Letters in Peptide Science, 7: 229-237,2O00.KLUWEMESCOMO 2001 Kluwer Academic Publishers. Printed in the Netherlands.

229

I

tI

Peptide synthesis containing a B-cell and a T-cell epitope on dextran beadsand evaluation of humoral response against bead-peptide construct

Luis J . Cruza*, Rub6n Padr6na, Luis J . Gonzillezu, Julio C. Aguilarb, Enrique Iglesiasb, HildaE. Garaya, Viviana Falc6na, Eulises Rodriguezu & Osvaldo Reyesaa Divisiones de Quimica Fisica; b Vacunas, Centro de Ingenierta Gendtica y Biotecnologia Apdo 6162, 10600Ciudad Habana, Cuba

Received 23March 2000; Accepted 30 November 2000

Key words; hydrophilic solid supports; solid-phase peptide synthesis; synthetic peptides; HIV-1; V3 loop.

Summary

Cross-linked dextran beads were chemically modified with Fmoc-BAla to give an amino functionalized supportsuitable for solid-phase peptide synthesis. On this support, a peptide comprising B-cell and T-cell epitopes intandem was synthesized manually by Fmoc procedure monitoring the coupling reaction by bromophenolblue pro-cedure and standard Kaiser test. The quality of peptide synthesis was verified by RP-HPLC and mass spectrometry.The size and morphologic characteristics of dextran beads were conserved after peptide synthesis. Furthermore, weanalyzedimmunogenicity in mice of the T-B peptide on dextran beads compared with T-B peptide immunogenicitywhen administered in CFAIIFA. In both cases, titers were high and there was not a significant difference in antibodytiters between groups (p<0.05). But in contrast CFA they are biocompatible and did not induce any adverse reactionat the site of injection. This experiment suggests that the peptides synthesis on dextran beads can be used to raisethe immunogenicity of synthetic peptide in vaccines or therapeutics.

Abbreviations: CFA, complete Freund's adjuvant; DIPEA, diisopropylethylamine; DIPC, I,3-diisopropylcarbodiimide; DCM, dichoromethane; DMF, N,N-dimethylformamide; DTT, dithiothreitol;EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ionization; Fmoc, 9-fluorenylmethoxycarbonyl;HOBI l-hydroxybenzotnazole; IFA, incomplete Freund's adjuvant; i.p., intraperitoneally; MS, massspecffometry; NMI, l-methylimidazole; OPD, orthophenylenediamine; PBS, phosphate-buffered saline;TB TU, 2- ( 1 H-ben zotrrazol- 1 -yl) - 1, 1, 3, 3 -tetramethyl-uronuim tetrafl uoroborate.

Introduction

Synthetic peptide vaccines are an interesting approachin vaccinology because they are safe, cheap and easyto store and handle. However, this methodology haslimitations, i.e. poor immunogenicity of small syn-thetic peptides to trigger an immune response. Theymust be coupled to macromolecular carriers such asproteins or synthetic polymers [1, 2], or associated to

x To whom correspondence should be addressed. Divisi6nde Quimica-Fisica, Centro de Ingenieria Gen6tica y Biotecno-logia, P. O. Box 6162, Cubanacdn, Habana 10600, Cuba.Phone: 53-7-218008, 218466. Fax: 53-7-214764, 218070. e-mail:ljcruz@cigb.edu.cu

particulate systems such as liposomes [3] or immunos-timulating complexes (e.g. ISCOMs) [4]. Another ma-jor obstacle for the development of this new strategy invaccination is the requirement of non-toxic adjuvants,which in most cases are required to stimulate efficientimmune responses. For the last few years, many newmolecules able to stimulate various components of theimmune system have been developed for such pur-pose. However, most of the new adjuvants found to beactive in experimental animal models are not allowedfor human applications due to safety concerns [5, 6].

Dextran, a biocompatible and biodegradable nat-ural polysaccharide, holds promise as an inexpensiveand attractive adjuvantforpeptides. Dextran, its sulph-

230

ate and diethyl aminoethyl derivatives are reported tohave adjuvant properties [7, 8]. Dextrans have beenused as carriers for many enzymes to increase the cir-culation half-life [9, 10] and to improve their stabilityto thermal and proteolytic degradation [11]. Recently,dextran beads covalently linked to tetanus toxoid havebeen used as a controlled delivery for immunogensystem [12].

Different applications of synthetic peptides areoften advantageously carried out with the peptides co-valently attached to a polymeric support [13]. Suchapplications include solid-phase binding studies inan ELISA format, resin screening of peptide librar-ies, isolation and purification of peptide acceptors onaffinity resins [14]. Besides, peptide synthesis on poly-meric adjuvants offers a direct way to vaccination [15].Up to now, peptides synthesized on dextranbeads havenot been used as immunogen.

In the present article, we describe the peptide syn-thesis on dextran beads using the Fmoc chemistry andtudy of humoral response of the V3 loop from gpI20of HIV-1 JYI isolate. The selected B-cell epitopeplays a key role in HIV-1 infection and pathogenesis

[16]. Furthennore, the V3 loop has been reported tobe essential for both virus infectivity and tissue trop-ism [17]. This region contains the main neutralizationdomain of HIV-1 gp 120 tl81.

Experimental

Materials

Fmoc-protected amino acids used for peptide syn-thesis were purchased from Bachem (Switzer-land). Dexffan beads cross-linked with epichlorhydrin(Sephadex G 150) were purchased from Pharmacia Bi-otech (Sweden). Solvents were obtained from Merck(Germany). TAB9 protein was obtained from CIGB(Cuba). It is a recombinant Multi-Epitope Polypeptide(MEP) including V3 regions from six divergent HIV-1isolates LR150, JY1, RF, MN, BRVA and IIIB, in thisorder; fused to the amino terminal 47 residues of theP64k protein of Neisseiia meningitidis U9l.

D etermination of functionalizate d dextran b eadssubstitution degree

The substitution degree (mmoVg of Fmoc-aminoacid-supports) was determined by deprotection of aweighed aliquot of dry Fmoc-amino acid-resin (5-10mg) with 2O7o piperidine in DMF for 20 minutes.

After filtering off the support, the solution containingN-(9-fluorenylmethyl) piperidine was measured at 300nm. Molar extinction coefficient 7800 [20].

Amino -functionalization of dextran b e ad s

The dried dextran beads were chemically modified tointroduce suitable anchor functions for the subsequentpeptide synthesis. In the first step, one gram of dex-ffan beads was treated for 24 hours'with 4 mL ofa 0.4 mol/L Fmoc-B-alanine solution (activated with0.48 mol/L DIPC and 0.8 mol/L NMI in DMF) toachieve an even distribution of amino functions byesterification of hydroxyl groups [21]. The remain-ing hydroxyl functions were acetylated by a treatmentwith 1O7o acetic anhydnde/2O7o NMI in DMF for 30minutes. After washing three times for 5 minutes withDMF, the Fmoc protecting group was cleaved with207o piperidine in DMF for a 2O-minute. Quantitat-ive Fmoc-determination gave loadings between 90 and100 tr^cmol per gram of dried dextran. In the secondstep, another B-alanine residue was coupled subequi-valently using 40 pmol Fmoc-B-alanine solution (ac-tivated with 40 prmol TBTU, 40 p,mol HOBT and l2Opr.mol DIPEA in DMF) 1227. After a 3-hour reactionat room temperature, the free amino groups were acet-ylated by a treatment with 207o acetic anhydridel2%oDIPEA in DMF for 15 minutes. The final substitutiondegree of the support was about 30 p,moUg.

Peptide design

The peptide selected contains B-cell and T-cell epi-topes in tandem (T-B peptide). The B-cell epitopecomprises the central 15 amino acids (Arg-Gln-Ser-Thr-Pro-Ile- Gly-Leu- Gly- Gln-Ala-Leu-Tyr-Thr-Thr)from the V3 loop of the gp I20 of JYI-HIV isol-ate 1231. The T-cell helper epitope belongs to region830-844 (Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu) of tetanus toxoid l24l.B and Thepitopes were spaced by a cathepsin like processingsite [25]. The designed peptide has the following se-quence: I Arg-Gln-Ser-Thr-Pro-Ile-Gly-Leu-Gly-Gln-Ala-Leu-Tyr-Thr-Thr-Lys -Ly s - G1n-Tyr-Ile-Ly s -Ala-

Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu- B Ala-

BAla3a.

Assembly of the T-B peptide

The synthesis was carried out on a 30 tr"cmol scale usingthe Fmoc/tBu chemistry [26]. Side chain protectinggroups used were tert-butyl (tBu) for Glu, Thr, Ser and

Tyr; Trityl (Trt) forGln and Asn;ter-butyloxycarbonyl(Boc) for Lys and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for Arg. Coupling of all amino acidswas carried out under standard conditions with 0.2mol./L Fmoc-amino acid solution (activated with 0.2mol./L HOBT and 0.2 mol/L DIPC in DMB. Thecoupling reaction was followed by bromophenol blueprocedure 1271 and standard Kaiser ninhydrin test

[28]. Recouplings were carried out in most cases using0.3 mol/L Fmoc-amino acid solution (activated with0.3 mol/L TBTU, 0.3 mol/L HOBT and 0.9 mol./LDIEA in DMF). After every coupling, the cappingsteps were performed with 47o acetic anhydride andlvo DIEA in DMF. The Fmoc group was cleaved bya 2O-minute treatment of the resin with 207o piperid-ine in DMF. The Fmoc groups were quantified spec-trophotometrically in order to determine the currentsubstitution degree of the resin (as described above).

Deprotection of side-chain groups andp eptide - support cleav age

After the last coupling cycle, protecting groups of theside-chains were removed by a 3-hour treatment with50Vo tdfluoroacetic acid containing l7o phenol, 27oethanedithiol, 2Vo tiophenol and 27o water in DCM.Then peptide-beads were washed four times withDCM, DMF and methanol respectively. Every peptidefragments were cleaved from the dextran beads with0.1 mollf- NaOH for 15 minutes. Next, they were neut-ralized and analyzed by RP-HPLC. The main peaksfrom each profile were collected and characterized bymass spectrometry analysis.

Hi gh- Perfownanc e Liquid Chromato graphy

The peptide was analyzed by reversed-phase, high-performance liquid chromatography (RP-HPLC) on aPharmacia LKB (Sweden) dual pump chromatographmodel 2150, coupled with an UV detector at 226 nm(Knauer Variable Wavelength Monitor). A C-18 Vy-dac column (4.6 mm x 100 mm), was used witha linear gradient (5Vo to 607o B in 40 minutes) ofacetonitrile (mobile phase B) in water (mobile phaseA), containing 0.05Vo and 0.17o, ffifluoroacetic acidrespectively, at a flow of 0.8 mllmin. Data wereprocessed by the BioCrom program (CIGB, Cuba).

Amino-acid analysis

Peptide hydrolysis was carried out for 24 hours invacuum-sealed ampoules, with 6 mol,/L HCI contain-

23r

ing 0.L%o phenol and O.l%o B-mercaptoethanol. Afterevaporation, free amino acids were dissolved in the ad-equate buffer and loaded on an automatic amino-acidanalyzer Alpha Plus 4151 (Pharmacia-LKB, Sweden),using a sodium buffer system for amino-acid separa-tion and ortho-phthalaldehyde derivatization for fluor-escence detection. The sample was analyzed in threereplicas.

Mass spectrometry

The ESI-MS and ESI-MSA4S spectra were obtainedusing a hybrid quadrupole orthogonal accelerationtandem mass spectrometer QTOF from Micromass(Manchester, UK) fitted with a Z-spray nanoflow elec-ffospray ion source 1291. The mass spectrometer wasoperated with a source at 80oC and a drying gas flowof 50 Lftr. The peptides purified by RP-HPLC werelyophilized and dissolved in a 50Vo waterlacetonitrile(v/v) solution containing l7o acetic acid to reach anapproximate concentration of 5 pmotelp,L. The pep-tide solution infused into the mass spectrometer byusing a syringe pump from Harvard Apparatus (Mas-sachusetts, USA) at3 pLlmin. The capillary and conevoltage were set to 3 kV and 35 volts, respectively.

The MSAvIS spectra were obtained by selecting theprecursor ion in the first quadrupole within a windowof 4-5 Th. A pressure of -3 x l0-2 Pa collision gas(argon) was used in the hexapole collision cell to yieldthe fragment ions. Appropriate collision energy wasused to reduce the intensity of the precursor ion tomore than half of its original intensity. Data acquisi-tion and processing were performed using a Masslynxsystem from Micromass.

Particle size and morphology

Dextran beads containing the assembled peptide andfree dextran as negative conffol were dusted on sup-port previously covered with glue, and overlappedwith a 40 nm gold layer in an ION SPUTTER (JFC-1100). Later, they were screened on a scanning elec-tron microscope (JEOL JSM-T330) at 15 kv, 200x.The samples from different synthesis were analyzedin three replicas.

Immunizati on p rot o c o I s

Female Balb/c mice (eight weeks old) were providedby CENPALAB, Havana, Cuba. Groups of eight micewere inoculated on days 0,14 and28 with approxim-ately 40 pg of peptide in each case: group A, peptide

232

35.0

30.0

25.0

20.0

15 .0

10 .0

5.0

10 15 20 25 30 34Couplings(reverse sequences)

Figure 1. Quantification of the Fmoc-groups after every five couplings during the manual synthesis of the T-B peptide on modified dextranbeads. Synthesis scale was 30 pr.mol.

tr.9oC)

Fc(E=C'(toEIL

oE?

0.0

synthesized on dextran beads resuspended in PBS i.p;group B, free peptide in PBS i.p; group C, peptideusing CFA/IFA i.p; group D, dextran alone (as a neg-ative conffol) i.p; group E, peptide in PBS via oraland group F, peptide synthesized on dextran beadsresuspended in PBS via oral administration. The in-oculation volume was 200 p,L in all cases. Mice werebled 10 days after the last inoculation.

En zyme - linke d immuno ab s o rb e nt a s s ay ( E LI S A ) fo rtitration of mice sera

A conventional indirect ELISA was performed.Briefly, the plates were coated with 4 1t gltttL of TAB9protein at 37 oC for three hours. After three washeswith 0.057o tween 20 rn distilled water, they wereblocked with LVo milk in PBS (blocking solution) forone hour at 37 oC. Then they were washed again, thesamples diluted in 0.05Vo tween 20, 57o sheep serumin blocking solution and incubated with an anti-mouseIgG-peroxidase conjugate for one hour at 37 "C. Fi-nally, the reaction was developed for 10 minutes usingorthophenylenediamine (0.05Vo) substrate and H2O2(0.05Vo). OD values were read at 492nm. The samplesera were diluted 1:100, 1:1000 and 1:10000 andtheir OD values were interpolated in a curve using astandard serum of known titer.

Statistical analysis

All ELISA OD values were transformed to the nat-ural logarithm for statistical analysis in the immunized

groups. An F-test was performed to assess variancehomogeneity between groups and a Studentps t-testwas done (p<0.05 was considered statistically signi-ficant).

Results and discussion

Synthesis and characterization of the peptide ondextran beads

Primary amino functions were introduced into dex-tran beads by esterification of hydroxyl groups withthe carboxyl functions of the first amino acid. Thisreaction was carried out in a Fmoc-B-alanine solu-tion activated with NMI and DIPC. The remaininghydroxyl functions were acetylated with acetic anhyd-ride and l-methylimidazole to avoid the formation ofdeletion sequences. The ester bond is stable enoughfor standard Fmoc peptide synthesis [30]. Thus, thesynthesis of a mixture of peptides linked via amideand ester bonds offers the possibility of cleaving theesters selectively after completion of the synthesis byalkaline hydrolysis, which would allow the qualitycontrol of the synthesized peptide while the amide-linked peptide remains on the resin [31]. Generally,amino group loads of 80-150 pmol per gram of drymaterial were obtained, which is comparable to sev-eral commercial resins. For the synthesis of the T-Bpeptide, it was preferred to reduce the load of thepolymer, due to a poor solvating of the dextran beadswith respect to the traditional polymers such as cross-

233

Ec

.El\l$t

GI

tl,

{

I=.tt

ttoL't(l

aoct

Ed

(ol\JtloIJ

F

o

d

Time (min)

Time (minl

(IIFIstoTJ

F

oo

E{754 .

mlz

Time (minl

Figure2. Reverse-phase HPLC analysis of the crude peptides obtained in the manual synthesis of lArg-Gln-Ser-Thr-Pro-Ile-Gly-Leu;Gly-Gln-

Ala-Leu-Tyr-Thr-Thr-Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu-BAla-BAla34. A, (30tnr-BRla34); B,(25t-ys-P*fa); C, (15itr-'Px*\;b,1lerg-pnta3a). conaitions: Vydac column C18 (4.6 mm x 100 mm),linear gradient over40 min of 5to607oCH3CN/0.05VoTFAnH2O/0.1Vo TFA,flowrate0.8ml/min.Detection at226nm.InsetofRP-HPLCprofiles,anexpandedregionofthe ESI-MS spectra of the main fractions are shown (see also table 1).

751.63

234

Thble 1. Summary of the results obtained by mass spectrometry analysis of the peptide synthesized on dextran beads

Segment Profiles Fraction # Exp. mlz Charge Theor.

state mJz

Assignment

30-3425-341.5-34r-34

ABCD

Main fractionMain fractionMain fraction123a3b45a5b6I

891011 (main fraction)T2T3

546.28 +0.01

552.82 4.02756.09 +0.02485.77 +0.01716,38 +O.02913.52 -O.03

757.42 -0.03

805.97 +0.03756.09 +0.01812.79 0.00653.36 +0.01809.46 +0.02596.33 +0.03948.20 4.02837.78 4.0275r.22 0.001093.85 4.02976.54 4.02

3016r-p6634zsLys-pAla3a1s15"-9663411,.g-Gtye28cty-pttu3alAtg-L"u82Glo-Leu81571u-11"27LsTltr-Bp;.#4lAtg-Lyr2123 Asn-pAla3azort"-ppttfa24ser-pAt;t410Gh_BAla3415 pro-p1r"261A"g-pAh3asPro-BAlfaz6p1o"_p61fa

546.27s52.80756.1,r485.767t6.409r3.49757.39806.00756.108r2.79653,37809.48596.30948.18837.7875r.221093.83976.52

1+2+3+1+1+1+1+2+3+3+2+2+2+3+3+5+3+1+

ABFigure 3. Analysis by electron microscope before and after the peptide synthesis on dextran beads: A; dextran (negative control), B;peptide-dextran. Magnifi cation 200x.

linked polystyrene and polyamide. For that, the secondFmoc-B-alanine linker was coupled subequivantly byactivation with TBTU. Remaining amino groups of thesupport were capped by a treatment with acetic an-hydride. The substitution degree of the dextran beadswas determined photometrically at 300 nm. The valuewas about 30 pmol per gram of dry resin. The amino-functionalized material can be used in this form for

immobilized protein or antibodies for potential applic-ations such as affinity purifications or binding assays.In our case, it was used for immunization of peptidesynthesized on biodegradable solid support.

The assembly of the peptide on dextran beads wascarried out by Fmoc chemistry. The couplings wereperformed manually using HOBT and DIPC in DMF.Double couplings were carried out in most cases and

235

6.00

5.00

4.00

3.00

2.OO

1.00

0.00F

Group

Figure 4. Antibody titers of Balb/c mice immunized with: (A), 40 pr.g of the peptide synthesized on dextran beads in PBS i.p; (B), 40 p'gfree

peptide in PBS i.p; (C), 40 ttg of peptide emulsified in Freund's adjuvant i.p; (D), dextran alone (as a negative control) i.p; (E), 40 ttg peptide

in PBS oral administration; (F), 40 lt g peptide synthesized on dextran beads oral administration. Titers are expressed as the geometric mean I

standard desviation.

eE

.=F

E )oJ

reaction times were 1-2 hours using TBTU activa-tion method. The capping step with acetic anhydridewas performed. The amino group load of the dextranbeads was determined indirectly every five couplingby Fmoc quantification. It showed a downward trendover the course of the synthesis, which was more sig-nificant at the end of the synthesis (Fig. 1). Similarbehaviour has been observed in different syntheses ofthe same peptide with this resin (data not shown).

After the side chain deprotection, all the peptideswere cleaved from the support by alkaline hydro-lysis. The quality of the crude peptide was verifled byRP-HPLC. The analytical profile for each peptide isshowed in Figure 2. As it can be observed, the qualityof the crude peptides was acceptable up to couplingtwentieth. The last profile (Figure 2,D) showed moreside products. Besides, as it was expected the qualityof the crude peptide synthesized on dextran beads wasnot as good as the quality of the crude peptide syn-thesized on MBHA (4-methylbenzhydrylamine resin)

t321.The main fraction from each profile were char-

acteized by electrospray ionization mass spectro-metry (ESI-MS) so that, the expected molecular massmatched very well with the theorical mass. A sum-mary of these results is given in Table 1. In the RPHPLC profile of the final crude peptide (Figure 2,D), all the undesirable products were identified byESI-MS and ESI-MSA{S. The peptides contained infractions 1 (1Arg-Glye), 3a (lArg-Leu*), 3b (2cln-

Leu8), 4 1lsThr-rlez7y, 5b (1Arg-Lyr21), 10 (lsPro-

Phe26) could be originated by the low stability of theester bond linker to dextran beads [33]. The deletionsequences corresponding to the fractions 2 128Gly-BAla34), 5a (lsThr- BAla34), 6 (23Asn- BAla3a), 7

120lre-Bxru34), 8 (2aser-BAla34), 9 110cln- BAla34),12 (sPro-BAla3a1, 13,126Phe-Bxla34) could be given

by the poor solvating of the dextran beads in thecoupling conditions or probably by sluggish coup-lings due to a folding of the sequence in this support.The amino-acid composition of the main fraction wasdetermined after a total acid hydrolysis. The amino-acid analysis of three replicates of the peptide (lAtg-

BAla3a) showed the expected composition (data notshown).

Characterization of particle size and morphology

The cross-linked dextran beads were analyzedby elec-tron microscope before and after the peptide synthesison dextran beads. In both cases, the particles had asimilar size of 5-50 trcm in diameter (Figure 3 A andB). A high degree of aggregation (100*300 pm in dia-meter) was observed in the peptide-dextran particles.This may be possible by peptide-chain interactions.

Immunogenicity of peptides on dextran beads

Groups of eight Balb/c mice were inoculated to evalu-ate the adjuvant properties of dextran beads carryingpeptides in the induction of anti-peptide antibodies.

236

After three doses the sera were evaluated by ELISAto determine the reactivity towards the JYl epitope,efficiently exposed by TAB9 protein, used for platecoating. Figure 4 shows the titers obtained for theimmunization groups. Antibody responses were onlydetected in groups A (Peptide-dextranlPBS, i.p.) andC (peptide inoculated in CFA/ICA, i.p.). Both anti-gens were highly immunogenic and generated specificantibodies against the JY1 epitope. There was not astatistically significant difference between groups Aand C (p<0.05). Antibody responses were not detec-ted for a dilution 1:100 in groups E and F orallyinoculated, and in group B intraperitoneally inoculatedwith the peptide in PBS. But, the last result is lo-gical, considering the poor immunigenicity of peptideswhen inoculated in PBS. Immunizations with peptide-dexfran neither had serious adverse reactions on theanimals nor caused granulomas at the site of injection,although proper toxicology studies are wiuranted todraw conclusions.

Conclusions

We have demonstrated the performance of the highlyhydrophilic and use-compatible dextran beads insolid-phase peptide synthesis using the Fmoc protocol.The T-B peptide was synthesized on dextran beadsand charactenzed by HPLC, mass spectrometry andamino-acid analysis. The limitation of the synthesisof peptide on this support is clearly indicated in thisarticle. Thus, it could be more suitable for the peptidesynthesis up to twenty amino acids in the sequence.

After the synthesis, the size and morphology ofbeads were unaltered. Furthermore, the adjuvant activ-ity of dextran beads carrying synthetic peptides inmice was demonstrated. This result agreed with pre-vious studies showing the immunenhancing effect ofdextran when it was conjugated to the tetanic tox-oid [12]. This strategy may be used to present otherB-cell epitopes of different pathogens and is poten-tially a peptide-based vaccine candidate consideringthe strong adjuvant effect comparable to the FCA andthe demonstrated safety of their use [12].

Acknowledgements

The authors wish to thank Dr. Lila Castellanos andOrlando Alvarezfor critical reading of the manuscript.

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