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Toxicon 48 (2006) 64–74

www.elsevier.com/locate/toxicon

North and South American Loxosceles spiders:Development of a polyvalent antivenom withrecombinant sphingomyelinases D as antigens

Alejandro Olveraa, Blanca Ramos-Cerrilloa, Judith Estevezb, Herlinda Clementa,Adolfo de Roodtc, Jorge Paniagua-Solısb, Hilda Vazquezb, Alfonso Zavaletad,

Marıa Salas Arruzd, Roberto P. Stocka,�, Alejandro Alagona

aDepartamento de Medicina Molecular y Bioprocesos. Instituto de Biotecnologıa,

Universidad Nacional Autonoma de Mexico. Av. Universidad 2001, Cuernavaca, Morelos 62210, MexicobDireccion de Investigacion. Laboratorios Silanes S.A. de C.V. 83100 Mexico City, Mexico

cSeccion Farmacologıa, Facultad de Ciencias y Filosofıa. Universidad Peruana Cayetano Heredia. Av. Honorio Delgado 430 Lima, PerudNational Institute for the Production of Biologicals, Administracion Nacional de Laboratorios e Instituto de Salud ‘‘Dr. Carlos G. Malbran’’,

Buenos Aires, Argentina

Received 25 January 2006; received in revised form 17 April 2006; accepted 24 April 2006

Available online 3 May 2006

Abstract

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles

laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active

recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared

in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in

rabbits and F(ab0)2 preparations in animals used for commercial antivenom production (horses). We performed studies on

in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins.

We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit

and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our

conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-

Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic

groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Loxosceles; Recombinant sphingomyelinase D; Enzymatic activity; Antivenom

e front matter r 2006 Elsevier Ltd. All rights reserved

xicon.2006.04.010

ng author. Tel.: +52 777 329 1602;

7 2388.

ss: rstock@ibt.unam.mx (R.P. Stock).

1. Introduction

The development of therapeutic antibodies fortreating envenomation can be conveniently dividedinto three distinct stages: first, the use of wholehyper-immune serum from immunized animals,

.

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–74 65

known since the end of the XIX century and still inuse very occasionally to this day; the second stagecorresponds to the development of efficient meansto enrich the immunoglobulin fraction from hyperimmune sera, ideally separating the protectivefraction from other serum components known tobe responsible for adverse effects such as serumsickness and distinct types of (hyper)sensitivityreactions; thirdly, the development of methods forthe production of very pure antigen-binding mono-or divalent immunoglobulin domains (Fab0 orF(ab0)2, respectively), of high neutralizing potencyand very well tolerated by the majority of patients(for a recent summary of antivenom productionstrategies, see Theakston et al., 2003).

Therapeutic antibodies, however, must still beobtained by immunization of suitable animals withvenom or venom-containing tissue preparations orfractions thereof. Many animal venoms are knownto be complex mixtures of high molecular weight(protein) and intermediate to low molecular weight(peptide, nucleotides) components with distincttoxic activities. In some instances, no individualconstituent is of exceptionally high toxicity, and thepotency of the venom is the result of synergybetween several components; therefore, protectiveantibodies must bind and neutralize a wide spec-trum of molecular species to be effective. Venoms oflow complexity, however, contain one or very fewmolecules responsible for the physiopathologicaleffects of envenomation. These venoms then, atleast regarding mammals, lack significant synergismbetween components, such that, by and large, thetoxicity of the whole venom amounts to that of aparticular individual component. If the toxin inquestion is a protein, as it often is, then hetero-logous expression might prove useful in theproduction of active toxins and/or toxoids whichcould be used for immunization of animals. Shouldthese recombinant proteins elicit protective anti-bodies of comparable potency to those elicited byimmunization with natural venoms, production andstandardization of antivenoms could be greatlyfacilitated, as one of the limiting steps in productionis the acquisition of large enough quantities ofquality venoms and/or venom fractions (venomapparatuses, venom glands) for immunization, titerevaluation and functional efficacy testing, particu-larly from small animals such as spiders. Theantivenoms obtained against recombinant immuno-gens, then, could be classified as ‘‘fourth genera-tion’’ therapeutic antibodies, in the sense that not

only are potentially harmful components eliminatedfrom the immunoglobulin fraction, but that thefraction is obtained by immunization with, and itsefficacy tested against, purified recombinant venomcomponents of species selected for their medicalrelevance.

In the case of spiders of the genus Loxosceles,

several enzymatic activities have been characterizedin their venom, and have been proposed to play apart in the physiopathology of envenomation(extensively reviewed in Hogan et al., 2004; com-ments in Isbister and Vetter, 2005). While several ofthese activities may contribute, in an as of yetundetermined way, to various aspects of envenoma-tion, sphingomyelinase D (SMD, a major venomcomponent; Kurpiewski et al., 1981; Binford andWells, 2003) seems to be responsible for lethalityand necrotic lesions characteristic of loxoscelism, ashas been shown for several species of this genususing either purified or recombinant SMD (Tam-bourgi et al., 1998; Fernandes Pedrosa et al., 2002;Ramos-Cerrillo et al., 2004; Tambourgi et al., 2004).It was also shown that experimental antisera raisedagainst the recombinant proteins protected againstenvenomation in susceptible animals (FernandesPedrosa et al., 2002; Araujo et al., 2003). In aprevious study, we reported the purification ofseveral SMD isoforms of Loxosceles boneti andLoxosceles reclusa and the sequence of some of thegenes encoding them, and proposed, in agreementwith others, that recombinant proteins could be ofuse in the study of the, apparently rather complex,mechanisms underlying local and systemic toxicity,as well as the molecular structure and enzymaticcharacteristics of this family of enzymes (Ramos-Cerrillo et al., 2004). Furthermore, the amino acidsequences of active SMDs are notoriously con-served within and between species, thus makingsignificant cross-reactivity between antibodies raisedagainst SMD from different species a reasonableexpectation (Ramos-Cerrillo et al., 2004; Tambour-gi et al., 2004). In fact, very high inter-speciesneutralization of dermonecrosis in rabbits wasobserved for antisera raised against dermonecroticfractions of the North American species Loxosceles

deserta and L. reclusa by Gomez et al. (2001) and ina monovalent antivenom against Loxosceles gaucho

(Barbaro et al., 2005).Specific antivenoms have been proposed to be of

value in the treatment of cutaneous (CL) andvisceral (VCL) forms of human loxoscelism (Sezer-ino et al., 1998), such that several antivenoms are

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–7466

available (Hogan et al., 2004). In this study weaddress the question of inter-species serologicalcross-reactivity by a systematic comparison ofneutralizing capacity of antibodies obtained usingrecombinant SMD and venom preparations asimmunogens in experimental animals. The strategywe chose makes use of both in vitro enzymaticactivity assays and in vivo neutralization assays byantibodies raised against purified recombinantSMD of L. boneti, L. reclusa and Loxosceles laeta

tagged at the N- or C-terminus, with the aim ofestablishing criteria for the generation of effectivepolyvalent antivenoms against SMDs from differentspecies of Loxosceles. We also present a phyloge-netic analysis which frames our observations,and those of others, in the context of the diversityof Loxosceles sphingomyelinases with knownsequences.

2. Materials and methods

2.1. SMD sequence information

The GenBank accession numbers for the L. laeta

sequences from Peru reported in this work areDQ369999 (isoform 1, Ll1) and DQ370000 (isoform2, Ll2). The rest of the sequences were obtained atthe NCBI web server with the following accessionnumbers: L. boneti isoform 1 (Lb1, AY559844), L.

boneti isoform 3 (Lb3, AY559845), L. reclusa

isoform 1 (Lr1, AY559846), L. laeta from Brazil(LlH17, AAM21154) Loxosceles intermedia P1(LiP1, P83045) and P2 (LiP2, P83046), L. gaucho

(Lg, AAY42401) and Loxosceles arizonica (La,AAP44735).

2.2. Cells and cultures

The Escherichia coli strains XL1-Blue (Strata-gene) and BL21trxB-DE3 (Novagen) were used inthis work, the former for general molecular biologyand the latter for expression of soluble rSMD. Allcultures for plasmid preps and cloning were done inLB medium supplemented with antibiotics whenappropriate, and except for expression of solublerSMD, at 37 1C.

2.3. Primers for amplification of SMD for expression

in E. coli

The primers listed below were used for amplifica-tion of mature SMD sequences and introduction of

appropriate restriction sites for in-phase directionalcloning in the expression vectors for E. coli. Designof the primers for L. boneti and L. reclusa was basedon the sequences listed above.

Name

Sequence Site Vector

50 North-N/C

AAA GGA TCCGCG AACAAA CGC CCGGCG TGG ATCATG

BamH I

pQE30-60

30 North-N

GGGG GTCGAC TTA ATTCTT GAA TGTTTC

Sal I

pQE30

30 Lb1-C

GGGG AGATCT ATT CTTGAA TGT TTCCCA

Bgl II

pQE60

50 South-N/C

AAA GGA TCCGCT GAT AACCGT CGT CC

BamH I

pQE30-60

30 Ll1-N

GGGG GTCGAC CTA ATTTTT AAA AGTCTC

Sal I

pQE30

30 Ll2-C

GGGG AGATCT ATT TTTAAA AGT CTCCCA TG

Bgl II

pQE60

2.4. General molecular biology and generation of

expression constructs

The general strategy was to amplify by PCR thesequences of the mature toxins with the primersdescribed above and directional cloning into depho-sphorylated pQE vectors (Qiagen) digested with theappropriate enzymes. The sequences of the L. boneti

and L. reclusa SMDs were from our previous study(Ramos-Cerrillo et al., 2004), and the template usedfor reverse transcription and cDNA amplification ofthe L. laeta SMDs was total venom gland RNA ofL. laeta from Peru and the design of the primers foramplification of SMD from L. laeta was based onthe sequences published by Fernandes Pedrosa et al.(2002) for Brazilian specimens. The two distinctsequences amplified from these specimens werecalled L. laeta isoforms 1 and 2. The PCR-amplified

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–74 67

fragments were first cloned in Topo 2.1 (Invitrogen)sequencing vectors, sequenced and subcloned intothe appropriate expression vectors using the intro-duced restriction sites. When the PCR-amplifiedSMD coding sequence was cloned into plasmidpQE30, a recombinant fusion protein with a 6-histidine (His)6 tag at the amino terminus isproduced, whereas plasmid pQE60 codes for afusion protein with the (His)6 tag at the C-terminalend. Both plasmids have IPTG-inducible promo-ters, a start codon and appropriate selectionmarkers. The sense (50) primer used for L. boneti

and L. reclusa was the same (50 North-N/C), and theantisense (30) primers were chosen for each C- or N-tagged construct. The same is true for the L. laeta

expression constructs. The sequences amplified andcloned for expression were: SMD isoform 1 from L.

boneti (expressed as one N-tagged and one C-taggedconstruct, Lb1N and Lb1C, respectively); SMDisoform 1 from L. reclusa (N-tagged, Lr1N), SMDisoform 1 from L. laeta (N-tagged, Ll1N) and SMDisoform 2 from L. laeta (C-tagged, Ll2C).

2.5. Expression of soluble recombinant SMD

E. coli strain BL21trxB-DE3 was used forexpression of soluble and active forms all recombi-nant proteins. Cells were grown in LB mediumsupplemented with 100 mg/ml ampicillin at 20 1C for16 h in agitation after induction with 100 mM IPTG.Cells were harvested in phosphate-buffered saline(PBS) and lysed by sonication on ice, clarified bycentrifugation (10min at 11,300g) and proteinpurified by passage through a metal affinity columnNi-NTA-Sepharose (Qiagen) as described by themanufacturer. Washes were performed with varyingconcentrations of imidazole in PBS and elution with250mM imidazole in the same buffer. The recom-binant proteins were dialyzed against PBS andanalyzed by SDS-PAGE (12.5%) according tostandard protocols (Laemmli, 1970) under reducingconditions and stained with Coomasie Blue.

2.6. Protein determination and measurement of

SMD activity in venom preparations and recombinant

proteins

Venom preparations (gland extracts, GE) fromvenom glands were done as previously described(Ramos-Cerrillo et al., 2004). Protein quantitationwas done using the Bicinchoninic acid reagent(BCA, Pierce) using the protocols and standards

supplied with the reagents. SMD activity ofrecombinant proteins was measured by a coupledassay using the substrate AMPLEX (MolecularProbes) as previously described (Ramos-Cerrillo etal., 2004).

2.7. Immunization of rabbits

NZW white rabbits (3–4 kg) were immunizedsubcutaneously with increasing doses of eachrecombinant SMD (Lb1N, Lb1C, Lr1N, Ll1N andLl2C), starting with 30 mg/dose and ending with100 mg/dose (total of 8 doses for L. boneti and L.

reclusa, and 9 for the L. laeta isoforms). The firstimmunization was always done with each immuno-gen emulsified with an equal volume of CompleteFreund’s Adjuvant (CFA) and subsequent doseswere given emulsified in Incomplete Freund’sAdjuvant (IFA). Additional rabbits were alsoimmunized with each of the following antigens inPBS: whole venom gland homogenates (9 doses withincreasing doses of 20–250 mg of protein) of L.

reclusa and L. boneti. Rabbits immunized with L.

boneti gland extract received a final boost with 4doses with of 60–100 mg of protein from an SMD-enriched chromatographic fraction previously de-scribed (Fraction 2, Ramos-Cerrillo et al., 2004).The rabbits were immunized over a period of 3months.

2.8. Immunization of horses and F(ab0)2 preparation

Besides the monospecific experimental rabbitantisera generated against each recombinantSMD, five horses were hyperimmunized subcuta-neously with the following mixture of recombinantSMDs: Lb1C (1/3), Lr1N (1/3), Ll1N and Ll2C (1/6each) starting with a dose of 15 mg total protein inCFA and increasing the doses up to 60 mg totalprotein over a period of five months with alternatingIFA and aluminum hydroxide as adjuvants. F(ab0)2fragments were prepared following the standardprotocol (US Patent 6,709,655 B2) at the InstitutoBioclon S.A. de C.V (Mexico City, Mexico).

2.9. Inhibition of smd enzymatic activity by F(ab0)2

fragments

The extent of inhibition of enzymatic activity ofrecombinant SMD by antibodies was measured usingthe standard activity assay with a fixed amount ofprotein (well within the linear range of the assay) and

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–7468

increasing amounts of antibodies in 96-well micro-titer plates. Briefly, 100ng each of Lb1C, Lr1N andLl1N, or 50ng of Ll2C, were preincubated for 30minat 37 1C with 8 serial dilutions (1:3) of F(ab0)2starting at 180mg/well (lowest amount 0.08mg/well)in 100ml reaction buffer (100mM Tris–HCl pH 7.5containing 4mg/ml Bovine Serum Albumin). In thecase of natural venom (as venom gland extracts,GE), the amounts of protein were 1mg for L. boneti,2mg for L. reclusa and 0.5mg for L. laeta. Theenzymatic reaction was initiated by addition of 100mlof substrate solution as indicated in the AMPLEXkit (Molecular Probes) and incubated for a further30min at 37 1C. Controls consisted of enzyme withirrelevant antibody (100% activity) and a blank withno enzyme. Absorbance at 570 nm was recorded in amicroplate reader and, after blank subtraction, theabsorbance plotted as a function of the amount ofF(ab0)2. The results were analyzed by non-linearregression (sigmoidal dose–response) using the soft-ware package Prism 4.0 (GraphPad Software, Inc.)to calculate the ID50, that is, the amount of F(ab0)2that inhibits 50% of enzyme activity, and convertedto the amount of protein (in mg) neutralized per mgof antibody. In all cases over 99% total inhibitionwas observed.

2.10. In vivo assays

Median lethal doses of venom preparations orrecombinant SMD (LD50) were determined onBALB/c mice (18–20 g, four mice per group) byintraperitoneal injection according to standardprotocols (World Health Organization, 1981).

Fig. 1. (A) Nucleotide and amino acid sequences of the N- and C-termin

work. Vector sequences are in lower case, SMD coding nucleotides are i

N–tagged constructs in pQE30 and Bgl II site for the C–tagged construc

isoform 1, C-tagged; Lr1N, L. reclusa isoform 1, N-tagged; Ll1N, L. la

SDS-PAGE analysis of the affinity-purified recombinant SMDs, reduc

Neutralization potency (as ED50) of rabbit antiseraor equine F(ab0)2 were done by intraperitonealinjection of 3�LD50 of venom gland homogenatesor recombinant SMD preincubated with varyingamounts of antivenom as previously described (deRoodt et al., 2004). ED50 values were converted tomg of recombinant toxin or gland extract proteinneutralized per ml of rabbit antiserum or mg ofF(ab0)2 polyvalent antivenom.

2.11. Sequence analysis and alignment

Mature SMD sequences reported in this study, aswell as those reported by other groups were alignedusing the Multiple Sequence Alignment software ofthe Gene Works v. 2.5 package (Intelligenetics, Inc.)and the dendrogram generated using standardparameters.

3. Results

3.1. Cloning, expression and purification of soluble

recombinant His-tagged SMDs

The PCR-amplified fragments of mature SMDfrom L. boneti (Lb1N), L. reclusa (Lr1N) andL. laeta (Ll1N) were cloned into the expressionvector pQE30. The same isoform (Lb1) of L. boneti

SMD and the second isoform of L. laeta werecloned into pQE60 (obtaining Lb1C and Ll2C,respectively). The 50 ends of the fusion proteinsobtained from the constructs in pQE30, as well asthe 30 ends of the fusion proteins from pQE60 areshown in Fig. 1A. Induction of expression was

i of the constructs for expression of His-tagged SMDs used in this

n bold upper case, and cloning sites are overlined (BamH I for the

ts in pQE60. Lb1N, L. boneti isoform 1, N-tagged; Lb1C, L. boneti

eta isoform 1, N-tagged; Ll2C, L. laeta isoform 2, C-tagged. (B)

ing conditions, 12.5% gel.

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–74 69

initially done using XL1-Blue cells, however, undercarefully controlled conditions of inducer concen-tration, time of induction and temperature, nosoluble recombinant SMDs could be obtained.Soluble recombinant SMD of all three species,however, could be induced at low IPTG concentra-tion (100 mM) at relatively low temperature (20 1C)and longer induction time (16 h) in E. coli strainBL21, and purified by metal affinity chromatogra-phy. The purity of the fractions was greater than90%, as shown by SDS-PAGE analysis (Fig. 1B).The proteins were soluble at physiological pH andionic strength, requiring no detergents, and theywere used for production of antibodies in rabbitsand horses, enzymatic activity determinations andtoxicity assays.

3.2. SMD activity and toxicity (as LD50) of natural

venom preparations and recombinant toxins

All soluble proteins purified as above wereenzymatically active. Table 1 shows a comparisonof the specific SMD activities of preparations fromwhole venom gland homogenates (GE) of L. boneti

(Lb), L. reclusa (Lr) and L. laeta (Ll), as could beexpected, the specific activity of all recombinantSMDs of L. boneti and L. reclusa were higher thanthat of whole gland homogenates, consistent withthe fact that SMDs (active and inactive) in venomor homogenate represent a significant, but byno means the only, part of the venom. Also, theN-tagged and C-tagged isoform of L. boneti hadsimilar specific activity, indicating that the place-ment of the histidine tag was of minor signifi-cance, at least in this case. L. laeta SMD isoformswere considerably more enzymatically activethan those of the North American species.Once the enzymatic activities of soluble rSMD

Table 1

Specific activity and toxicity (as LD50) of gland extracts and recombin

Preparation Specific activity (Um

Gland extract-Lb 8.071.7

Lb1N 31.578.6

Lb1C 32.577.4

Gland extract-Lr 8.671.0

Lr1N 18.373.8

Gland extract-Ll 40.775.3

Ll1N 56.879.9

Ll2C 228.2758.7

Activity was measured using the coupled AMPLEX assay (7SD of tri

were determined, and knowing from previousreports that enzyme activity was a requisite fortoxicity and dermonecrotic activity, we proceededto evaluate their lethality in mice. The results arealso summarized in Table 1. All enzymatically activerecombinant toxins had LD50 (for mice) values inthe microgram range, and the most toxic wasisoform 2 of L. laeta (Ll2C).

3.3. Amino acid sequence similarity between SMDs

A multiple sequence alignment (Fig. 2A) of allavailable mature SMD sequences was performedand a dendrogram of phylogenetic distances (asmeasured by frequency of amino acid replacements,Fig. 2B) was generated. Two branches and threemain groups for active SMDs can be distinguished:one that encompasses all the SMD from NorthAmerican species, that is, L. boneti isoform 1 (Lb1),L. reclusa isoforms 1 and 2 (Lr1 and Lr2) andL. arizonica (La); another group within this branchthat includes the South American species L. gaucho

(Lg) and the two isoforms P1 and P2 ofL. intermedia (Li); the second branch defines a,so far, monospecific group encompassing the threeL. laeta (Ll) sequences (two from our Peruvianisolate, Ll1 and Ll2, and one from Brazil, LlH17).The outlying branch corresponds to the enzymati-cally inactive isoform of L. boneti (Lb3).

3.4. Immunological cross-reactivity between

recombinant SMDs

In Table 2 we summarize the results of in vivoneutralization of lethality experiments using mono-valent rabbit antisera. The monospecific rabbit anti-L. boneti obtained using recombinant SMD orwhole gland extracts (GE) neutralized the toxicity of

ant SMD preparations

g�1) LD50 in mg/mouse (95% c.i.)

30.5 (23.8–39.1)

5.00 (4.96–5.04)

4.00 (3.80–4.20)

21.0 (20.0–22.0)

3.50 (3.48–3.52)

21.7 (19.7–24.7)

11.3 (11.2–11.4)

2.37 (2.36–2.38)

plicate determinations) as indicated in Section 2.

ARTICLE IN PRESS

Fig. 2. (A) Alignment of deduced amino acid sequences of mature SMDs. Asterisks indicate cysteine residues; the five amino acid insertion

of L. laeta SMDs is overlined (accession numbers in Section 2). (B) Dendrogram of phylogenetic distance (as measured by amino acid

variation) between reported sequences of SMD derived from the sequence alignment.

A. Olvera et al. / Toxicon 48 (2006) 64–7470

L. boneti gland extract (GE-Lb) and recombinantSMD (Lb1N and Lb1C), and also comparablyneutralized the toxicity of L. reclusa gland extract(GE-Lr) as well as recombinant SMD (Lr1N). Theconverse was also true for the antisera raised againstL. reclusa gland extract or recombinant SMD. Inthe case of the two L. laeta isoforms, they were noteffectively neutralized by any of the anti-L. boneti

or anti-L. reclusa antisera and were only neutralizedby the specific antisera and significant cross-protec-tion was observed between the two isoforms (Ll1Nand Ll2C).

3.5. Neutralization of lethality and SMD activity by

F(ab0)2 fragments

The polyvalent horse F(ab0)2 preparation raisedagainst the mixture of recombinant SMDs effec-tively neutralized the lethality of all three venompreparations (Table 3). Neutralization per mgF(ab0)2 was highest for the North American species(2.48mg of L. boneti gland extract protein, equiva-lent to 81.3 LD50, and 2.96mg of L. reclusa protein,equivalent to 140.9 LD50), and lower for L. laeta

isoforms (1.16mg, equivalent to 53.5 LD50). The

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–74 71

equine F(ab0)2 also effectively inhibited SMDactivity of all recombinant proteins and inhibition(measured as ID50, see Section 2) was highestagainst the North American SMDs, and loweragainst L. laeta enzymes, as shown in Table 4.

4. Discussion

4.1. Expression of soluble SMD

In our hands, it was difficult to produce recombi-nant soluble and active SMD, which we achieved

Table 2

Neutralization of lethality by rabbit monovalent antisera raised

against different recombinant SMD and venom gland extract

(GE) when assayed against recombinant SMD and venom gland

extract

Serum against mg of protein neutralized/ml of antiserum

GE-Lb GE-Lr Lb1N Lb1C Lr1N Ll1N Ll2C

GE-Lb 40.1 ND ND ND ND ND ND

GE-Lr ND 47.1 ND ND ND ND ND

Lb1N ND ND 50.5 65.5 60.0 ND ND

Lb1C 123.0 120.0 46.0 60.0 107 — —

Lr1N 103.0 122.5 50.0 46.0 60.1 — —

Ll1N ND ND — — — 65.8 45.2

Ll2C ND ND — — — 23.3 51.9

Numbers in bold face indicate the specific responses. (—)

indicates very low or undetectable neutralization; ND, not

determined.

Table 3

Neutralization of lethality of natural venom preparations (gland

extracts—GE) of the three species of Loxosceles by the horse

F(ab0)2 antivenom obtained against recombinant SMD of the

three species of Loxosceles (Lb1C, Lr1N, Ll1N and Ll2C)

mg of protein neutralized/mg of F(ab0)2 (95% c.i.)

GE-L. boneti GE-L. reclusa GE-L. laeta

2.48 (2.19–2.88) 2.96 (2.34–3.74) 1.16 (0.90–1.62)

Table 4

Neutralization of enzymatic activity of venom preparations and recom

mg of protein neutralized/mg of F(ab0)2 (95% c.i.)

GE-Lb GE-Lr GE-Ll Lb1C

2.95

(2.52–3.45)

3.43

(2.60–4.53)

0.656

(0.485–0.887)

0.243

(0.199–0.297

only after careful study of the culture conditions andselection of an appropriate E. coli strain. To avoidformation of inclusion bodies and the production ofinsoluble recombinant enzymes, very gentle induc-tions using low amounts of IPTG (100mm) and slowgrowth rates at low temperature (20 1C) for longtimes were an absolute requirement. The reducingpotential of the intracellular environment seems to beimportant for successful heterologous expression ofSMD (at least for the North American enzymes) inE. coli, as under the conditions in which BL21trxB-DE3 cells (which are thioredoxin reductase deficient)produced soluble active recombinant SMDs, thesecould not be produced in XL1-Blue cells, in whichinvariably insoluble enzymes were produced. We didobtain antisera against these (using guanidiniumhydrochloride-solubilized insoluble recombinantSMD of L. boneti), and although the antiserarecognized denatured SMD by ELISA and Westernblotting, and to a lesser extent native SMD fromvenom gland preparations by ELISA, they were notcapable of neutralizing the toxicity of L. boneti

venom, indicating the crucial importance of con-formational epitopes present in the active SMD ineliciting a protective humoral response (data notshown).

4.2. Enzymatic activity and toxicity of recombinant

SMD

Enzymatic activity of the recombinant enzymes ofL. boneti and L. reclusa were comparable to those ofnatural purified SMDs (Ramos-Cerrillo et al., 2004).When the specific activities of recombinant SMDs arecompared to venom gland extracts within eachspecies, they correspond to their estimated relativeabundance in the preparations, which has beenproposed to be between 20–40% of total venomprotein (Binford and Wells, 2003; Ramos-Cerrillo etal., 2004). Although enzymatic activity seems to berequired for necrotic activity and lethality, it isnoteworthy that enzymatic activity as measured in

binant SMDs by the horse F(ab0)2 antivenom (as ID50)

Lr1N Ll1N Ll2C

)

0.281

(0.245–0.321)

0.043

(0.035–0.052)

0.049

(0.044–0.053)

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–7472

our assay—which uses egg sphingomyelin as sub-strate—was not correlated to toxicity, an interestingfact that has been recently pointed out (de Oliveira etal., 2005). For example, both L. laeta recombinantenzymes had a significantly higher specific activity inthe in vitro assay but their lethal potency was notproportional to their higher activity. One reasonablepossibility is that the egg sphingomyelin used,although a substrate for SMD, is not the substrate(or group of substrates) relevant to the in vivophysiopathology. It has been suggested that necrosisby SMD may be related to the production ofpowerful signaling molecules such as a phosphocer-amides, or the metabolites derived from its action onlysophopholipids (LPL), which may initiate thecellular events leading to inflammation and necrosisin CL (Huwiler et al., 2000; Van Meeteren et al.,2004). Although the natural targets of SMD are notknown, it is reasonable to hypothesize that differen-tially acylated sphingomyelins/LPL could be thetargets from which release of particular products bySMD may trigger the proinflammatory response thatleads to local necrotic damage (for a review of thephysiological effects of sphingosine metabolites oncell stress see Hannun and Obeid, 2002). Anotherpossibility, not necessarily exclusive, is that in vivoeffects reflect not so much particular substratepreferences by the different enzymes as idiosyncraticdistributions, in patients or wound sites, of relevantsubstrates for SMD. Moreover, other parametersaffecting enzymatic activity, such as cofactor depen-dency or stability of the enzymes, may be at work invivo. More studies are needed at least at two levels:first, on the substrate specificity of SMD of differentspecies and isoforms within a given species; second,on the physiological effects on different cell types ofthe phosphoceramides or other metabolites producedby SMDs acting on different substrates.

4.3. Antigenic cross-reactivity between recombinant

SMD and paraspecific protection

In the species we tested, two of which were NorthAmerican (L. boneti and L. reclusa) and one SouthAmerican (L. laeta), two clear antigenic groupscould be defined by in vivo neutralization oflethality using the experimental rabbit monovalentantisera, as antisera against either North AmericanSMD (recombinant or natural) protected mice fromlethal challenges with SMD from both species withcomparable efficiency, indicating their antigenicrelatedness. In addition, antisera against either

N- or C-tagged L. boneti recombinant SMDneutralized the toxicity of both tagged recombinantSMDs of L. boneti, as well as that of N-tagged L.

reclusa SMD, with comparable efficiency, indicatingthat the position of the histidine tag does notsignificantly affect protective epitopes. This obser-vation is consistent with the sole crystallographicstructure available for SMD, which shows that theN- and C-termini are not part of the catalytic site(Murakami et al., 2005).

None of the antisera raised against the NorthAmerican spider SMD preparations could signifi-cantly neutralize the toxicity of either recombinantL. laeta isoform. This was also true for antibodiesraised against the L. laeta SMDs when testedagainst the North American SMD preparations.L. laeta SMDs were effectively neutralized by thespecific antisera and, interestingly, a degree of cross-neutralization between the two L. laeta isoformswas detected as anti-Ll1N significantly neutralizedLl2C.

4.4. Amino acid sequence similarity and antigenic

cross-reactivity

As the dendrogram shown in Fig. 2B illustrates,SMDs from L. gaucho and L. intermedia, bothSouth American species, are more similar to, andare consequently grouped with, the SMDs of theNorth American species than to those of the SouthAmerican L. laeta. They form, however, a distinctbranch, as proposed in a recent study forL. intermedia SMDs which, however, did not includethe L. gaucho enzyme (Tambourgi et al., 2004).

Recently, two enlightening and timely studieswere published which address the twin issues ofantigenic cross-reactivity and the development of awide spectrum anti-Loxosceles antivenom. One ofthese, by Barbaro et al. (2005) contained a detailedstudy of neutralization profiles of two Brazilianantivenoms against Loxosceles. The first antivenomwas obtained against venom of the South Americanspecies L. gaucho (as well as Phoneutria nigriventer

and Tityus serrulatus, with which cross-reactivitywould be expected to be minimal and can thereforebe considered a monovalent anti L. gaucho) and thesecond one against a mixture of venoms of L. laeta,L. intermedia and L. gaucho (all South American).Interestingly, their results show extensive cross-protection, as the anti-L. gaucho antiserum neutra-lizes SMD enzymatic and dermonecrotic activity ofall other venoms tested (including the North

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–74 73

American L. reclusa), albeit at different titers. Theseresults are consistent with the fact that, by seq-uence, L. gaucho falls into a branch which al-though grouped with North American toxins,seems to define an intermediate group betweenNorth and South American SMDs which couldshare neutralizing antigenic determinants for bothgroups. In fact, alignment of the deduced aminoacid sequence of L. gaucho SMD shows that interms of its conserved cysteines it is more similar tothe L. laeta group (only two cysteines in closeproximity), but in terms of overall sequence it lacks,for example, the five amino acid insertion char-acteristic of all L. laeta SMD isoforms. By contrast,both L. intermedia SMD isoforms share the cysteinepattern of the North American SMDs but are,overall, more similar to the L. gaucho enzyme(particularly the P1 isoform) and also lack theL. laeta insertion.

The second study, by Tambourgi et al. (2004),reported the cloning of the two SMD isoformsof L. intermedia mentioned above, their activityprofiles and immunochemical reactivity with mono-valent antisera raised against the recombinantSMDs and the venom of L. laeta, L. gaucho andL. intermedia. Their results demonstrate a certainlevel of in vitro cross-reactivity between the L.

intermedia P1 isoform and venoms of L. gaucho andL. laeta, and a more limited reaction between theantiserum raised against recombinant P2 and thesame venoms, consistent with the grouping shownin Fig. 2.

The two studies mentioned do not address in vivoneutralization of lethality, a most important para-meter for the evaluation of the efficacy of anti-venoms, although the neutralization of SMDactivity correlates very closely. It would be veryinformative to explore more species/isoform-specificvenom–antiserum combinations, thus completingthe correlation that our analysis of sequencesimilarity, in conjunction with the other studiesdiscussed, suggests. For example, it should be mostinteresting to study the patterns of neutralization oflethality of a monospecific antiserum againstL. intermedia, to assess whether its cross-reactivitywould fall in the L. laeta/L. gaucho group, or in theL. boneti/L. reclusa (and, possibly, L. arizonica)group or, like L. gaucho its cross-reactivity spansboth North and South American groups of Lox-

osceles.Thus, a so far rather consistent picture seems to

be emerging for the phylogenetic and antigenic

clustering of SMDs. Our data, when viewed in thecontext of the study on the antiserum againstL. gaucho venom suggests that the importantsequence divergence of SMD of L. laeta accountsfor the lack of recognition in vitro and neutraliza-tion in vivo by the antisera raised against the SMDsof North American spiders. The converse, that is,lack of neutralization by anti-L. laeta of NorthAmerican SMDs, is also true. Overall, threephylogenetic groups based on the primary sequenceof SMDs can be defined for Loxosceles in theAmericas: one comprising the North Americanspecies L. boneti, L. reclusa and L. arizonica (andvery likely L. deserta); another, so far monospecificgroup, comprising solely SMDs from the SouthAmerican L. laeta (genes cloned from Brazilianand Peruvian isolates are grouped together andshare distinct characteristics), and another, appar-ently intermediate, group comprising L. gaucho andL. intermedia SMDs.

In this study, we establish the feasibility ofproducing a F(ab0)2-based polyvalent antivenom ofhigh neutralizing potency using recombinant anti-gens. This antivenom, as gleaned from our studiesand those of others, may be effective for treatmentof envenomation by a large range of Loxosceles.

Antivenoms produced using recombinant SMDmay be easier and cheaper to produce, control andmodify as needed—for example, to adapt them tolocal species or sub-species or to expand their degreeof coverage—than those up to this point obtainedby traditional means. On an anecdotal level, whenwe started immunization of the horses used forantivenom production (we originally used two) westarted the immunization scheme with just 100 mg ofthe recombinant SMDs—what we thought a reason-able amount of antigen based on LD50 measure-ments in the mouse model and immunization ofrabbits—with the result that one horse died within36 h with signs of massive hematuria, and the otherwas very ill although finally recovered. This eventhas two interesting implications: first, that horsesare extremely sensitive to SMD and that, on aweight-corrected basis, their sensitivity may reflecthuman sensitivity to Loxosceles envenomation, as aspider may inject microgram quantities of SMD in asingle bite, which may lead to extensive necroticlesions and the sometimes fatal manifestations ofVCL; second, that recombinant expression of SMDmay allow the production of toxoids (possibly bysingle amino acid replacement) devoid of enzymaticactivity that may nonetheless trigger protective

ARTICLE IN PRESSA. Olvera et al. / Toxicon 48 (2006) 64–7474

humoral responses, thus minimizing envenomationand suffering of animals used in anti-Loxosceles

antivenom production.An important question that remains, which will

be without doubt answered soon, is whether a true‘‘pan-American’’ anti-Loxosceles antivenom shouldinclude some member of the intermediate groupcomprising L. gaucho and L. intermedia, as can beinferred from the study by Barbaro and colleagues(2005), besides the North American species andL. laeta recombinant toxins we used in the genera-tion of the polyvalent equine F(ab0)2 antivenom wepresent in this study. Another issue of interest iswhether other Loxosceles antigenic clusters can bedefined for the Americas and whether Old Worldspiders have evolved distinct sequences that woulddistinguish them from those of the New World.

Acknowledgements

We wish to thank Maricela Olvera for sequen-cing, and Penelope Magana and Felipe Olvera fortechnical assistance. This work was supported inpart by Grant PAPIIT-UNAM IN230203 (Mexico).

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