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Chicken antitoxin neutralizes ricin in vitro and in vivo
Tenth European Symposium
527
backbone amide proton of Gly-39 and Cys-40. Dendrotoxin I is based on a oeatrel antiparallel ß-sheet and two small helices located at the N-aad the Gterminal extremities. According to the disulfide bridges positions and the long-range nuclear Overhauser effects (NOE) observed, these secondary structural elements are folded in a similar manner than for bovine pancreatic trypain inhibitor (BPTI) for which dendrotoxin I aluu+ea 33Y. of sequence homology . Prior to structural calculations using NMR derived constraints, this similarity allows the gross localization of solvent exposed charged amino acids by auperimpo:ing the sequeax of dendrotoxin I to the fold of BPTI. The spatial repartitioa of the charged amino acids concerned strongly singla~out dendrotoxin I from BPTI sad form potential aitea of interaction with the voltage sensitive potassium channels for which dendrotoxin I is a strong inhibitor. In addition, the structural homology between the two structursa reinforce the idea that dendrotoxin I would derive from a Kïmitz-type inhibitor as genetic ancestor .
Chicken mttltox5t nerurallzes rkin in vitro mtd ia vivo. P. V. L.»ay~tr,' P. Axetv~rn~,' D. Cae/slu,' J. Rtrvo,' A. OTitm.t.' and D. C. Sr~moau' ('Twuxot oov Drvtatotv, U.S . Atesnr Mi~tc~t. Res~+acx I~rrrtrre ot" Irv~trrtotls Dt~w.~s, I~yt1iDFJUCIC, MD 21702-5011, U.S.A .; aad=Orz~ux PHearrec~tmc~s, Mennotv, WI 53711, U.S.A.). Ax evurr antitoxin wan investigated for its ability to provide passive immunoprophylaxis against ricin intoxication . Laying hens were hyperimmunized with ricin toxoid and antibody was purified from egg yolks. After an affinity-purification step, the antibody wan characterized both in a sensitive in vitro all assay and !n vivo by using a mouse model. When the In vitro ricin challenge was set at 100 ng/ml ( > 10 x ~, the ammity-purified chicken antitoxin neutralized > 98'/. of the rien at a 4 :1 antibody : toxin ratio. When toxin challenge (i .v .) was titrated against a 100'rg i.v. protective antibody, the avian antibody protected all animals challenged with 5.0 pg of toxin ( ~ 175 pglkg; > 60 x t.o~). Thin was significantly better than previously characterized mammalian antitoxins. F~rlrmrcenrent ojTmrdB responses agabrst a snake toxin by primtreg with a synthetic T-cell stinpdathrgpeptide. M. L~oxerrt, S., Ltatov, H. M~F, J. Carrox, H. L~xx, G. Moor and A. M~vez (Département d'In®énierie et d'Etudea des Protéines, CE-Saclay, 91191, Gif/Yvette, Cedex, France). Stvw,s~ aeurotoxins bind to the nicotinic axtylcholine inceptor, thereby inducing flaccid paralysis and provoking death by respiratory failure. We previously showed that the immunodominant T-cell stimulating region of the neurotoxin a from Nq/a ntgricoUis is comprised betwxn residues 24 and 41, in BALB/c mix. To improve the quality of existing antieeraa we investigated the effect of primingmin with the fragment 24-41 on toxin a-specific T and B raponsa. Mix received various doeea of peptide a~ were boosted with toxin a, 2l days Inter. In control experiments min were primed with native or unfolded toxin a and subsequently boosted with native toxin. A dwadepeadent priming effect wes seen with peptide 24-41 which, however, was a leas potent priming immunogen than unfolded toxin which in turn, was leas potent than native toxin. Antitoxin antibodies raised by priming with peptide 241 were present in sera of mice more than 3 months after the boosting injection, however, titres progreuively declined with time. The priming effect by the peptide was long-lasting sinx both toxin-specific T a~ B responses could be boosted more than 5 montlu slier the priming injection . Prrrlfrcatiore mid characterizatlorr of two high molecular weight hentorrhagk toxins front Crotalus viridis viridis verrorn ruing morroclorwl mrtibodies. Q. Lt, T. R. Ctx.eeaa and C. L. OwNav (Department of I~ysiological Sciences, Oklahoma State University, Stillvater, OK. 74078, U.S .A.) . Two xiax mol. wt hemotrhagic toxins were purified from Crotahrs virldis vb~ir venom using HPLC anion exchange and affinity chromatography following initial separation by HPLC with a preparative DEAE column. Fraction 4 with the highest lremorrhagic activity wan used to immunize BALB/c min for production of monodoaal antibodia. MAb 17e recognized a single protein band of fraction 4 in native PAGE, and was purified and conjugated to activated membranes for amity purification of the antigen from fraction 4. Proteins eluted from the amity membranes showed flues bands in SDS-PAGE with an apparent mol. wt of 70,000, 62,000 and 30,000 . The 70,000 and 62,000 proteins were further purified using as analytical DEAE anion exchange column, and purity was demonstrated by the prssenx of a single band in SDS-PAGE with Coomasaie blue sad a single pr+eelpitation arc against antibodies to Crotahu viridir vir/dir venom in immunoelectrophore.~is. The isoekctric points were 8.5 and 4.1 for the 70,000 and 62,000 toxins, respectively. Both toxins are 1>ighly hemorrhagic, both cleaved N,N-dimethyk~sein and fibrinogen, and their enzymatic activity wes completely inhibited by 0.25 M EDTA, indicating their metalloproteinase nature. Both toxins are glycoproteins with the name galactoao-ß(1-4}N-axtylgluoosamine aide chains which are Gnked to asparagine residues of the proteins. Trlat
~1t5-B
527
backbone amide proton of Gly-39 and Cys-40. Dendrotoxin I is based on a oeatrel antiparallel ß-sheet and two small helices located at the N-aad the Gterminal extremities. According to the disulfide bridges positions and the long-range nuclear Overhauser effects (NOE) observed, these secondary structural elements are folded in a similar manner than for bovine pancreatic trypain inhibitor (BPTI) for which dendrotoxin I aluu+ea 33Y. of sequence homology . Prior to structural calculations using NMR derived constraints, this similarity allows the gross localization of solvent exposed charged amino acids by auperimpo:ing the sequeax of dendrotoxin I to the fold of BPTI. The spatial repartitioa of the charged amino acids concerned strongly singla~out dendrotoxin I from BPTI sad form potential aitea of interaction with the voltage sensitive potassium channels for which dendrotoxin I is a strong inhibitor. In addition, the structural homology between the two structursa reinforce the idea that dendrotoxin I would derive from a Kïmitz-type inhibitor as genetic ancestor .
Chicken mttltox5t nerurallzes rkin in vitro mtd ia vivo. P. V. L.»ay~tr,' P. Axetv~rn~,' D. Cae/slu,' J. Rtrvo,' A. OTitm.t.' and D. C. Sr~moau' ('Twuxot oov Drvtatotv, U.S . Atesnr Mi~tc~t. Res~+acx I~rrrtrre ot" Irv~trrtotls Dt~w.~s, I~yt1iDFJUCIC, MD 21702-5011, U.S.A .; aad=Orz~ux PHearrec~tmc~s, Mennotv, WI 53711, U.S.A.). Ax evurr antitoxin wan investigated for its ability to provide passive immunoprophylaxis against ricin intoxication . Laying hens were hyperimmunized with ricin toxoid and antibody was purified from egg yolks. After an affinity-purification step, the antibody wan characterized both in a sensitive in vitro all assay and !n vivo by using a mouse model. When the In vitro ricin challenge was set at 100 ng/ml ( > 10 x ~, the ammity-purified chicken antitoxin neutralized > 98'/. of the rien at a 4 :1 antibody : toxin ratio. When toxin challenge (i .v .) was titrated against a 100'rg i.v. protective antibody, the avian antibody protected all animals challenged with 5.0 pg of toxin ( ~ 175 pglkg; > 60 x t.o~). Thin was significantly better than previously characterized mammalian antitoxins. F~rlrmrcenrent ojTmrdB responses agabrst a snake toxin by primtreg with a synthetic T-cell stinpdathrgpeptide. M. L~oxerrt, S., Ltatov, H. M~F, J. Carrox, H. L~xx, G. Moor and A. M~vez (Département d'In®énierie et d'Etudea des Protéines, CE-Saclay, 91191, Gif/Yvette, Cedex, France). Stvw,s~ aeurotoxins bind to the nicotinic axtylcholine inceptor, thereby inducing flaccid paralysis and provoking death by respiratory failure. We previously showed that the immunodominant T-cell stimulating region of the neurotoxin a from Nq/a ntgricoUis is comprised betwxn residues 24 and 41, in BALB/c mix. To improve the quality of existing antieeraa we investigated the effect of primingmin with the fragment 24-41 on toxin a-specific T and B raponsa. Mix received various doeea of peptide a~ were boosted with toxin a, 2l days Inter. In control experiments min were primed with native or unfolded toxin a and subsequently boosted with native toxin. A dwadepeadent priming effect wes seen with peptide 24-41 which, however, was a leas potent priming immunogen than unfolded toxin which in turn, was leas potent than native toxin. Antitoxin antibodies raised by priming with peptide 241 were present in sera of mice more than 3 months after the boosting injection, however, titres progreuively declined with time. The priming effect by the peptide was long-lasting sinx both toxin-specific T a~ B responses could be boosted more than 5 montlu slier the priming injection . Prrrlfrcatiore mid characterizatlorr of two high molecular weight hentorrhagk toxins front Crotalus viridis viridis verrorn ruing morroclorwl mrtibodies. Q. Lt, T. R. Ctx.eeaa and C. L. OwNav (Department of I~ysiological Sciences, Oklahoma State University, Stillvater, OK. 74078, U.S .A.) . Two xiax mol. wt hemotrhagic toxins were purified from Crotahrs virldis vb~ir venom using HPLC anion exchange and affinity chromatography following initial separation by HPLC with a preparative DEAE column. Fraction 4 with the highest lremorrhagic activity wan used to immunize BALB/c min for production of monodoaal antibodia. MAb 17e recognized a single protein band of fraction 4 in native PAGE, and was purified and conjugated to activated membranes for amity purification of the antigen from fraction 4. Proteins eluted from the amity membranes showed flues bands in SDS-PAGE with an apparent mol. wt of 70,000, 62,000 and 30,000 . The 70,000 and 62,000 proteins were further purified using as analytical DEAE anion exchange column, and purity was demonstrated by the prssenx of a single band in SDS-PAGE with Coomasaie blue sad a single pr+eelpitation arc against antibodies to Crotahu viridir vir/dir venom in immunoelectrophore.~is. The isoekctric points were 8.5 and 4.1 for the 70,000 and 62,000 toxins, respectively. Both toxins are 1>ighly hemorrhagic, both cleaved N,N-dimethyk~sein and fibrinogen, and their enzymatic activity wes completely inhibited by 0.25 M EDTA, indicating their metalloproteinase nature. Both toxins are glycoproteins with the name galactoao-ß(1-4}N-axtylgluoosamine aide chains which are Gnked to asparagine residues of the proteins. Trlat
~1t5-B