INFECTION AND IMMUNITY, Dec. 1990, p. 4136-41410019-9567/90/124136-06$02.00/0Copyright © 1990, American Society for Microbiology

Vol. 58, No. 12

An Intact Interchain Disulfide Bond Is Required for theNeurotoxicity of Tetanus Toxin

GIAMPIETRO SCHIAVO,' EMANUELE PAPINI,' GIOIA GENNA,2 AND CESARE MONTECUCCO1Centro Consiglio Nazionale delle Ricerche Biomembrane e Istituto di Patologia Gener-ale,Universitca di Padova, Via Trieste 75, 35121 Padua,' and Sclavo Spa, 53100 Siena,2 Italy

Received 28 June 1990/Accepted 29 September 1990

Tetanus toxin is composed of a heavy chain (100 kDa) and a light chain (50 kDa) held together by a singleinterchain disulfide bridge. An additional intrachain disulfide is present in the carboxy-terminal part of the heavychain. Reduction of the two disulfide bonds in tetanus toxin with both chemical and proteinaceous reducing agentswas studied. Dithiothreitol and 2-mercaptoethanol cleaved both the inter- and intrachain disulfide bridges of thetoxin, while glutathione and cysteine were ineffective. Specific reduction of the single interchain disulfide link wasachieved with the thioredoxin-thioredoxin reductase system, thus indicating that this bond is exposed at theprotein surface. Also, dead or permeabilized cells were able to reduce the toxin. Such reduced toxin bound toneuronal membranes as well as the native toxin but was not neurotoxic. These findings open the possibility thatreduction by cytoplasmic agents released by dead cells contributes to detoxification of tetanus toxin. Moreover,together with the notion that the light chain is the active form of the toxin in the cytoplasm, these results suggestthat the interchain disulfide bond of tetanus toxin plays a role in nerve cell penetration.

Tetanus toxin is produced by toxigenic strains of Clostrid-ium tetani and is responsible for the clinical symptoms oftetanus (13, 35). It induces spastic paralysis by blockingrelease of neurotransmitters at the level of inhibitory inter-neurons of the spinal cord (35). Tetanus toxin is a protein of150,700 daltons (10, 11) composed of two chains, the heavy(H; 100 kDa) and light (L; 50 kDa) chains, held together bynoncovalent interactions and a single interchain disulfidebridge (Fig. 1) connecting Cys-438 of the L chain withCys-466 of the H chain (19). There is an additional intrachaindisulfide bond linking Cys-1076 with Cys-1092 of the Hchain, while the residual six half-cystine residues are presentin the sulfhydryl form (5, 19).A disulfide bond linking subunits or domains that play

different roles in the intoxication process is a commonfeature of protein toxins with intracellular targets, termedA-B toxins (R. Rappuoli and M. G. Pizza, in J. E. Alouf andJ. H. Freer, ed., Structure, Regulation, and Activity ofBacterial Protein Toxins, in press). Their cellular mechanismof action can be divided into three different steps (32):binding, membrane translocation, and target modification. Ithas been suggested that, by analogy to diphtheria toxin,tetanus toxin is taken up by receptor-mediated endocytosisand that the low pH present in the endosomal lumen causesthe toxin to insert itself into the lipid bilayer and cross themembrane to reach the cytosol (6, 13, 23, 32).A common requirement for activation of several intracel-

lularly acting toxins is reduction of their interchain disulfidebridge (1, 8, 12, 27; Rappuoli and Pizza, in press). Fortetanus toxin, it has been shown that the L chain, freed byreduction, is the active form of the toxin in the cytoplasm (2,4, 22). Moreover, very recently, mRNAs that encode the Lchain or for carboxy-terminal deletion mutant proteins havebeen generated by in vivo transcription. When microinjectedinto the presynaptic cholinergic neurons of the buccal gan-glia of Aplysia californica, mRNAs encoding L-chain deriv-atives that lacked the cysteine residue produced inhibition

* Corresponding author.

similar to that produced by an mRNA that encodes theauthentic L chain (H. Kurazono, S. Mochida, T. Binz, U.Eisel, B. Poulain, L. Tauc, and H. Niemann, submitted forpublication).

It is not clear whether reduction of tetanus toxin has totake place outside the cell, as appears to be the case forcholera toxin (30, 33), or inside the cell, as for diptheria toxinand for some plant protein toxins (3, 26. 37, 38). This has abearing on the mechanism of cell penetration of these toxins,because while reduction inhibits entry of diptheria toxin andplant toxins into cells, splitting of the interchain disulfidebond appears to be a prerequisite for membrane insertion ofcholera toxin (C. Montecucco, E. Papini, and G. Schiavo, inJ. E. Alouf and J. H. Freer, Struicture, Regulation, andActivity of Bacterial Protein Toxins, in press).

MATERIALS AND METHODSDithiothreitol (DTT), cysteine, glutathione, 2-mercapto-

ethanol, octylglucoside, and iodoacetamide were fromSigma (Munich, Federal Republic of Germany). Purifiedstreptolysin 0 (220 to 240 hemolytic units/jig of protein) wasa kind gift of Dr. Fabbiani (Sclavo, Siena, Italy).

Tetanus toxin. The single-chain toxin was purified from aculture of C. tetani (Harvard strain) by the extractionprocedure of Ozutsumi et al. (29). The crude toxin solutionwas chromatographed on DEAE-cellulose (Whatman) andUltrogel ACA-34 (LKB) (21), followed by high-performancesize exclusion liquid chromatography analysis on a TSKG4000SW column (0.75 by 60 cm; LKB) equilibrated with0.1 M sodium phosphate (pH 6.8). The tetanus toxin con-centration was determined spectrophotometrically at 280 nmwith an £j mg/ml of 1.55. This value was determined asdescribed by Whitaker and Granum (36), and we found it tobe the average value of protein determinations by amino acidanalysis and enhanced alkaline copper protein assay (20).The dichain form of tetanus toxin was obtained by nickingsingle-chain toxin with tosylsulfonyl phenylalanyl chloro-methyl ketone-treated trypsin (Serva) at 25°C for 60 min witha toxin-to-protease ratio of 1,000:1 (wt/wt). Proteolysis wasterminated by addition of soybean trypsin inhibitor at a final

4136

NEUROTOXICITY OF TETANUS TOXIN 4137

311 lSo law a)NH*430

/77= 7777777777771ZJ~IrMFI7,77>.

FIG. 1. Schematic structure of dichain tetanusinter- and intrachain disulfide bridges and theremaining six free cysteine residues.

protease-to-inhibitor ratio of 1:4 (wt/wt). Th4at 4°C, or after being frozen in liquid nitrogeat -80°C at a protein concentration of 2 tomM sodium phosphate buffer (pH 7.4) or4-(2-hydroxyethyl)-piperazine-1-ethanesulfoiThe toxin contained about 107 mouse lethalprotein, determined as described below. Ictoxin to a specific activity of about 10 mCi/Lperformed with Bolton-Hunter reagent (Arnresidual toxicity higher than 85%.

Reduction of tetanus toxin with chemicaltetanus toxin was diluted to 0.5 mg/ml wit]mM sodium phosphate-1 mM EDTA (pHcontaining various amounts of DTT (1 to 25 j

toethanol (10 mM), glutathione (10 mM),mM) and incubated at 37°C under N2. Rdenaturing conditions was performed withdissolved in buffer A. At different times, samremoved and the reaction was blocked by aacetamide at a final concentration three tijreducing agent. The mixture was incubated fin the dark and prepared for sodium dodecypolyacrylamide gel eletrophoresis by addinchloride-8% SDS (pH 6.8). The samples100°C for 3 min and loaded onto 8% polyagels (17). After staining with Coomassie Idestaining, the relative amounts of reducedtetanus toxin in each sample were evaluatedric analysis at 600 nm with a Shimadzu CSlength densitometer.

Reduction of tetanus toxin with thioredoxinus toxin (0.5 mg/ml in buffer A) was incubh0.2 mM NADPH (Sigma) and 2 ,uM Escheridoxin (IMCO, Stockholm, Sweden). The ereaction was started by adding an ammoniurrsion of E. coli thioredoxin reductase (IMconcentration of 0.1 ,uM (15). After 5 to 60 mN2, the reaction was stopped by incubation0.5 mM (final concentration) iodoacetamicwere analyzed by SDS-polyacrylamide geland densitometry as described above.

Reduction of tetanus toxin with cells. From106 Vero cells (African green monkey kidwere washed twice with deaerated Hank:solution, and after centrifugation, they were100 R1 of the same medium. They were incubof 125I-labeled tetanus toxin with or withouttamide, streptolysin 0 (80 hemolytic uniloctylglucoside at 37°C for 3 h under nitrogenand octylglucoside were then added sequ4concentrations of 10 mM and 0.4%, respectples were centrifuged, the pellets were trealTris-acetate-0.5 mM EDTA-8% SDS-105

electrophoresis sample buffer), and the supernatant wasL (50 kDa) treated with 50% trichloroacetic acid to a final concentration

of 6.5%. The trichloroacetic acid-precipitated material wasrecovered by centrifugation and treated with gel electropho-resis sample buffer. After neutralization with 2 M Tris base,

H (100 kDa) all samples were boiled for 2 min and loaded onto a 10%polyacrylamide gel and subjected to electrophoresis (17).The dried gels were exposed to Kodak X-Omat AR films,and the amount of reduced toxin was determined by densi-

toxin showing the tometric scanning of the developed films.positions of the Titration of free thiol groups by DTNB. Tetanus toxin

samples (150 ,ug) obtained by reduction with chemical orproteinaceous agents were precipitated with trichloroacetic

e toxin was kept acid (6.5% final concentration) and centrifuged at 15,000 x g-n, it was stored for 3 min. Pellets were washed twice with 0.5 ml of 6.5%10 mg/ml in 10 trichloroacetic acid and then with 0.5 ml of deaerated10 mM sodium distilled water. Precipitates were dissolved by adding 0.8 mlnate (pH 7.4). of 50 mM Tris-glycine-1 mM EDTA-2% SDS (pH 8.0). Afterdoses per mg of 15 min at room temperature, the protein concentration was)dination of the estimated spectrophotometrically. Subsequently, 30 p1l ofmg of toxin was 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (5 mg/ml in 50iersham) with a mM Tris-glycine-1 mM EDTA, pH 8.0) was added to the

samples, and the A410 was determined against that of a blankagents. Dichain without toxin. The sulfhydryl content was calculated on theh deaerated 100 basis of a molar absorptivity of 13,600 M-1 cm-' at 410 nm7.5) (buffer A) for 2-nitro-5-thiobenzoate anion and expressed as moles ofmM), 2-mercap- free thiol groups per mole of tetanus toxin.or cysteine (10 Neurotoxicity determination. Dichain tetanus toxin wasLeduction under treated with thioredoxin (200 jiM NADPH, 2 jiM thiore-6 M guanidine doxin, 0.1 jiM thioredoxin reductase; 60 min, 30°C) or DTTples (8 jig) were (10 mM; 90 min, 37°C) and then diluted with deaerated bufferiddition of iodo- A containing iodoacetamide (final concentration, 10 mM). Ames that of the control sample of nonreduced tetanus toxin was carboxy-For 5 min at 25°C methylated with iodoacetamide under the same conditions.4I sulfate (SDS)- After dialysis against 100 mM sodium phosphate-1 mMg 62.5 mM Tris EDTA (pH 7.5), the samples were serially diluted withwere heated to phosphate-buffered saline (pH 7.4) containing 0.1% bovineicrylamide-SDS serum albumin and injected intraperitoneally (100 jil ofblue R-250 and tetanus toxin per g of body weight) into 40-day-old mice.and nonreduced Time before death was recorded, and mouse lethal dosesby densitomet- were calculated as described by Boroff and Fleck (7, 34).

,-930 dual-wave- Tetanus toxin binding to synaptosomal membranes. Synap-tosomal membranes were prepared from whole rat brain (28)

in. Dichain teta- or bovine brain cortex (9). Samples (10 ng) of these mem-ated at 4°C with branes were suspended in 80 pJ of 12 mM sodium phos-'chia coli thiore- phate-1.5 mM potassium phosphate-118 mM sodium chlo--nzymatic chain ride (pH 7.4) and preincubated with different amounts ofa sulfate suspen- unlabeled or thioredoxin-reduced tetanus toxin at 10-12 toICO) to a final 10-6 M. After 30 min at 4°C, 15,000 cpm of 125I-labeledin at 30C under tetanus toxin (3 fmol) was added to each sample underfor 15 min with stirring. After 90 min of incubation at 4°C, unbound 1251_de and samples labeled tetanus toxin was eliminated by centrifugation atelectrophoresis 15,000 x g for 3 min and the pelleted membranes were

washed once with 1 ml of cold buffer. Samples were counted0.5 x 106 to 3 x in a Packard Auto-Gamma 800, and the results, after sub-Iney fibroblasts) traction of unspecific binding, were expressed as percents balanced salt binding in the absence of unlabeled tetanus toxin (28).resuspended in

iated with 100 ng RESULTS5 mM iodoace-

ts/ml), or 0.4% Reduction of tetanus toxin with chemical reductants. Teta-. lodoacetamide nus toxin contains 10 cysteine residues (5, 10, 11). Six ofentially to final them are present in the thiol form (residues 26, 185, 198, 311,ively. The sam- 868, and 1300); Cys-438 and Cys-466 form a disulfide bondted with 10 mM linking the two toxin chains, while Cys-1076 and Cys-1092Mo glycerol (gel are involved in an interchain disulfide link (19).

VOL. 58, 1990

4138 SCHIAVO ET AL.

FIG. 2. Kinetics of reduction of the interchain disulfide bridge oftetanus toxin by DTT. (A) Coomassie blue-stained SDS-8% poly-acrylamide gel of a tetanus toxin sample treated with DTT at 37°C;the upper part shows the corresponding densitometric scan. (B)Kinetic curves of tetanus toxin (TeNT) reduction at different DTTconcentrations (U, 1 mM; 0, 2.5 mM; *, 10 mM; 0, 25 mM). Thecurves were obtained by plotting the logarithm of the unreducedtoxin concentration versus the reaction time. Points are averages ofthree separate sets of experiments. Standard deviation bars neverexceeded 10% and were omitted for clarity.

Reduction of the interchain disulfide of tetanus toxin canbe monitored quantitatively and conveniently by densitom-etry of Coomassie blue-stained gels. Tetanus toxin wasreduced rapidly with DIT, and by graphic plotting, a sec-ond-order rate constant for DTT reduction of 12 min-' M`was estimated (Fig. 2A and B). However, this assay does notprovide information about the reduction of the intrachaindisulfide in the H chain. Free sulfhydryl groups associatedwith tetanus toxin before and after treatment with DTT,2-mercaptoethanol, and glutathione were titrated with Ell-man reagent (14). Both the interchain and intrachain disul-fide bonds were broken by DTT and 2-mercaptoethanol(alone or with 6 M guanidine) because their actions gener-ated four additional thiols (Fig. 3); glutathione and cysteinewere ineffective. In contrast to diphtheria toxin (18), itappears that selective cleavage of the disulfides of tetanustoxin cannot be achieved with chemical reductants.Enzymatic reduction of tetanus toxin. To test the possibility

that the two disulfides of tetanus toxin differ in degree ofexposure, which would allow obtainment of a specificallyreduced toxin, we used a well-established method based onuse of the thioredoxin-thioredoxin reductase system (15, 16).

Z S E E E

(ao 0 la0 1--0

NATIVECONDITION

GUANIDINE6 M

FIG. 3. Titration of free thiol groups of tetanus toxin (TeNT)before and after treatment with different reducing agents. Tetanustoxin was reduced with glutathione (GSH), 2-mercaptoethanol(ME), and DTT. SH, Sulfhydryl.

Thioredoxin is a ubiquitous protein with an active centercomposed of two disulfide-linked Cys residues that pro-trudes out of the protein globular structure. Thioredoxin isreduced by thioredoxin reductase and NADPH, and reducedthioredoxin can cleave only exposed protein disulfide bonds(15, 16). The reaction can be conveniently monitored spec-trophotometrically by measuring NADPH oxidation at 340nm or by titration of newly formed free thiol groups withDTNB after inactivation of thioredoxin.

Thioredoxin was also very effective in reducing the inter-chain disulfide link of tetanus toxin, and parallel to this chainsplitting was the appearance of titratable sulfhydryl groups,with a final plateau level of two additional thiols (Fig. 4). Thesame result was obtained with the single-chain toxin (datanot shown), thus indicating that nicking did not influence thedegree of exposure of the interchain disulfide. Exposure ofthe interchain disulfide bridge also was not influenced by thepresence of bovine brain lipids-cholesterol vesicles at neu-tral pH under conditions in which tetanus toxin interactswith the head group of phospholipids (25). At pH 4.5,although thioredoxin is active (26), this disulfide bond couldnot be reduced. Under these conditions, tetanus toxin un-

dergoes a structural change with exposure of hydrophobicsurfaces that allows its insertion into the membrane (6, 24,31). When neutral pH was restored, the reducibility of theinterchain disulfide was completely recovered (data notshown).Tetanus toxin with a cleaved interchain disulfide was

stable, did not precipitate for a period of weeks at 4°C, andshowed a retention time in high-performance size exclusionliquid chromatography equal to that of unreduced toxin. InSDS-polyacrylamide gel electrophoresis, after blocking of

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INFECT. IMMUN.

NEUROTOXICITY OF TETANUS TOXIN 4139

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FIG. 4. Comparison of kinetics of reduction of the interchaindisulfide bridge of tetanus toxin with thioredoxin determined bydensitometric scanning of SDS-polyacrylamide gel electrophoresisgels and titration with DTNB. Open bars refer to percentages of thedichain form of tetanus toxin as determined by densitometricscanning, while hatched bars refer to the increase of thiol groupsdetermined with DTNB. The averages from three different sets ofexperiments are reported, together with standard deviation bars.SH, Sulfhydryl.

the activity of thioredoxin with iodoacetamide, the H chainof thioredoxin-reduced toxin showed 9% lower mobility thanthe DTT-reduced sample. This was due to the presence of anintact intrachain disulfide, because treatment in the presenceof SDS with DTT or thioredoxin, which is still active in thepresence of the detergent, shifted the band to the position ofthe fully reduced sample (data not shown).

Reduction of tetanus toxin by cells. It is possible thatpermeabilized or dead cells in a necrotic anaerobic areareduce tetanus toxin by releasing cytoplasmic reducingagents. To test for this possibility, Vero cells were treatedunder anaerobiosis with radioiodinated tetanus toxin in thepresence of (i) buffer, (ii) iodoacetamide (to block anyreductant released by cells), or (iii) a permeabilizing reagentsuch as octylglucoside or streptolysin 0.

While no reduction took place in the presence of cells andiodoacetamide, cells alone reduced 16 to 20% of the toxinadded. In the presence of streptolysin 0- and octylglucoside-permeabilized cells, 12 to 15 and 11 to 15%, respectively, ofthe radioiodinated toxin was reduced. In contrast, the sametreatments under aerobic conditions caused no reduction ofthe toxin. Thus, it appears that the single interchain disulfideof tetanus toxin may be cleaved efficiently by reducingagents released by dead cells. This effect may contribute atleast partial to neutralization (see below) of the toxicity oftetanus toxin in vivo.

Neurotoxicity of reduced tetanus toxins. To assay theneurotoxicity of the reduced toxin, the possibility of S-Sreforming by oxidation was prevented by carbamidomethy-lation with iodoacetamide. This treatment does not influencethe enzymatic activity of other A-B toxins and blocks thesulfhydryl groups generated by reduction with either DTT or

thioredoxin, as determined with DTNB (data not shown).While carbamidomethylation did not affect the toxicity oftetanus toxin, in agreement with previous findings (5), re-

duction of the single interchain disulfide bridge was sufficientto abolish the neurotoxicity of tetanus toxin (Table 1). In

TABLE 1. Neurotoxicity of different forms of tetanus toxin

Mean ± SD

Form of tetanus toxin MLD" (ng oftetanus toxin/kg of body wt)

Native .............................. 4 + 0.5Carboxymethylated ........... ................... 5 + 0.5Thioredoxin reduced, carboxymethylated ........... ..... 50 ± 5DTT reduced, carboxymethylated .......................... 320 ± 30

a MLD, Mouse lethal doses.

fact, the residual activity of thioredoxin-reduced toxin canbe accounted for by a residual amount of unreduced toxinthat was traced after gel electrophoresis; this is in line withthe fact that DTT-reduced tetanus toxin contained lessunreduced toxin and was less active than thioredoxin-re-duced toxin.Thioredoxin-reduced toxin can be defective at any of the

different steps of the process of cell intoxication. Theintracellular step can be ruled out, because it has beendemonstrated that the reduced toxin and the isolated L chainare fully active (2, 4); the possibility that carboxymethyla-tion of Cys-438 influences the intracellular activity of the Lchain is unlikely on the basis of the above-mentioned results,showing that a mutant chain truncated before Cys-438 is asactive as the intact chain (Korazano et al., submitted). Onthe other hand, interchain-reduced tetanus toxin and thenative toxin appeared to be identical in the ability to competewith 125I-labeled tetanus toxin for binding to bovine braincortex and rat whole-brain synaptosomal membranes (Fig.5).

DISCUSSIONThe present study investigated the reducibility and acces-

sibility of the two disulfide bonds in the tetanus toxinmolecule. We have provided evidence that the interchainS-S bond is exposed at the protein surface and that it can becleaved specifically by a proteinaceous reducing agent, such

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FIG. 5. Binding of '251-labeled tetanus toxin to synaptosomalmembranes from bovine brain cortex (A) and rat brain (B). Differentconcentrations of unlabeled, thioredoxin-reduced, and carboxy-methylated (A) or native (0) tetanus toxin fromt 10-11 to 10-6 Mwere added, followed by 10,000 to 15,000 cpm of '25I-labeled tetanustoxin per sample, and samples were treated as described in Materi-als and Methods. The results are expressed as percent binding in theabsence of unlabeled tetanus toxin after subtraction of unspecificbinding (28). The points are averages of at least three differentsamples, and the bars represent standard deviations.

w _r.H

VOL. 58, 1990

4140 SCHIAVO ET AL.

as the coupled thioredoxin-thioredoxin reductase system. Incontrast, the intrachain S-S bond in the H chain is notaccessible to thioredoxin, while it is cleaved by muchsmaller chemical reductants, such as DTT and 2-mercapto-ethanol. It is interesting that other chemical reducing agentsabundant in the cell cytosol, such as glutathione and cys-teine, are ineffective in a period of hours. Since the activeform of tetanus toxin in the cell cytosol is the reduced one (2,4, 22), the latter finding raises the question of the nature ofthe agent that cleaves and activates the toxin in the cytosol.The present results, the ubiquitous presence in the cyto-plasm of protein-reducing systems such as thioredoxin-thioredoxin reductase, and the knowledge that full activationof tetanus toxin in the cytoplasm of A. californica occurswithin 1 h (22) are consistent with the possibility of enzy-matically catalyzed cleavage of the interchain disulfide bondof tetanus toxin.Tetanus toxin reduced at the interchain disulfide does not

release free chains and is stable in the cold for weeks: thetwo chains appear to be held together efficiently by nonco-valent forces. However, with specific reduction of the inter-chain disulfide, tetanus toxin loses its neurotoxicity. In thisrespect, it is noteworthy that dead cells are able to reducetetanus toxin because it could have relevance in vivo. Infact, it is known that C. tetani proliferates and releases thetoxin in the anaerobic conditions reached in necrotic tissues,where reducing agents released by dead cells could at leastpartially detoxify tetanus toxin by reduction and/or prote-olysis.The loss of neurotoxicity of reduced tetanus toxin is due

neither to lack of binding, as shown by its ability to competefor binding to synaptosomal membranes, nor to inactivation,because the isolated L chain is active in the cytoplasm (2, 4,22). Since reduction does not influence the cell-binding andintracellular neuroexocytosis-blocking activities of tetanustoxin, we are left with the possibility that cleavage of theinterchain disulfide alters a property of tetanus toxin in-volved in the second step of the cell intoxication pathway,i.e., membrane translocation (32). With respect to the role ofthe interchain disulfide link, this situation resembles morethat of diptheria toxin, in which reduction blocks cell pene-tration (37), than that of cholera toxin, in which reductionappears to cause membrane insertion (Montecucco et al., inpress).

ACKNOWLEDGMENTSWe are grateful to 0. Rossetto for experimental help and R.

Rappuoli for critical reading of the manuscript.This research was supported by grants from the C.N.R. Target

Project on Biotechnology and Bioinstrumentation and by the con-tract Programma Nazionale di Ricerca per la Chimica: tema 3,Linfochine e Vaccini Sintetici, granted to Sclavo by the ItalianMinistry of Scientific Research and Technology.

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