.rchives of Medical Research Volllme 27, No. 3, pp. 275-283, 1996Printed in Mexico

Original Article

Genetic Manipulation of Vibrio cholerae for VaccineDevelopment: Construction of Live AttenuatedEl Tor Candidate Vaccine Strains

JORGE A. BENITEZ,* ANISIA J. SILVA,** BORIS L. RODRIGUEZ,*RAFAEL FANDO,* JAVIER CAMPOS,* ALMA ROBERT,* HILDA GARCIA,***LUIS GARCIA,*** JOSE LUIS PEREZ,*** REYNALDO OLIVA,***CARIDAD A. TORRES,*** and TALENA LEDON*

* Grupo de Genética del Centrol Nacional de Investigaciones Científicas, Havana, Cuba** Departamento de Microbiología, Facllltad de Biología de la Universidad de La Habana, Havana, Cuba*** Instituto Finlay, HGI'ana,Cuba

Received for pllblication JlIly 14, 1995; accepted Febmary 28, 1996 (95/90).

AbstractThe recent spread of El Tor cholera to Americaaugments the need for an effective, safe andeconomical vaccine. In the present paper we describethe construction oflive attenuated V.c/lOleraestrainsby specifically deleting the genes encoding choleratoxin and other putative toxins from the bacterialchromosome. To maximize the likelihood of exposingprotective antigens relevant to currently circulatingvibrios we selected for genetic manipulation recent

KEY WORDS: Víbrío cholerae; Virulence cassette; Vaccine development

Introduction

Cholera is an acute diarrheal discase caused byserogroups 01 and 0139 ofV. cholerae. The 01 vibriosare divided into the c1assical and El Tor biotypes. Incontrast to the former pandemic characterized by thepredominant isolation of c1assical vibrios, the currentand seventh pandemic, that arrived in South America in1991, is typified by the isolation of El Tor vibrios (1).RecentIy,anon-O1vibriocapableofproducingepidemic

Correspondence to:Jorge A. Benítez, Grupo de Genética del Centro Nacional deInvestigaciones Científicas, Havana, Cuba. Tel: (537) 21-8066 Ext.248; FAX: (537) 33-0497.

epidemic V. eholerae isolates from Pero. The mutantstrains did not produce cholera toxin in vitro and invivo. Deletion of the virulence cassette wasaccompanied by marked attenuation in the infantmouse cholera modeI. A selected El Tor Ogawacandidate vaccinestrain was refractory to acquisitionof foreign genes by conjugation with toxigenicvibrios. (Areh Med Res 1996; 27:275)

cholera has been isolated in the Indian continent (2).Thenew serotype, 0139 (synonym Bengal), although notagglutinated by 01 antiserum, shares many features incommon with the El Tor vibrios (3-7).

In order to cause disease, V. cholerae must adhere tothe cells liningthe surfaceof the human smallbowel. TheEl Tor and 0139 vibrios express several putativeadhesins such as the toxin co-regulated pilus (TCP)(8,9), the mannose-sensitive hemagglutinin (MSHA)(10-12) and the core encoded pilus (cep) (13). Mutationin these genes causes an overall defect in bacterialintestinal colonization in the infant mouse model.

The c1inicalsymptoms of cholera gravis is due to thesecretion by vibrios of cholera toxin (CT) (1). Thecholera holotoxin is formed by one A subunit (CTA), a

275

};.-\D'.ADPribosyltransferase,andfiveB subunits(CTB)iliarbind to theGMl gangliosidereceptorin the gut(14).

Expression of CT, and several other operonsencoding(be TCP assembly line, auxiliary colonization factorsand some outer membrane proteins are undertranscriptional control by ToxR (15). In addition to CT,v. cholerae produces other toxins like the lOnulaoccludens toxin (zot) (16,17) and the accessory choleraenterotoxin (ace) (18). These toxins have been definedby their effect on rabbit ileal tissue mounted in Ussingchambers (16-18). Their role in pathogenesis, if any,remains to be elucidated.

In V.cholerae, thegenesforCTA (ctxA), CTB (ctxB),zot, ace, an open reading frame caBedorfU, and cep areclustered in the bacterial chromosome defining a core orTox region flanked by direct repeat sequences (RS) oftype RS1amI/orRS2. This transposon-like structurehasbeen caBed the virulence cassette (19,20) (Figure lA).

Dcspite cholera being anancientdisease we stilldo nothavean effective, econornicaland safe vaccineagainstit.The recognition that protection against V. choleraeinfcction is of mucosal rather than systemic nature hasled to the concept of an oral vaccine (21).An inactivatedwhole cell oral vaccine has been extensively tested infield trials (22). Although this vaccine is safe and welltolerated,itiscostly,requiresmultipledoses andprovidesless than optimal protection particularly to children.Theobservation that a clinical or experimental episode ofcholera confers solid long term protection supports theproposal of a livegenetically attenuatedvaccine (23-25).The yet experimental, genetically engineered, livingTox attenuated mutants are more promising from thestandpoint of economy and potential efficacy.CVD103HgR, a ctxA-ctxB+derivative of the classicalInaba strain 569B, has been extensively tested involunteers (26). This vaccine is safe, non-reactogenicand highly protective. However, CVD103Hg is lessprotective against mild diarrhea due to challenge withElTor vibrios and does not provide protection againstinfection with 0139 vibrios (27).

An effort is currently being made to develop a suitablevaccine againstEI Tor cholera. It isof critical importanceto develop live attenuated strains from recent clinicalisolates of the predominating serogroups and serotypes.Unfortunately, the live attenuated El Tor candidatevaccines sofarunder investigationdo notmeet theaboverequirement. In the presentpaperwe describe the geneticattenuation of El Tor vibrios of both serotypes startingfrom virulent epidemic strains recently isolated fromPeru.

Materials and Metbods

Strains and Media. Bacterial strains utilized are listedin Table 1. Strains C7258 and C6706 were used as wild

type starting strains for genetic manipulation. V.cholerae

BENITEZ, SIL VA, RODRIGUEZ, ET AL.

and E. coli strains were routinely grown in LB mediumand conserved in the same medium supplemented with20% glycerol at -70°C. For in vitro toxin production V.cholerae was streaked in blood agar plates andsubsequently cultivated by the AKI procedure (39).Antibiotics were added at the foBowing concentrations(in micrograms per milliliter): ampicillin (Ap) 100,kanamycin (Km) 50, tetracycline (Te) 12.5 (E. coli) or 5(V. cholerae), rifampicin (Rf) 50, polymyxin B (PolB)100 units/m1.

Plasmid Construction and Gene Transfer

Technology. Plasmid pBB6 (17) was used as a source ofCt, lOt and ace genes. This plasrnid contains a 5.1 kbPstI-EcoRI insert coding ace, lOt, CTA and CTB from V.cholerae 569B cloncd in pBR322 (17). Due to the lackof a 3' RS element in this CT operon the EcoRI at the 3'end of the insert lies within vibrio chromosomal DNA of

undefined function. Plasmid pBB6 was modified bydeleting an internal ScaI fragment (Figure lA) to createplasmid pBSCT5 containing amutantcoreregion (mTox).Plasmid pBSCT5 was linearized with PstI, made bluntwith T4 DNA polymerase and self-ligated in presence ofa 100-fold molar excess of 5' phosphorylated EcoRIoctameric linker to create plasrnid pBSCT64. The mToxregion was finally extracted as an EcoRI fragment,ligated to EcoRI digested pGP704 (40) and transformedinto E. coli SY327Apir. Plasmid DNA from the coreectclone, pAJF, was purified and transformed into E. coliSM 10Apir for mobilization to V. cholerae. Derivativesof pBR322 and pUC vcctors were transfen'ed to V.cholerae by filter mating with E. coli HB 10lIpRK20 13transformed with the test plasmid (29). Conjugativetransfer of pAJF DNA to V. cholerae was achieved byfilter mating its SM 1OApirtransformant with V. choleraeand selection for exconjugants in LB plates containingAp and poIB (31). Exconjugants were characterized bySouthern blot hybridization analysis using a DIG-DNAlabelling and detection kit (Boehringer Mannheim). V.cholerae chromosomal DNA was prepared as describedin Reference 41. Southern blots were probed withdigoxigenin-labeIled Pst- HincII fragment extracted frompBB6 (17). Alternatively, Southern blots werehybridizedwith an RS probe derived from vector pUJ~EV. Plasmidp3083 (32) was used as a source of RS sequences. Thisplasmid contains an 8.3 kb PstI insert encoding ace, lOt,CTA! CTB and RS (see Figure lA) cloned in pBR328(32). Plasmid pUMEV was constructed by subcloningan XbaI-PstI fragment from p3083 in pUC19 andsubsequently deleting the EcoRV fragment 3' to the RSelement. Plasmid pUJ~EV encodes CTA C-terminalsequences, cTB and RS sequence up to its EcoRV site(seeFigure lA). A digoxigenin-labeBedHincIIfragmentfrom pUJ~EV encoding mostIy RS sequences was usedas hybridization probe.E. coli was transformed accordingto Refcrence 28. Plasmid DNA minipreparations were

performed as described by Bimboim and Doly (42).DNA restriction and modification enzymes were fromBoehringer Mannheim and used according tomanufacturer' s instructions.

Determination of CT. CT was dctermined using aGMl ganglioside-dependent enzyme-linkedimmunosorbent assay (ELISA) (43). Monoclonalantibodies (Mab) 4EIG5 and lGlOG5 directed againstCTB and CTA, respectively, were used as primaryantibody and peroxidase-conjugated anti-mouse IgG(whole molecule, Sigma Chemical Co.) as secondaryantibody.

Ligated Rabbit Ileal Loop Assay. Ilealloops were

277

performedas described inReferences44 and45. Briefly,New Zealand adult rabbitswere fasted for 24 h, the smallintestine withdrawn and ligated approximately 10 cmfrom the appendix. The intestine was divided into 5- 6cm segmentsby ligatures andinjectedwith 108cfuoflivevibrios in 0.5 mI ofphosphate buffered saline. After 16- 18 h the animals were sacrificed and loop length andfIuid volume readout. Results are expressed as FA =fIuidaccumulated (ml)/length (cm).

Determination of LDso' LDso was determined usingthe infant mouse model. Dilutions containing from 103to107 vibrios in 20 111phosphate-buffered saline wereorally inoculated to groups of six to ten 2- to 3-day-oldnewbom mice. Mortality was determined after 72 h andLDso caIculated as described by Reed and Muench (46).

Filter Mating Experiments. At least 109cells ofgenetically marked donor and recipient V. choleraestrains grown in liquid LB medium were mixed,concentrated on a 0.45 11mmembrane filter and the filterincubatcd 4 h on the surface of an LB plate. Finally, thecells on the filter were resuspended in LB medium andplated on selected medium. An aliquot of each matingpartner was manipulated in the same way and plated onselectedmedium toestimatethefrequencyofspontaneousmutation. The genetic crosses were designed so that theorigin of colonies growing on selectivemedium could beinvestigatedby slideagglutinationwithspecificantiserum(0139,01, Inaba, Ogawa). For studying the capacity ofattenuatedstrainstoacquireplasmid-bomegenes,plasmidpJS752-3 (47), encoding Apr and a recombinant CTB,was mobilized from HB 10l/pRK20 13 to thecorresponding V. cholerae donor strain.

Results

The strategy for construction of V. cholerae deletionmutants is shown in Figure lB and C. Plasmid pAJF, aderivative of pGP704, contains the R6K origin ofreplication (40). The R6K origin requires for its functiona protein caBed1tencoded by the pir gene. The1tproteinis supplied in trans to E. coli host strains by a prophageApir (30,31). Plasmid pAJF also contains the RP4 mobregion (40). Strain SMlOApir contains RP4 transferfunctions integrated in its chromosome and mobilizespAJF to V. cholerae (31). Since V. chlolerae does notcontain the pir gene, pAJF can only give rise totransformants by homologous recombination andintegration with the bacterial chromosome to form anAprresistantco-integrate.Thisco-integratewasidentifiedby Southem blot hybridizatíon analysis of chromosomalDNA digested with restriction enzyme HindIlI (Figure2, lanesb, c). In the absenceof antibioticselection the co-integratecangiverisetoApSrecombinantsbyhomologousrecombination between Tox genes (Figure 1C,1), or by

LIVE ATIENDA TED CANDIDATE CHOLERA VACCINES

Table 1BacterialStrains

IStrain Relevant genotype andlor phenotype Ref.

E. coli

HBIOl F- hsdS20(rb' mb')recA13 leuB6 ara-14 proA2 28

lacYl galK2 rpsL20 (Sm') xyl-5 mll-l supE44A.-HBI01l HBlOl transformed wilh mobilizing plasmid 29

pRK2013 pRK2013SY327A (lac pro) argE(Am) rif natA recA56 (ApirR6K) 30

Apir

SMIOApir thi thrleu lonA lacY supE 31

recA::RP4-2Tc::Mu (ApirR6K) Km'-

V. cholerae

C7258 Wild Iype, al, El Tor, Ogawa, Pero 1991 32

C6706 Wild Iype, al, El Tor, Inaba, Pero 1991 32

81 clxA- clxB' zol' ace' orill' cep'mulanl from C7258

82 clxA- clxB' zol' acc' orill' cep'mulanl from C7258

88 ctxA- ctxB' zol"ace' orill' cep'mulanl from C7258

413 clxA- clxB' zol' ace' orill' cep'mulanl from C6706

417 clxA' clxB' zol' ace' orill' cep'mulanl from C6706

81R!' Rifampicin resislanl mUlanlfroÍn 81569B Wild Iype al, classical, Inaba 33

569BP+ 569B conlaining V. cholerae P faclor 34

569BRfb Rifampicin resistant mulanl frol11569B569BP+Rf Rifampicin resisl.mt mulanl from 569BP+395 Wild type al, classical, Ogawa, India 1964 35

N16961 Wild type al, El Tor, Inaba, Bangladesh 1975 36

1837 Wild type 0139, Bangladesh 1993 37

CVDlO9 clxA' clxB' zol' ace' orill' cep' mulanl from 48

El Tor Ogawa E7946JBK70 clxA- clxB' mulanl frol1lNI6961 36

JBK70 al, El Tor, Inaba ClxA'clxB' I1Ish::Tn5(Km') 38

::Tn5

- -

Bi.clllI

Sea!iEcoRV¡i Xbal11 ¡ScaII! I ~iDcIIli , : B¡¡LIl!' I ¡ i EcoRV11 i: i EcoRV

EcoRV H

I

I¡ I tRinelIAval!l I I I MP,d

B¡¡1lI I¡ PatI J: :i : ~ HindlIlwas .ceporl1hc8 aot CTACTB as

HindlIl RS

(B) L {

Tox RS HindlIl

] J chromosome

,,¡, bomolocoul recombination

Hi.dIlI RS mTox Amp-R HindIll Tox RS

~Hind1ll

(C) ResolutioD by recombination 01 Amp-R co-inte,rate

(1) (11)

RS Tox

mTox RS

,¡.RS

-c=::J--

Figure 1. (A)Structure of V. cholerae chromosomal regioncontaining the virulence cassette consisting of a core or Toxregion flanked by RS sequences; (B) integration byhomologous recombination of suicide vector pAJF leading toco-integrate formation; (C) resolution 01 co-integrate byhomologous recombination between Tox genes (1)or byhomologous recombination between RS elements (11).Pctx,cholera toxin promoter.

homologousrecombinationbetweenRS elementsleadingto deletion of the entire Tox region (Figure 1C,U).AnApr co-integrate from strain C7258 was allowed tosegregate in LB medium and Ap' recombinants weredirectly analyzedby Southern blot hybridization using aDNA probe consisting of a PstI-HincIl fragmentcomplementary to ctxB, ctxA, zot, ace and part of orfU(Figure lA). All Aps recombinants failed to hybridizewith the PstI-HinclI fragment suggesting that RS-mediated deletion of the entire core region took place(Figure 2A, lanes e, f, and g). Similar results wereobtained when the above procedure was applied to the

BENITEZ, SIL VA, RODRIGUEZ, ET AL.

Inaba strain C6706to generaterecombinants413 and417 (Figure2B, lanes a,b). Additional evidencethat RS-mediated recombination was responsible for deletion ofthe entireTox region comes from the observation thatallthe Tox' mutants retained a single array ofRS elementsin their chromosome (Figure 2C, lanes a-g).

The mutants did not produce cholera toxin whenassayedby GM1-ELISA (Figure 3).Toxinproductioninvivo was assayed by measuring fluid accumulation inligated rabbit ilealloops. Deletion of toxin genes led toa substantial decrease in the capacity of these strains toinduce fluid accumulation (Figure 4). The effect of theTox'mutationonvirulencewasinvestigatedbycomparingthe LDso of Tox- mutants and several Tox+ virulentstrains. In Table 2 we show that deletion of the Toxregion in the attenuated strains led to a 10- to 100-foldincrease in LDsoin the infant mouse cholera model.

The above Tox' strains lack approximately 7.2 kb ofDNA from their chromosome and cannot revert tovirulence,-HoW&v8f;-itremains, at least theoreticallypossible, that they could regain toxic genes from othervibrios in the environment. To assess the likelihood ofthis outcome we performed a set of genetic crossesbetween strain 81 or its Rf derivative with 01 c1assical,01 El Tor and 0139 toxigenic strains. Strains weregeneticallymarked with plasmid-borneor chromosomalresistance mutations for recognition of putativeexconjugants (Table 1). In crosses 1 and 2 coloniesappearedin the crossing plates ata frequencyidentical tothe spontaneous mutation frequency of the receptorstrain and were, as expected, all of serotype Ogawa. Incross 3, in which a Rf derivative of 81 was mated withthe 0139 strain 1837bearing aplasmidmarker (pJS752-3, Apr, CTB), no colonies appeared in the crossingplates. In cross 4 the Aprgene of pAJF was integratedbyhomologous recombination within the RS elementsflanking the Tox region of strain 1837 and the resultingstrainmatedwith81Rf. Coloniesappearedinthecrossingplatesatafrequencyidenticaltothespontaneousmutationfrequency of the donor and, as expected, all reacted with0139 antiserum. No potential cxconjugants could berecovcred from cross 5.

Discussion

Deletion ofthe core orTox regionfrom virulent strainsof recent isolation from Peru was achieved using thesuicide vector strategy shown in Figure 1.The deletionresulted from homologous recombination between RSelements (Figures 2A, B). The recombination eventoccurred with high frequency in Apr co-integrates ofstrain C7258 and C6706. The mechanism by whichinsertion mutations in the Tox region promotes highfrequency recombination between RS sequences isunknown. It should be noted that spontaneous deletion .

of the Tox region is rare in the normal vibrio. On the

RS Toxo/

C.:' J-,¡.

RS Tox RS-1 I I ....

orRS mTo1 RS

-t I I t--

LIVE ATIENUA TED CANDIDA TE CHOLERA VACCINES

MW(kb)

23,13

9,426,564,36

2,322,03

a 9

Figure 2. Scanned images of Southern blot hybridizationexperiments. (A) Lane a. molecular weight markers; lane b.C7258 digested with Hindlll; lane c. Ap' co-integrate digestedwith Hindlll; lane d, C7258 digestedwith Pstl; lanes eto g. Apsrecombinants 81, 82, and 88 digested with Pstl; (8) lanes aandb. Aps recombinants 413 and 417 digested with Pstl; lanec. C6706 digested with Pstl; (C) lane a, C6706 digested withPstl; lanes b and c. 413 and 417 digested with Pstl; Jane d.C7258 digested with Pstl; lanes e - g. recombinants 81, 82and 88 digested with Pstl. Panels A and 8 were hybridizedwith a digoxigenin-Iabelled Pstl-Hincll extracted from p886.Panel C was hybridized to a digoxigenin-Iabelled Hincllfragment extracted from plasmid pUJDEV complementary tothe virulence cassette RS element.

contrary, RSelements havebeen suggested to participatein the amplificationofTox genes during animal passage(19). The RS element retained in the deletion strainsisolatedfromC7258 werepresentin restrictionfragmentsof different sizes indicating that they originated fromdistinct recombination events (Figure 2C, lanes e, f andg). Since many El Tor strains contain tandem repeats ofRSl amI/orRS2 elements flanking the Tox region (19),the restriction fragment length polymorphism observedin Figure 2C could be explained by retention in themutants of single RS elements or tandem repeats of RS1amI/or RS2 elements.

GeneticallyattenuatedV.choleraestrainsareprornisingas irnmunizing agents against cholera (26) and as apotential antigen delivery system to the gut associatedlymphoid tissue (48). Unfortunately, the geneticallyengineered candidate vaccine strains so far developedhavebeen reactogenic and/or provided less than optimal

279

protection against infection with El Tor vibrios (26). Inorder to attach the second problem we selected forgenetic manipulation Ogawa and Inaba V. choleraeepidemic strains isolated recently during the spread ofthe seventh pandemic in South America. In contrast tothe similar El Tor Ogawa candidate vaccine strainCVD110,isolated from E7946 (Bahraim, 1978)(49), theones developed in the present work are expected toexpose protective antigens and virulence factorscharacteristic of currently circulating vibrios. Recently,the El Tor Inaba candidate vaccine strains Peru-3 andPeru-14 have been developed (50). However, althoughthe appearanceof the seventhpandernicinLatinAmericawas initially typified by Inaba strains, Ogawa isolatesbegan to appear about the seventhmonth of the epidernicand have subsequently become predominant in manyareas suggesting that the epidemic strain had undergoneserotype conversion, possibly because of immunepressure in the population (51,52). Thus, the concept ofdriving vaccines from epidemic strains may already notbe applicablc to Pern-3 and Peru-14. Differently fromthe strains developed in thiswork andbased on ascarcelydocumented synergistic effect between anti-toxic andanti-bacterial immunity, other living attenuated Tox V.cholerae strains have been manipulated to preserve orre-introduce the ctxB gene (49,50). Experiments involunteers with the atoxigenic strain JBK70 havedemonstratedthat aneffectiveprotectioncanbe achievedin the absence of anti-toxin immunity (26). Addition ofCTB to the inactivated vibrio oral vaccine during a fieldtrial inBangladeshshowedanegativeeffectonprotectiveefficacy at the third year of a follow-up (1). On the otherhand, CT, apowerful mucosal immunogen and adjuvant,is likely to encounter broader applications in non-livingoral or parenteral vaccines. Recently, it has been shownthat small amounts of CTB induce systernic tolerance toconjugated antigens, a finding that may l~ad to thedevelopment of agents to prevent and treat discasescaused by tissue-damaging immune responses (53),Considering the potential of CT and its B subunit formore general applications in vaccinology and therapy,theinductionof anti-toxinimmunityofdoubtfulnecessityvia cholera vaccine is highly debatable.

The attenuatedstrains derived from C7258 and C6706were atoxigenic in vitro and in vivo (Figures 3 and 4).Deletion of the Tox genes increased their LDsoin theinfant mouse cholera model (Table 2). However, theseresults donot exclude potential reactogehicity inhumansdue to expressionofunknown secretagoguesorintestinalcolonization.Reactogenicityoflive geneticallyattenuatedV. cholerae vaccines has been correlated with differentlevels of intestinal colonization (49,50), a complex,multifactorial and strongly strain dependent trait. Sincecurrent animal models are incapable of predictingreactogenicity in humans (49), work is in progress todetermine the side effects of these strains in volunteers.

Table 2

Derennination ofLDso in !.heInfant Mouse Cholera Model

Suain Properties LDsaVirulent strains

Classical, OgawaClassical, InabaEl Tor, InabaEl Tor, OgawaEl Tor, Inaba0139

Attenuated strains

El Tor, OgawaEl Tor, OgawaEl Tor, OgawaEl Tor, Inaba

3955698N16961C1258

C67061837

6.1 x 1()32.3 x lOS1.7 x 1041 x lO"'3 x lO"'

3.7 x lQ3

818288413

1.3 x 1Q69.5 x lOS2.5 x lOs5 x lOS

Concerning the safety of the potential vaccine strains,preliminary data shown in Table 3 suggest that transferof plasmid-borne or chromosomal markers between V.cholerae strains is a rare event. The rationale of this

experiment consisted in that the detection lirnit of genetransfer and recombination is the spontaneous mutationfrequency of each mating partner. In addition, if coloniesgrowing on crossing plates were mutants rather thanexconjugants, one would expect the ratio of donor/receptor, judged from scoring an unselected marker likeserology, to reflect the frequency of spontaneous mutationof each mating partner. On the basis of these criteria, wecan conclude that under the present experimentalconditions gene transferdid not occur despite the physicalcontact of potential mating partners at a high populationdensity in a rich medium. Similar mating experiments aswell as in vivo matings performed in the suck1ing miceintestine with the c1assical vaccine strain CVDI03HgRhave suggested thatreacquisition oftoxin genes, althoughtheoretiéally possible, is in practice unlikely (54). In

0.1

a b d e

Strain

g he

_ Mab 1G10G5 Anti-CTA _ Mab 4E1G5 Anti-CTB

Figure 3. Toxin production in vitro measured by GM1-ELlSA.a, C7258; b, 81; c, 82; d, 88; e, C6706; f, 413; g, 417; h, JBK70(negative control).

BENlTEZ, SILVA, RODRIGUEZ, ET AL.

contrast to E. coli, V. cholerae lacks a high frequencyrecombination (Hfr) system (55). Although a P factoranalogous to E. coli fertility factor is present in some V.cholerae strains (34), its occurrence is extremely rareanditscapacityto mobilizechromosomalgenesis lirnitedby its poor homology to the bacterial chromosome (55).P factor-mediated reacquisition of Tox genes has onlybeen observed in an experiment using a Tn5-marked Pfactor mobilized ÍDto a strain harboring a deletion-insertion of an Apr gene in the Tox region (54). Thepresence of P factor did not enhance gene transfer tostrain 81 (compare cross 1 and 2). RS-mediatedreacquisition of their associated Tox genes has beendemOlistraterl(13).However, theseexperimentsrequiredthe use of heavily engineered E. coli strains formobilization of suicide vectors to V. cholerae (13). NoRS-mediated transfer of an Apr gene to strain 81 wasobserved in the vibrio-vibrio cross 4. Tn5 has beenshown to insert itself in the V.choleraechromosome andcreate mutationsin target genes (38).However,wecouldnot score exconjugants in cross 5 suggesting the lack ofa gene mobilization system in the potential conjugants.Accordingly,no transfer of aplasrnidmarkerto strain 81could be detected in cross 3.

Conjugative reversion of a vaccine strain wouldgenerate a strain already existing in nature. Theenvironmental effect of this outcome is doubtful in thefate of an epidemic where a singleperson can excrete 108- 109virolentvibriosper day. Instead,more concernshould be given to recombination between V. choleraestrains in their natural reservoirs. We suggest that theintroduction of accessory mutations in our candidatevaccine strains to decrease their potential for geneticrecombinationisnotnecessary. Moreover,furthergeneticmanipulation could have counterproductive effects onprotective efficacy, the main variable in vaccinedevelopment. It has been demonstrated that a recA-

FA (m L/cm)1.8

a b g hd e

Stra!n

e

Figure 4. Toxinproduction in vivomeasured in ligated rabbitilealloops. a, C7258; b, 81; c, 82; d, 88; e, C6706; f, 413; g,417; h, CVD109 (negative control).

Table 3

Potential for Acquisition of Foreign Genes by Candidate Vaccine Strains

Selection CDa CRbRf + polB <5 x 10-1\ 2 X10-9Rf + polB <3 x lO-lO 2 X10-9Ap + Rf <1 X lO-lO <1 X 10-9Ap + Rf 5 X lO-lo <1 X 10.9Km + Rf 7 X 10-1\ <3 X10-12

LIVE ATIENUA TED CANDIDATE CHOLERA VACCINES

No.12345

Donor XReceptor569BRf' x 81569BRf'P+ x 81

1837/pJS752-3 x 81Rf'1837::pAJF x 81Rf'JBK70::Tn5 x 81 Rf'

281

DxR<

2 X 10-9

2 X 10-9

<1 x 10.10

5 X lO-lo

<3 x 10.12

aCD = frequency of spontaneous mutation for donor strain.bCR =frequency of spontaneous mutation for the receptor strain.<Dx R = frequency of colonies appearing on crossings plates.

mutation decreases intestinal colonization andimmunogenicity of classical and El Tor vibrios (56,57).

The development of genetically attenuated El Torstrains from epidemic strains is likely to provide asuitablestartingmaterialforfurtherattenuationorgeneticmodificationthatcouldultimatelyleadtothedevelopmentof an effective, well tolerated and affordable vaccineagainst cholera. The El Tor Ogawa live attenuatedcandidate vaccine strain 81 has been recently shown tobe highly immunogenic and protectivein the adultrabbitmodel developed by Cray et al. (58).

Acknowledgments

We are grateful to Dr. Carlos Gutiérrez Calzado andDr. Gustavo Sierra González for their support,encouragement and many helpful discussions. We alsothankDr. Richard A.Finkelstein (UniversityofMissouri- Columbia School ofMedicine) for making the El Torstrains C7258 and C6706 availableto usand toDr. JamesB. Kaper (Center for Vaccine Development, UniversityofMaryland, Baltimore) for providing us with plasmidpBB6 used as source of toxin genes.

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