁇ against the LPS.
  • V. cholerae serotype Inaba, biotype El Tor, cholera toxin (CT) 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
  • LPS is detoxified by two methods.
  • For acid-hydrolysis LPS, 10 mg/ml in 1% acetic acid is o heated at 100"C for 90 min [59]. The reaction mixture is ultracentrifuged at 60,000 x g, 10"C, for 5 hr. and the supernatant passed through a sterile 0.22 micron filter (Nalge, Rochester, NY) and freeze-dried (designated as 0-SP) .
  • LPS at 10 5 mg/ml is treated with 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 ice bath until a precipitate formed 0 (approximately 90% acetone) and the reaction mixture centrifuged at 15,000 x g, 10"C, for 30 min.
  • the precipitate is dissolved 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 H 2 0 5 exhaustively, passed through a 0.22 micron filter, and freeze dried.
  • the protein and nucleic acid concentration of the 0-SP and the DeA-LPS are ⁇ 1%. LPS, extracted from acetone-dried V.
  • cholerae cells of El Tor biotype Ogawa serotype strain 3083-13 is used for inhibition of vibriocidal activity.
  • the LPS is subjected to various preliminary characterizations, using both in vitro and in vivo techniques. SDS-PAGE is used for detection of LPS [56].
  • LPS concentration, assayed by LAL, is expressed in endotoxin units (EU) related to the US standard [25].
  • EU endotoxin units
  • the molecular sizes of LPS, 0-SP and deacylated LPS (DeA-LPS) are estimated by gel filtration through Superose 12 in 0.2 M NaCl, 1 mM EDTA, 10 mM Tris, 0.25% deoxycholic acid, pH 8.0, using the dextran standards to calibrate the column.
  • KDO is measured by the thiobarbituric acid assay using KDO as a standard [5].
  • Double im unodiffusion is performed in 1% agarose in phosphate-buffered saline (PBS) .
  • Data for NMR spectra are recorded on a JEOL GSX-500 spectrometer. Each spectrum is acquired with broad-band 1 H decoupling at 90' 10-msec carbon observed pulse; 32,000 data points which are zero-filled to 64,000 points prior to Fourier transformation; 30 KHz spectral window (0.54 sec acquisition time); 3.0 sec delay between pulse cycles. Prior to Fourier transformation each free-induction-decay signal is exponentially multiplied so as to result in an additional 4Hz line-broadening in the frequency domain spectrum.
  • Immunodiffusion shows a single band of precipitation between the LPS and the hyperimmune LPS sera (Fig. 2) .
  • a less intense and more diffuse band is observed with the DeA-LPS which yields a partial identity reaction with the LPS.
  • the O-SP nor the CT precipitates with this hyperimmune serum.
  • the molecular sizes of the LPS, O-SP and DeA-LPS are estimated by HPLC on Superose 12 (Fig. 3).
  • the LPS and DeA-LPS show two peaks: the LPS has Kd values of 0.40 (16,000 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 exhibited only one peak corresponding to the second peak of the DeA-LPS (Kd 0.51, 5,900 d) . Because of its greater antigenicity and high molecular weight, DeA-LPS is preferably used as the saccharide for the conjugates. We cannot detect KDO in either the 0-SP or the DeA-LPS by the thiobarbituric acid assay [5,57].
  • the 13 C NMR spectra of the DeA-LPS and 0-SP are in agreement with previous reports [30,47]. Each spectrum shows 10 major signals with identical, or nearly identical, chemical shifts to those reported (Fig. 3).
  • Conjugation of DeA-LPS with proteins is performed using either of two methods.
  • method 1 the covalent attachment of the LPS to the protein is accomplished by using SPDP to thiolate both the protein and the DeA-LPS as described for the cell wall polysaccharide of pneumococci [52].
  • DeA-LPS (3 mg/ml) or protein (10 mg/ml) are dissolved in 0.15 M HEPES, 2 mM EDTA, pH 7.5. SPDP (20 mM in ethanol) is added dropwise at weight ratios of 0.5 for DeA-LPS and 0.2 for protein. The ° reaction mixture is stirred at ambient temperature for 1 hr.
  • DeA-LPS-SPDP is freeze-dried and the protein is concentrated by membrane filtration (Amicon, YM10) .
  • the extent of derivatization with SPDP in aliquots of DeA-LPS or the proteins is determined spectrophotometrically following reduction of the N-pyridyl disulfide bond with 40 mM DTT and assuming a molar extinction coefficient at 340 nm of 8.08xl0 4 [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 Sephadex in 0.2 M NaCl and the void volume fractions mixed with the SPDP derivative of the protein. This reaction mixture is stirred at room temperature for 2 hrs., passed through a 5x100 cm column of S-300 Sephacryl in 0.2 M NaCl and the void volume fractions pooled.
  • the conjugate synthesized by this method using Cholera Toxin as the protein component is designated as DeA-LPS-CTI.
  • An aliquot of DeA-LPS-CTI in saline is treated with 0.05 M EDAC at room temperature for 1 hr. at pH 6.0 to cross-link the conjugate. The non-reacted EDAC is removed by exhaustive dialysis against water.
  • DeA-LPS is derivatized with ADH as described for Haemophilus influenzae type b [9,48].
  • DeA-LPS 10 mg/ml in saline, is brought to pH 10.5 with 1 N NaOH and an equal weight of CNBr (1 g/ml in acetonitrile) is added. The pH is maintained between 10.0 and 11.0 with 1 N NaOH for 3 minutes.
  • An equal volume of 0.5 M ADH in 0.5 M NaHC0 3 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 overnight and passed through a 5x35 cm Sephadex G-25 column in H 2 0. Fractions from the void volume are pooled and freeze dried.
  • the DeA-LPS-AH derivative is dissolved in 0.15 M NaCl to 10 mg/ml.
  • An equal volume of protein (-10 mg/ml) is added and the pH adjusted to 5.5 with 0.1 M HCl.
  • EDAC is added to a final concentration of 0.05 M and the pH is maintained at 5.5-6.0 for 1 hr..
  • the reaction mixture 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 582) and CT (Lot rst) as the protein component are designated as DeA-LPS-CTII and DeA-LPS-CTIII.
  • the conjugates are characterized by several in vitro and in vivo methods.
  • the extent of derivatization of DeA-LPS with adipic acid hydrazide is measured by reaction with trinitrobenzene sulfonic acid (TNBS) using ADH as a standard [9].
  • TNBS trinitrobenzene sulfonic acid
  • Protein is measured by the BCA reagent using bovine serum albumin as a standard [17] .
  • Hexose is measured by the anthrone reaction using the 0-SP as the standard [55].
  • Endotoxin concentration is estimated as in Example 1.
  • Double immunodiffusion is also performed as in Example 1.
  • In vitro cytotoxicity of CT is measured by observation of elongation of CHO cells [15]. Pyrogenicity of the conjugate is assayed in rabbits using the method of Hochstein et al. [25].
  • Table 1 shows some of the results of characterization of the conjugates.
  • a representative double 0 immunodiffusion experiment shows that the serotype Inaba hyperimmune antiserum yields an identical line of precipitation with the DeA-LPS and DeA-LPS-CTII (Fig. 2) .
  • the CT and LPS antisera yields a line of identity with the DeA-LPS-CTII and the CT.
  • a faint spur 5 from the CT antiserum extends over the LPS antiserum and the conjugate, suggesting that there is a slight amount of unbound CT in this preparation.
  • the residual toxicity of the CT and the DeA-LPS in the conjugates as estimated by the in vitro and in vivo assays described above is o very low.
  • the DeA-LPS was not pyrogenic when injected at 1 mg/Kg rabbit body weight.
  • the endotoxin content of the conjugates was -2 EU/mg by the LAL assay.
  • CT induced elongation in CHO cells at 0.4 ng/ml.
  • the amount of CT, as a conjugated 5 form required to elicit the same degree of elongation was 10 3 to 10 10 greater.
  • DeA-LPS-CTIII a preparation intended for clinical use, had no detectable toxicity in CHO cell assay at 1.0 mg/ml and passed the general safety test in guinea pigs at 10 human doses (25 mg DeA-LPS per 0 dose) as described in the Code of Federal Regulations C.
  • Example 3 Comparison of efficacy of cellular and LPS-CT conjugate vaccines
  • Hyperimmune LPS antiserum is prepared by injecting c female, adult BALB/c mice with heat-killed V. cholerae strain 2524 [41]. Burro CT antiserum is prepared as described [13].
  • 6 eeks-old BALB/c or general purpose mice (NIH) are injected subcutaneously with 2.5 mg or 10 mg of DeA-LPS 5 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 mice are bled 7 days and 6 months later.
  • mice are immunized similarly with conjugates 0 adsorbed with 0.125 or 1.25 mg of aluminum hydroxide per dose.
  • Cellular cholera vaccine purchasable from Wyeth-Ayerst Laboratories, Marietta, PA
  • Mice are immunized with 0.1 or 0.2 ml of the vaccine.
  • Complement-mediated vibriocidal antibody is measured against Inaba and Ogawa strains [19,20].
  • Ten-fold serum dilutions are mixed with equal volumes of -1000 cells/ml diluted guinea pig serum and incubated at 37*C for 1 hr.
  • a hyperimmune serum is used as standard in each assay. o
  • the serum titer is expressed as the reciprocal of the highest dilution of serum that yielded 50% vibriocidal activity.
  • Some sera were assayed for vibriocidal antibodies against strains 569B and 075 of serotype Inaba: the titers of these sera were identical against 5 both strains. Therefore, vibriocidal activities of the sera are assayed with strain 569B. Inhibition of vibriocidal activity is assayed by mixing 100 mg/ml of LPS, DeA-LPS, 0-SP or CT with various dilutions of antisera at 37 * C for 1 hr. prior to the addition of the 0 bacteria [20].
  • LPS and protein antibody levels are determined by enzyme-linked immunosorbent assay (ELISA) using Immunolon 4 plates (Dynatech, Chantilly, VA) . The plates are coated with 100 ml per well of either LPS, 10 mg/ml, or 5 CT, 5 mg/ml, in phosphate buffered saline (PBS) .
  • LPS antibody levels are expressed in ELISA units using hyperimmune sera as a reference.
  • CT antibody levels are expressed in ELISA 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 the ELISA are assigned values of one-half of that level.
  • mice a Female general purpose mice, -6 wks-old, were injected s.c. with saline solutions of the antigen every week for three times and then were given a fourth injection 4 weeks later.
  • the mice were bled 7 days after each injection and then again 6 months after the fourth injection.
  • h vs f, h vs d, p NS;
  • h vs g, p 0.002,
  • d vs c, p 0.08;
  • conjugate elicits LPS antibodies after the first immunization.
  • DeA-LPS-CTII elicits IgG and IgM antibodies after the second injection.
  • Both conjugates elicit a significant rise of IgG antibodies after the third and fourth injections (P ⁇ 0.01).
  • the IgG levels after fourth injection are similar 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.
  • Similar levels of antibodies are elicited 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 conjugates in BALB/c mice are lower than those of the general purpose mice.
  • DeA-LPS-CTII elicits low levels of vibriocidal antibodies to the Inaba strain in general purpose mice after the first injection.
  • mice from the NIH were injected s.c. with 2.5 ⁇ g of DeA-LPS and their sera were pooled in equal amount for each group
  • Both DeA-LPS-CTI and DeA-LPS-CTII elicit booster responses after the next two injections.
  • LPS elicits the highest level of vibriocidal antibodies.
  • both conjugates elicit vibriocidal antibodies after the first injection; only DeA-LPS-CTII elicits booster responses following the second and third injections (Table 5) .
  • cholera a disease which is caused by a non-invasive organism, whose symptoms are mediated by an exotoxin and which is not accompanied by inflammation.
  • serum antibodies especially those of the IgG class, penetrate into the lumen of the intestine [28,59]. It is likely complement proteins are also present.
  • the walls of the intestine are in contact due to peristalsis.
  • the inoculum that survives the acid conditions of the stomach is probably approximately 10 3 V. cholerae [22,31].
  • V. cholerae have short polysaccharides on their LPS; this trait is associated with a high susceptibility to the complement-dependent action of serum antibodies [42].
  • the conjugates elicit higher vibriocidal activity to the homologous serotype (Inaba) than to the heterologous serotype (Ogawa) .
  • the cellular vaccine which contains both serotypes, induces higher levels of vibriocidal antibodies against Ogawa than Inaba.
  • the vibriocidal ° levels to serotype Inaba are elicited earlier and in higher titer by the cellular vaccine compared to the conjugates.
  • the vibriocidal levels to Inaba elicited by the whole cell and conjugate vaccines are similar.
  • All vibriocidal activity is 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 0 O-SP in contrast, removes approximately 90% of the vibriocidal activity 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 5 injected with either the conjugates or the cellular vaccines.
  • Adsorption of DeA-LPS-CTI, DeA-LPS-CTII or other conjugates onto alum has no effect upon their immunogenicity.
  • Table 6 shows the cholera toxin antibodies that are o 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.
  • the LPS-protein conjugate of the present invention may be administered to human or animal subjects in the form of a vaccine for the purpose of treating or preventing infections caused by organisms containing the LPS and/or protein antigen.
  • Such vaccines can 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 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 twelve month period.
  • Monoclonal or polyclonal antibodies of human or animal origin can be produced via the use of the above- described vaccines. These antibodies can be administered to animals and humans, alone or in combination with the LPS-protein conjugate vaccines of the present invention, for the prevention or treatment of infections caused by the organism(s) from which the LPS and protein components of the conjugate are obtained. These antibodies can be administered to a subject in need thereof, either alone, for the purpose of passive immunization, or in combination with the LPS-protein conjugate vaccines of the present invention, as an adjunct therapy. Such antibodies can take the form of serum or gamma globulin containing the antibodies of interest.
  • 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.

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Abstract

L'invention décrit une formulation de vaccin comprenant des conjugués composés de LPS détoxiqués et de protéines comprenant la toxine du choléra (CT). Le traitement à l'aide de l'hydrazine (DeA-LPS) réduit les caractéristiques endotoxiques du LPS à des niveaux admissibles, et produit une molécule plus importante, et à activité/antigénique accrue, par rapport au saccharide produit par hydrolyse acide. Des conjugués utilisant la toxine de V. cholerae sont également décrit, lesquels présentent de faibles niveaux de pyrogènes, aucune activité toxique par rapport aux cellules ovariennes du hamster chinois, et induisent des réponses de renforcement chez les anticorps vibriocides et les anticorps dirigés contre la toxine du choléra lorsqu'ils sont injectés par voie sous-cutanée sous forme de solutions salines dans des souris. Les conjuguées produits sous forme de vaccin contre le choléra induisent la formation des mêmes anticorps que les vaccins cellulaires à injection parentérale, mais présentent des caractéristiques immunologiques et une sécurité améliorées.
PCT/US1993/000253 1992-01-16 1993-01-14 Vaccin conjugue a base de toxine du cholera et de lypopolysaccharide (lps) detoxique, utilise pour la prevention du cholera WO1993013797A2 (fr)

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JP5512624A JPH07503238A (ja) 1992-01-16 1993-01-14 コレラ予防用の無毒化ｌｐｓ−コレラ毒素結合ワクチン
AU34696/93A AU678549B2 (en) 1992-01-16 1993-01-14 Detoxified LPS-cholera toxin conjugate vaccine for prevention of cholera
EP93903428A EP0623026A1 (fr) 1992-01-16 1993-01-14 Vaccin conjugue a base de toxine du cholera et de lypopolysaccharide (lps) detoxique, utilise pour la prevention du cholera

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US82145392A 1992-01-16 1992-01-16
US07/821,453 1992-01-16

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WO1998026799A1 (fr) * 1996-12-18 1998-06-25 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vaccin conjugue contre la salmonella paratyphi a
WO2001078787A2 (fr) * 2000-04-18 2001-10-25 Endobiologics, Incorporated Vaccin conjugue anti-sepsie
WO2002080964A1 (fr) * 2001-04-06 2002-10-17 Institut Pasteur Vaccin conjugue d'un fragment de polysaccharide de lipopolysaccharide du vibrio cholerae 0139 lie a l'anatoxine tetanique
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
WO2003094959A1 (fr) * 2002-05-09 2003-11-20 Biosynth S.R.L. Vaccins de polysaccharide et glycoconjugues ameliores
WO2004043489A1 (fr) * 2002-11-14 2004-05-27 Instituto Finlay. Centro De Investigacion-Produccion De Vacunas Y Sueros. Procede d'obtention de vaccins conjugues et de compositions vaccinales qui les contiennent.
US7749511B2 (en) 2000-04-18 2010-07-06 Endobiologics, Incorporated Anti-sepsis conjugate vaccine
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 (zh) * 2012-09-12 2012-12-19 北京民海生物科技有限公司 霍乱弧菌o1群多糖结合疫苗、其制备方法及应用
US8852605B2 (en) * 2000-09-01 2014-10-07 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vibrio cholerae O139 conjugate vaccines

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026799A1 (fr) * 1996-12-18 1998-06-25 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vaccin conjugue contre la 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
WO2001078787A2 (fr) * 2000-04-18 2001-10-25 Endobiologics, Incorporated Vaccin conjugue anti-sepsie
WO2001078787A3 (fr) * 2000-04-18 2002-10-10 Endobiologics Inc Vaccin conjugue anti-sepsie
US8337863B2 (en) 2000-04-18 2012-12-25 Endobiologics, Incorporated Anti-sepsis conjugate vaccine
US7014857B2 (en) 2000-04-18 2006-03-21 Endobiologics, Incorporated Anti-sepsis conjugate vaccine
US8128942B2 (en) 2000-04-18 2012-03-06 Endobiologics, Incorporated Anti-sepsis conjugate vaccine
US7749511B2 (en) 2000-04-18 2010-07-06 Endobiologics, Incorporated Anti-sepsis conjugate vaccine
US8852605B2 (en) * 2000-09-01 2014-10-07 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vibrio cholerae O139 conjugate vaccines
US9173932B2 (en) 2000-09-01 2015-11-03 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vibrio cholerae O139 conjugate vaccines
WO2002080964A1 (fr) * 2001-04-06 2002-10-17 Institut Pasteur Vaccin conjugue d'un fragment de polysaccharide de lipopolysaccharide du vibrio cholerae 0139 lie a l'anatoxine tetanique
US7588765B2 (en) 2002-05-09 2009-09-15 Biosynth S.R.L. Polysaccharide and glycoconjugate vaccines
WO2003094959A1 (fr) * 2002-05-09 2003-11-20 Biosynth S.R.L. Vaccins de polysaccharide et glycoconjugues ameliores
WO2004043489A1 (fr) * 2002-11-14 2004-05-27 Instituto Finlay. Centro De Investigacion-Produccion De Vacunas Y Sueros. Procede d'obtention de vaccins conjugues et de compositions vaccinales qui les contiennent.
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
US8753649B2 (en) 2003-08-06 2014-06-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Polysaccharide-protein conjugate vaccines
US8465749B2 (en) 2003-08-06 2013-06-18 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Polysaccharide-protein conjugate vaccines
US9198976B2 (en) 2003-08-06 2015-12-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Polysaccharide-protein conjugate vaccines
US9474795B2 (en) 2003-08-06 2016-10-25 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Polysaccharide-protein conjugate vaccines
CN102824632A (zh) * 2012-09-12 2012-12-19 北京民海生物科技有限公司 霍乱弧菌o1群多糖结合疫苗、其制备方法及应用

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AU678549B2 (en) 1997-06-05
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WO1993013797A3 (fr) 1993-10-28
AU3469693A (en) 1993-08-03
CA2128212A1 (fr) 1993-07-22

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