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Vaccine 27 (2009) 7073–7079

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

255 is a key residue for recognition by a monoclonal antibody which protectsgainst Yersinia pestis infection

im Hill a, Sophie Learya, Sophie Smithera, Angus Besta, Jonas Petterssonb, Ake Forsbergb, Bry Lingarda,lexandria Lipkac, Katherine A. Brownc, E. Diane Williamsona,∗, Richard W. Titballd

Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UKSwedish Defence Research Agency, Division of NBC-Defence, SE-901 82 Umeå, SwedenDepartment of Life Sciences, Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UKSchool of Biosciences, Geoffrey Pope Building, University of Exeter, Exeter, Devon EX4 4QD, UK

r t i c l e i n f o

rticle history:eceived 7 May 2009

a b s t r a c t

Mab7.3 to Yersinia pestis LcrV antigen (LcrVYpe) protected J774A.1 macrophages in vitro from killing bya Yersinia pseudotuberculosis strain expressing LcrVYpe. Of 4 site-directed mutations in the coiled-coil

eceived in revised form 2 September 2009ccepted 16 September 2009vailable online 26 September 2009

eywords:lague

region (148–169) and 7 mutations in the 225–255 sequence of LcrVYpe, only the mutation of N255 toD255, abrogated the binding of Mab7.3 and reduced its protective capacity against plague. Since theMab7.3 epitope in LcrVYpe (135–275) encompasses a region (136–180) thought to be exposed on theinjectisome, we suggest that Mab7.3 protects by binding to LcrVYpe and interfering with protein–proteininteractions necessary for type three secretion.

rotectiononoclonal antibody

. Introduction

Three Yersinia species cause disease in humans. Yersinia pestisauses plague, whereas the closely related Yersinia pseudotuber-ulosis and Yersinia enterocolitica cause normally non-fatal gastricllnesses [1]. DNA sequence analysis suggests Y. pestis recentlyvolved from a Y. pseudotuberculosis strain through a series of genenactivation, deletion, and acquisition events [2]. All of the human-athogenic Yersinia share a 70 kb plasmid (termed pCD1 or pYV1)hat encodes a type III secretion system (TTSS) exporting a varietyf Yersinia outer proteins (Yops), including LcrV [3]. Significantly,. pestis has acquired two additional plasmids (pPCP1 and pMT1)hat contribute to virulence but are not present in other human-athogenic Yersinia.

TTSS is an important virulence mechanism found in severalram-negative animal and plant pathogens. TTSS operons encode aecretion complex that mediates the secretion of effector proteinscross the bacterial inner and outer membranes and a translocation

pparatus for the delivery of effectors across host cell membranes.dditional regulatory proteins ensure optimal translocation of theffector proteins, causing pathogen-specific effects in target cells4].

∗ Corresponding author. Tel.: +44 1980 613895; fax: +44 1980 614307.E-mail address: dewilliamson@dstl.gov.uk (E.D. Williamson).

264-410X/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Ltd. All rioi:10.1016/j.vaccine.2009.09.061

Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved.

TTSS was first discovered in Y. pseudotuberculosis and Y. ente-rocolitica and has been extensively characterised [3–5]. Yersiniatranslocate effector proteins (termed Yops) that inhibit phagocy-tosis and induce apoptosis in host cells [3,4]. Proteins encodedby the lcrGVHyopBD operon play a key role in Yersinia TTSS.LcrV is reported to be functional both intra- and extra-cellularly.Intracellularly, it acts as a regulator, by removing the nega-tive regulation imposed by the LcrG protein [6]. Regions ofLcrV required for LcrG binding map to the �7 alpha helix ofLcrV [7,8]. LcrV has been shown to be present on the surfaceof Y. pseudotuberculosis prior to contact with J774A.1 cells [9],and as a component of a needle-like structure in Y. enterocolit-ica [10]. YopB and YopD mediate effector Yop translocation byforming a pore in host membranes, through which Yops are deliv-ered [10–13]. SycD (LcrH) negatively regulates Yop secretion andalso chaperones and stabilises YopB and YopD, before secretion[14].

Immunisation with LcrV protects animals against plague [16]and in combination with the F1 antigen, high levels of protectionhave been demonstrated [17,18] which correlated with the anti-body titre to F1- and V-antigens [19]. Passive protection againstplague with polyclonal serum or monoclonal antibody (Mab)

directed against LcrV has also been reported [20–22] and mice havebeen protected against pneumonic plague by the direct deliveryof antibody to the lungs [23]. Several mechanisms for antibody-mediated protection have been suggested including enhancedphagocytosis of Y. pestis [24] or neutralisation of TTSS and/or its

ghts reserved.

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Table 1Identification of anti-LcrV cross-reactive monoclonal antibodies by Westernblotting.

Mab Protective? Recognition of GST-fusion protein in Western blotsa

rVype rVyptb rVyen03 rVyen08

7.3 Yes ++ − − −108.3 No + − − −

1.3 No ++ + − ++15.3 No ++ +/− − +48.3 No ++ ++ − −66.3 No + ++ +/− −

102.3 No +++ +++ − −103.3 No ++ − + +107.3 No ++ + + −109.3 No ++ ++ + −110.3 No +++ +++ +/− +++120.3 No + + ++ −

11.3 No + ++ ++ ++16.3 No ++ ++ ++ ++17.3 No ++ ++ + +24.3 No +++ +++ ++ ++28.3 No +++ +++ +/− +++45.3 No +++ +++ ++ ++46.3 No +++ +++ +++ +++47.3 No ++ ++ ++ ++50.3 No + ++ ++ ++51.3 No ++ ++ + ++68.3 No +++ +++ +++ +++

101.3 No ++ ++ + +104.3 No ++ ++ ++ ++121.3 No +++ +++ +++ +++

074 J. Hill et al. / Vaccin

ocal immunomodulatory effects or blockade of the TTSS transolo-ation pore [25].

Several naturally occurring variants of LcrV have been reportednd these sequence differences have functional consequences.ajor sequence differences in LcrV occur in a hypervariable cen-

ral region (residues 225–232) [21]. The LcrV from Y. enterocoliticaerotype 03, Y. pestis and Y. pseudotuberculosis lacks a 9-residuensertion at position 228 in the Y. enterocolitica serotype 08 LcrV21]. In addition to this major difference, amino acid substitutionsave been reported between the LcrV’s from different species andtrains. Passive protection with anti-LcrV sera from Y. enterocol-tica serotypes 03 or 08 suggested these two forms of LcrV aremmunologically distinct [21]. However, a further study showedhat cross-protection could be attained [26]. These data suggestmportant immunological differences between LcrV proteins fromersinia species and serotypes but that they may share someommon protective epitopes. Furthermore, antiserum against Y.seudotuberculosis LcrV, then adsorbed with truncated forms ofcrV, protected mice against plague in passive protection stud-es and residues in the region 168–275 on LcrV were deduced toontribute to protection [22]. Interestingly, the relative ability ofhe different forms of LcrV to promote IL-10 production dependsn amino acid sequence differences in the region 31–57, and theropensity to interact with TLR2 [15]. LcrV proteins derived from. enterocolitica O:8 serotypes promote high levels of IL-10 produc-ion [27] in comparison to LcrV from Y. pestis which promotes theeast [15].

We have previously shown that Mab 7.3 to Y. pestis LcrVrotected mice against Y. pestis infection and recognised a con-ormational epitope between residues 135 and 275 [28]. Mab7.3as more effective at protecting cells against the cytotoxic effects

f Y. pestis than those of Y. pseudotuberculosis [29] in TTSS tis-ue culture assays. This observation suggested to us that Mab7.3ecognised an epitope that was distinct in Y. pestis LcrV. Here,e present evidence that Mab 7.3 binds to an epitope which

ncludes residue N255, but which requires adjacent regions for itsntegrity.

. Materials and methods

.1. Recombinant proteins and site-directed mutants

The sources of reagents are shown (Table 1). The recombi-ant LcrV protein of Y. pestis (strain GB) sequence (rVype) wasxpressed as a glutathione-S-transferase (GST) fusion in Escherchiaoli, cleaved and purified as previously described [30]. The deducedmino acid sequence of strain GB is identical to the sequence of LcrVrom strain CO92.

A Y. pseudotuberculosis YPIII LcrV deletion mutant was con-tructed as previously described [9]. Briefly, in order to express Y.estis lcrV within YPIII/pIB19, a region of lcrV together with ∼400 bppstream and downstream flanking sequences was amplified byCR. The resulting ∼1800 bp fragment was introduced into SalI/SacIigested pDM4 to generate a plasmid which was electroporated

nto S17-1�pir, then conjugated into YPIII/pIB19 to achieve a strainf Y. pseudotuberculosis that expressed Y. pestis lcrV.

The LcrV proteins of Y. pseudotuberculosis YPIII or Y. enterocolitica08 (serotype O3) or Y. enterocolitica NCTC 10938 (serotype O8)ere expressed in E. coli as GST-fusion proteins from pGEX-5X-2

nd cleaved with Factor Xa to yield rVyptb, rVye03 and rVye08. The

ite-directed mutants of rVype (Q225A, E, N; V226A, I, L; G238A, E,; S229A, G, T; D238A, E, N; G241A, N, S; N255A, D, Q) were cloned

nto pGEX-5X-2 and expressed in E. coli as fusion proteins with GST.he proteins were cleaved with Factor Xa and purified as previouslyescribed [30].

131.3 Not tested ++ ++ ++ ++

a Recognition of protein was assessed as strong (+++), moderate (++), weak (+),equivocal (+/−) or nonexistent (−).

2.2. Screening of Mab’s for binding to LcrV from Yersinia

A total of 27 supernatants from hybridomas secreting anti-LcrV Mab’s [28] was tested by Western blotting for recognitionand binding in vitro to purified r LcrV from Y. pestis (strain GB),Y. pseudotuberculosis (strain YPIII), or Y. enterocolitica (strains 108or NCTC 10938). The concentration of tissue culture supernatantswas determined by BCA assay (Perbio). Culture supernatant (350 �gtotal protein) containing individual Mab 104.3, 103.3, 108.3, 1.3,17.3, 47.3, 45.3, 51.3, 107.3, 15.3, 109.3, 101.3 or Mab 7.3 (pos-itive control) or culture medium only (negative control) wasadministered in 0.1 ml i.p. to Balb/c mice (6/group) 24 h prior tochallenge s.c. with 12 median lethal doses (MLD) (12 cfu) of Y. pestisstrain GB. The mice were observed for 14 days and any animalreaching a humane end-point within this period was humanelyculled.

2.3. Screening of combinations of culture supernatants in a gridarray

Culture supernatants from a further 16 Mab’s were pooled infours in a grid array. Each pool, comprising 350 �g of protein fromeach supernatant was administered i.p. to 6 Balb/c mice, 24 h befores.c. challenge with 12 MLD Y. pestis GB. The mice were subsequentlyobserved and any animal reaching a humane end-point within thisperiod was humanely culled.

2.4. ELISA

ELISA plates were coated with 5 �g/ml LcrV proteins(0.1 ml/well). Plates were blocked with 2% (w/v) skimmedmilk powder in phosphate buffered saline (Blotto; 2 h, 37 ◦C).Plates were washed with PBS plus 0.02% (v/v) Tween 20 beforeadding test Mab (1:100 in Blotto). Each Mab was serially diluted

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n Blotto and incubated (1 h, 37 ◦C). Plates were washed againefore the addition of anti-mouse IgG HRPO (Amersham) at 1:1000nd incubated (45 min, 37 ◦C). The substrate 2,2′-azino-bis(3-thylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma) was added toach well (0.1 ml) and absorbance measured (414 nm).

.5. Competitive ELISA

A competitive ELISA was used to determine the binding of aonstant concentration of Mab 7.3 to immobilized LcrVYpe in theresence of increasing concentrations (1–100,000 ng) of either sol-ble LcrV or the mutants N255A, Q or D. The assay was repeatedtimes. The concentrations of the mutant LcrVs which gave 50%

nhibition of Mab7.3 binding were calculated and mean data withtandard errors were derived.

.6. Screening of Mab’s for the inhibition of cytotoxicity ofersinia-expressed LcrV

A tissue culture assay that measures the cytotoxicity of Y.estis towards J774A.1 macrophages has previously been reported29,31]. This was modified as follows. Briefly, J774A.1 cells wereeeded (5 × 105 cells/ml) into 24-well tissue culture plates. Cellsere grown in DMEM + 5% FCS at 37 ◦C statically with 5% CO2. An

vernight culture of Y. pseudotuberculosis YpIII(pIB19)(ptrcVype)a Y. pseudotuberculosis LcrV mutant expressing the LcrV generom Y. pestis) was grown in 10 ml LB supplemented with ampi-illin (50 �g/ml) and kanamycin (25 �g/ml). The OD600 value ofhe overnight culture was determined and 30 �l of bacteria atD600 value 2.5 were added per 3 ml DMEM (without serum).acteria were incubated (26 ◦C, 30 min), then at 37 ◦C for 60 minith shaking at 200 rpm. J774A.1 cells were washed with PBS to

emove non-adherent macrophages. Bacteria were diluted 1/100n DMEM + 5% FCS, test antibody added at 1/10 (v/v), and incubated

ith bacteria for 5–10 min at 37 ◦C. The mixture was then added toashed J774A.1 cells. After 1 h, gentamicin was added (100 �g/ml)

nd the cells were incubated for a further 3–4 h. Yersinia-mediatedytotoxicity was visualised through a conventional staining process31] using mixed ethidium bromide and acridine orange (20 �g/mlach) which discriminates between live and dead cells on theasis of membrane integrity, since acridine orange can cross theell membrane to stain living cells green whilst ethidium bro-ide intercalates with DNA to stain dead cells orange, since it

s excluded by viable cells. Confocal microscopy was used to co-isualise the presence of the fluorescent dyes, by exciting at aiven wavelength and recording the emission of green fluores-ence by healthy cells at 488 nm or red fluorescence by dead cellst 630 nm.

.7. Site-directed mutation of LcrV

LcrV protein mutants were prepared based on the Y. pestis lcrVequence [21]. The lcrV gene, in plasmid pVG100, was initiallyub-cloned into bacteriophage M13MP19 vector as an Eco-RI–SalIragment to generate a single-stranded mutagenesis template.fter mutagenesis (Muta-Gene in vitro mutagenesis kit, Bio-Rad),

he selected mutants were sub-cloned back into the expression vec-

or, pGEX-5X-2, as an Eco-RI–SalI fragment and expressed in E. coliM109, under the same conditions as for the wild type LcrV protein16].

Subsequently all the plasmids containing a mutated lcrV genensert were assessed by restriction enzyme digestion and DNAequencing for correct insert size and to verify the presence of theesigned mutation, respectively.

2009) 7073–7079 7075

2.8. Recognition of LcrV variants and mutants by Mab’s

Standard Western blotting procedures were used. Briefly, pro-teins were separated by SDS-PAGE on 12.5% PhastGels (Pharmacia)and transferred to HybondN membranes as recommended by themanufacturer (AmershamPharmacia). Glutathione-S-transferase(GST) protein was used as a negative control. Anti-LcrV Mabs wereused as a primary antibody at 1:1000 dilution in PBS plus 0.1%Tween 20 (PBS-T), with shaking (25 ◦C, 90 min). Following threewashes with PBS-T, a secondary anti-mouse-HRP conjugate (Amer-shamPharmacia) was added (1:2000 in PBS-T, 45 min). After afurther three washes with PBS-T, ECL detection (AmershamPhar-macia) was used to visualise the recognition of LcrV by Mab’s asrecommended by the manufacturer.

2.9. Protective efficacy of N255 mutants

An in vivo study was designed to achieve a screening assay forgross differences in protective efficacy between LcrV and the N255mutants.

Mutant forms of Y. pestis LcrV (N255A, D or Q) were formu-lated in 0.26% (w/v) alhydrogel in PBS and each used to immunise4 groups of 6 Balb/c mice with 10 �g protein by the intra-muscularroute on days 0 and 21. Additionally, 1 group of 6 mice was immu-nised with wild-type LcrV and a further group of 6 mice remainedunimmunised. All groups of immunised mice were challenged onday 60 with Y. pestis GB by the s.c. route at dose levels in the range10–107 cfu in 0.1 ml (equivalent to 10–107 MLD) [31].

2.10. In silico analysis

A crystal structure for LcrV has been reported [7] and regions ofLcrV important for function and the induction of protective immu-nity can be mapped onto this structure. Entry 1R6F was obtainedfrom the Protein Databank (www.rcsb.org). All modeling and visu-alisation was carried out using SYBYL 7.1 (Tripos Ltd., MiltonKeynes, UK). Missing or incomplete sidechains were reconstructedfrom the standard sidechain conformation database. Residues priorto residue 28 and following residue 322 were not present in thecrystal structure and no attempt was made to model the N- or C-termini. A single chain model was created using SYBYL’s loop searchprocedure to model undefined loop regions as a precursor to energyoptimization and interpretation. Whilst modeling of the importantundefined loop 263–275 cannot be assumed absolute, the presenceof a feasible loop structure acts to anchor the region and stabiliselocal interactions. Interpretation of the model structure was used toelucidate regions of interaction for maintaining stability and under-standing the local interaction impact of mutations. Mutant N255structures were generated by sidechain replacement and local opti-mization to elucidate local interactions and to visualise effects onprotein surface properties that might relate to binding.

2.11. Statistical analysis

1-Way ANOVA of transformed data was used to determinethe statistical significance of differences in the mean protein con-centrations required to inhibit by 50% the binding of Mab7.3 toimmobilized LcrV in competitive ELISA, compared with LcrV.

3. Results and discussion

3.1. Screening of anti-LcrV Mab’s for binding and protectiveefficacy

A panel of Mab’s raised against recombinant Y. pestis LcrV(rVype) was tested for their recognition of variant forms of LcrV

7076 J. Hill et al. / Vaccine 27 (2009) 7073–7079

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ig. 1. Inhibition of the cytotoxicity of Y. pseudotuberculosis expressing Y. pestis Lcb) cells exposed to Y. pseudotuberculosis expressing Y. pestis LcrV. In Panels (c) andromide and acridine orange.

n different strains and species of the human-pathogenic YersiniaTable 1). All of the antibodies recognised rVypeand all of the Mabspart from 7.3 and 108.3 recognised at least one other form of LcrVs rVyptb, rVyen03, rVyen08 (Table 1). All of the Mabs were of IgG1sotype except 102.3 which was an IgG2.

Individual culture supernatants containing Mabs 104.3, 103.3,08.3, 1.3, 17.3, 47.3, 45.3, 51.3, 107.3, 15.3, 109.3, 101.3 or 7.3positive control) and each comprising 350 �g of total protein, weredministered to groups of 6 Balb/c mice prior to a challenge with. pestis. Mab7.3 protected 5 of 6 animals, but none of the otherabs conferred protection. Subsequently, pooled culture super-

atants were each administered to a group of 6 Balb/c mice, beforehallenge with Y. pestis. Only those pools which contained Mab 7.3rotected mice against plague. Thus Mab7.3 was of interest for fur-her study since it was the only Mab which was protective and waslso exclusively specific for Y. pestis V-antigen.

.2. Mab 7.3 protects cells from killing by inhibiting the functionf the TTSS

To devise an assay for measuring the protection afforded byab’s, without the need to use Y. pestis, we exploited the func-

ig. 2. Alignment of the predicted amino acid sequences of Y. pestis LcrV (Vype; accessiseudotuberculosis YPIII (Vyptb; accession AAA27645 ), Y. enterocolitica 108 serotype O3 (ccession CAA65589 ). Residues targeted for mutagenesis in this study are shown in bold

ards J774A.1.1 macrophage like cells. Panel (a) shows uninfected cells and Panellls were pre-treated with MAb 7.3 or Mab 110.3. Cells were stained with ethidium

tional conservation of the lcrGVHyopBD operon between Y. pestisand Y. pseudotuberculosis. A Y. pseudotuberculosis lcrV mutant(YpIII (pIB19)) was shown to be effective at killing macrophages.J774A.1.1 cells were protected against killing in vitro by Mab7.3,but not by Mab 110.3 (Fig. 1). Previously it has been shown thatantiserum to LcrV which is able to protect mice from a Y. pestischallenge also inhibits cytotoxicity in this assay [9,29], whethersourced from immunised humans or macaques [31].

3.3. N255 is critical for Mab 7.3 binding

We have previously shown that Mab7.3 recognises a fragmentof LcrV encompassing residues 135–275 [28]. As Mab7.3 did notrecognise the rVyptb, rVyen03 or rVyen08 variants of LcrV, we spec-ulated that residues within this region are important for binding.By comparing the LcrV amino acid sequence of residues 135–275 in4 homologues from Y. pestis strain GB, Y. pseudotuberculosis strain

YPIII, Y. enterocolitica serotypes 03 and 08 (Fig. 2) and knowing thatMab7.3 bound only the Vype sequence (Table 1) it was possible topredict that 7 key residues in the region of greatest sequence vari-ability (225–255) could influence the binding of Mab7.3 to LcrV.These residues in Y. pestis LcrV are Q225, V226, G228, S229, D238,

on CAB54908 ) residues 135–275 with the corresponding regions of LcrV from Y.Vyen03; accession CAA65591 ) or Y. enterocolitica NCTC 10938 O8 serotype (Vyen08;and underlined.

J. Hill et al. / Vaccine 27 (

Fig. 3. Panel I: Western blot of N255 variant proteins (N255A, D and Q), GST or wildtype LcrV (N255) with polyclonal antibody to LcrV (polyclonal �-Vag), Mab107.3or Mab7.3. Proteins were diluted to 0.5 mg/ml, run on 8–25% SDS Phast gels andWestern blotted. Panel II: Western blot of site-directed mutants in the coiled-coilregion of the �7 alpha helix of LcrV, with Mab7.3. WT = LcrV wild type; lane 1:LLP

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N255 is located at the N-terminal end of a short �-sheet (�6) that

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crV L153R; lane 2: LcrV L157R; lane 3: LcrV L160R; lane 4: LcrV L164R: lane 5:153R/L157R/L160R/L164R. Proteins were diluted to 0.5 mg/ml, run on 8–25% SDShast gels and Western blotted.

241 and N255. The gene encoding Y. pestis LcrV was mutated toeplace each of these residues with alanine or with a conservativeubstitution. All of the mutant proteins (Q225A, E or N; V226A, I or; G228A, E or N; S229A, G or T; D238A, E or N; G241A, N, or S and255A, D or Q) were recognised by Mab7.3 by Western blotting,xcept N255A, D and Q (Fig. 3, Panel I).

A competitive ELISA was performed to assess the impact thathanges at N255 had on antibody binding based on quantifyinghe amount of LcrV protein required to inhibit binding of Mab 7.3o immobilized rVype by 50%. As anticipated, higher amounts of255A, D or Q compared with LcrV were required to inhibit binding

f Mab7.3 (Table 2) and these were significant for N255D (p < 0.05)nd for N255Q (p < 0.01) by 1-way ANOVA of transformed data. Thus255 plays a key role in Mab7.3 binding and it is conserved in all

eported sequences of LcrV from different strains of Y. pestis.

able 2ompetitive binding of LcrV and mutants to Mab7.3 and protective efficacy in Balb/c mic

LcrV or mutant protein Mean protein concentration ± standard error of the mean (�of Mab7.3 to immobilized LcrV in competitive ELISA (signific

None –LcrV 0.08 ± 0.03

N255A 1.2 ± 0.8 (NS)

N255D 5.0 ± 3.0 (p < 0.05)

N255Q 20.0 ± 15.0 (p < 0.01)

2009) 7073–7079 7077

3.4. Protective efficacy of N255 mutants

No gross differences in protective efficacy were found for micewhich had been immunised with mutated forms of LcrV (N255A,D or Q) compared with LcrV, and all were protected against a sub-sequent challenge with 105 MLD Y. pestis. However, at the higherchallenge level of 107 MLD, mice immunised with N255D or Q werenot completely protected (Table 2) although future work to com-pare the protective efficacies of N255 mutants statistically, wouldrequire enlarged group sizes.

Previous passive protection studies have shown that the LcrVfrom Y. pseudotuberculosis and Y. enterocolitica have common pro-tective B cell epitopes [26]. Antiserum raised against a ProteinA-LcrV fusion protein (PAV) containing residues 68–326 of LcrVfrom Y. pseudotuberculosis strain 995 passively protected miceinfected with Y. pestis KIM or Y. pseudotuberculosis PB1/+, but not Y.enterocolitica WA, a highly virulent O:8 serotype strain [22]. Theseworkers also demonstrated that protective B cell epitopes sharedby Y. pseudotuberculosis and Y. pestis mapped to residues 168–275,highlighting the importance of this central region of LcrV for pro-tection against plague [22].

3.5. Prediction of structural effects of N255 mutation

The minimal LcrV fragment (residues135–275) recognised byMab 7.3 includes the �7 alpha helix (residues 148–182) thatcontains a coiled-coil (residues 148–169). The coiled-coil has pre-viously been shown to be important for the binding of LcrV to LcrGand for LcrV–LcrV interactions [8] and a truncated form of LcrV, con-taining residues 168–326 from which most of the coiled-coil hadbeen deleted, retained only weak binding for Mab7.3 [8]. In thisstudy, the influence of point mutations in the coiled-coil (residues148–169) as well as in the hypervariable region (residues 225–241)has been examined by Western blotting. Since point mutations inthe coiled-coil region did not significantly affect Mab7.3 binding(Fig. 3, Panel II) and neither did the mutation of residues other thanN255 in the hypervariable region, residue N255 was singled out forfurther study.

In the crystal structure of a monomeric form of LcrV [7] residue

pairs in an anti-parallel manner with another short �-sheet (�3),that follows the �7 helix. There is evidence that LcrV exists as ahomodimer or heterodimer with LcrG in solution [8]. These dimericforms of LcrV are believed to be associated with the regulatory func-

e immunised with LcrV mutants.

g) required to 50% inhibit the bindingant difference compared with LcrV)

Y. pestis GB (MLD) Survivors/group

10 0/610 6/6103 6/6105 6/6107 6/6

10 6/6103 6/6105 6/6107 6/6

10 6/6103 5/6105 6/6107 4/6

10 6/6103 5/6105 6/6107 4/6

7078 J. Hill et al. / Vaccine 27 (2009) 7073–7079

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ig. 4. Model structure of the LcrV protein. Panel I: LcrV 28–322 with structural elore and upper (N255 shown). Panel II: coordination around N255 and beta strands �.3 binding face.

ion of LcrV within the bacterial cell, and it is not known whetherimeric LcrV (or LcrV–LcrG heterodimers) occur extra-cellularly.

t is not clear whether N255 would be accessible to antibody in

imeric LcrV or in LcrV–LcrG heterodimers, but evidence suggestshat this region is accessible to Mab7.3. The data presented herehow that soluble N255 variant proteins show only a modest reduc-ion in ability to compete with wild type immobilized LcrV forinding to Mab7.3, compared with wild type soluble LcrV. Also,

s labelled, long helices (�7 and �12) to rear and proposed Mab 7.3 binding face to�6 with hydrogen bonds displayed as green dotted lines, viewed to proposed Mab

Tito et al. [32] have previously reported evidence from mass spec-troscopy for the binding of Mab7.3 to LcrV dimer in solution.

Of greater functional relevance to the role of antibody to LcrV

in protective immunity is the possible structure of LcrV at the tipof the injectisome complex [10,33]. This structure, proposed fromscanning transmission electron microscopy (STEM) imaging andfrom data obtained with hybrids of LcrV, PcrV (Pseudomonas aerug-inosa) and AcrV (Aeromonas salmonicida), suggests that LcrV exists

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J. Hill et al. / Vaccin

s a pentamer at the injectisome tip [33]. This orientation wouldxpose the proposed Mab7.3 binding site. Further insights into theedundancy or otherwise of residues in the epitope for Mab7.3 areeing gained by the systematic removal of residues from the N- and-termini of the sequence [34].

Overall our data indicate that the epitope in LcrV for Mab7.3s conformational. It may be speculated that the C-terminal por-ion of helix �7 and the residue N255 are crucial to maintaininghe spatial integrity of the protective epitope for Mab7.3. Given theverlap between regions of LcrV thought to be important for TTSSresidues 136–180) [8], antigenicity (residues 135–245) [28] and forrotection (135–275) [28] (Fig. 4) along with these new data pre-ented here, we suggest that Mab7.3 mediates protection against. pestis by interfering with protein–protein interactions importantn TTSS. This may occur in vivo through disruption of LcrV–LcrV

ultimerization, by blocking binding of LcrV to other componentsf the Yersinia TTSS, or LcrV–host interactions. This is supportedy the observations that passive protection can be achieved withntibody delivered late in infection [23] and that protection wasimilar in wild type and IL-10 deficient mice [15], suggesting thatntibody did not neutralise the immunosuppressive properties ofcrV, but may have the effect of blocking Yop delivery to host cells,chieved by binding to exposed LcrV at the injectisome tip. The needo use significantly increased quantities of those proteins mutatedt N255, to compete with binding of Mab7.3 to LcrV, underlines themportance of this residue in the protective epitope. However, thisoes not rule out the possibility that the epitope on LcrV definedy Mab7.3 is not the single major or only protective epitope oncrV.

cknowledgements

We are grateful for the excellent technical support of Jasonewer. This work was funded by the UK Ministry of Defence and

he Biotechnology and Biological; Sciences Research Council.

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